Research ArticleThe Parenteral Vitamin C Improves Sepsis andSepsis-Induced Multiple Organ Dysfunction Syndrome viaPreventing Cellular Immunosuppression

Yu-Lei Gao Bin Lu Jian-Hua Zhai Yan-Cun Liu Hai-Xia Qi Ying YaoYan-Fen Chai and Song-Tao Shou

Department of Emergency Medicine Tianjin Medical University General Hospital Tianjin 300052 China

Correspondence should be addressed to Yu-Lei Gao gaoyulei828126com and Song-Tao Shou stshou66sinacom

Received 11 October 2016 Revised 21 December 2016 Accepted 27 December 2016 Published 22 January 2017

Academic Editor Mirella Giovarelli

Copyright copy 2017 Yu-Lei Gao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cellular immunosuppression appears to be involved in sepsis and sepsis-induced multiple organ dysfunction syndrome (MODS)Recent evidence showed that parenteral vitamin C (Vit C) had the ability to attenuate sepsis and sepsis-inducedMODS Herein weinvestigated the impact of parenteral Vit C on cellular immunosuppression and the therapeutic value in sepsis Using cecal ligationand puncture (CLP) sepsis was induced in WT and Gulominusminus mice followed with 200mgKg parenteral Vit C administration Theimmunologic functions of CD4+CD25+ regulatory T cells (Tregs) and CD4+CD25minus T cells as well as the organ functions weredetermined Administration of parenteral Vit C per se markedly improved the outcome of sepsis and sepsis-inducedMODS ofWTand Gulominusminus mice The negative immunoregulation of Tregs was inhibited mainly including inhibiting the expression of forkheadhelix transcription factor- (Foxp-) 3 cytotoxic T lymphocyte associated antigen- (CTLA-) 4 membrane associated transforminggrowth factor-120573 (TGF-120573m+) and the secretion of inhibitory cytokines [including TGF-120573 and interleukin- (IL-) 10] as well as CD4+

T cells-mediated cellular immunosuppressionwhichwas improved by parenteral Vit C inWTandGulominusminus septicmiceThese resultssuggested that parenteral Vit C has the ability to improve the outcome of sepsis and sepsis-induced MODS and is associated withimprovement in cellular immunosuppression

1 Introduction

Sepsis which is defined as the life-threatening organ dysfunc-tion caused by the dysregulated host response to infectionis the leading cause of MODS and death among criticallyill patients [1ndash4] There was a significant loss of immuno-cytesmainly includingBT-lymphocytes and gastrointestinalepithelial cells even at the beginning of sepsis [5ndash8] Ithas been noted that patients gradually enter into a stateof immunosuppression after primary hyperinflammatoryresponse especially cellular immunosuppression defined asimmunoparalysis which could be a significant cause ofthe exacerbation of MODS or even death out of sepsis[2 4] More and more researches showed that modulatingthe immunosuppressive stage might be a promising inter-ventional strategy to improve the outcome of sepsis [9]With sepsis Tregs subdued the process of inflammation

and tissue damage while also causing immune dysfunctionincluding induction of T-lymphocytic apoptosis inhibitionof CD4+CD8+ T-lymphocytic function and mediation ofshifting from the helper T cell (Th) 1 to Th 2 response viathe expression of CTLA-4 and TGF-120573m+ as well as anti-inflammatory cytokines (IL-10 and TGF-120573) [10ndash15]

Vit C characterized as essential endogenous trace ele-ment and important physiological antioxidant is involvedin the incidence of sepsis and sepsis-induced MODS evendirectly with the survival of septic patients [16 17] Vit Cis identified as an essential component of the immuno-logical response in humanity and experimental animalswhich augmented a variety of immune reaction such asthe development of mouse bone marrow derived progenitorcells to functional BT-lymphocytes the balance of Th1Th2response and the increased serum levels of IgA and IgM aswell as regulating the balance of proliferation and apoptosis

HindawiMediators of InflammationVolume 2017 Article ID 4024672 12 pageshttpsdoiorg10115520174024672

2 Mediators of Inflammation

of T cells [18ndash21] In addition treatment with Vit C wascorrelated with the expression of Foxp-3 the demethylationof Foxp3-TSDR (Treg-specific demethylated region) and thesuppressive capacity of Tregs in vitro [22] Yet direct effectsof Vit C on sepsis-induced cellular immunosuppression arenot known Thus further investigating the impact of Vit Con sepsis-induced cellular immunosuppression will providea new evidence for understanding the mechanisms of Vit Cin the treatment of sepsis Humanity completely powerlesslysynthesizes Vit C on account of the deficiency of L-gulono-120574-lactone oxidase (Gulo) which is the pivotal enzyme inthe biosynthesis of Vit C therefore we conducted studies intransgenic mice lacking Gulo 4 (Gulominusminus) to investigate theimpact of parenteral Vit C on cellular immunosuppression insepsis and sepsis-induced MODS

In the present study using the classical septic model thatis CLP we demonstrated that administration of parenteralVit C per se markedly improved the outcome of sepsis andsepsis-inducedMODSofWTandGulominusminusmiceThenegativeimmunoregulation of Tregs was inhibited mainly includingdecreasing the expression of Foxp-3 CTLA-4 and TGF-120573m+and the secretion of inhibitory cytokines (TGF-120573 and IL-10) as well as improving CD4+ T cells-mediated cellularimmunosuppression by parenteral Vit C in WT and Gulominusminusseptic mice These results suggested that parenteral Vit Chas the ability to improve the outcome of sepsis and sepsis-induced MODS and associate with improvement in cellularimmunosuppression

2 Materials and Methods

21 Animals The Gulominusminus C57BL6J mice were propagatedfrom heterozygous Gulo+minus C57BL6J mice which werepicked up fromTheMutantMouse Regional Resource Center(httpswwwmmrrcorg No 000015-UCD) andweremain-tained on a C57BL6J background (The Laboratory AnimalCenter of Chinese Academy of Medical Sciences numberSCXK-Jing-2009-0007 Beijing China)TheWT andGulominusminusC57BL6J mice 6ndash8 weeks old 20 plusmn 2 g were maintainedin a specific pathogen-free condition They were maintainedfor about 1 week with Vit C supplementation (33 gL pickedup from Luwei Pharmacy Jinan Shan Dong China) intheir drinking water All procedures were undertaken inaccordance with the National Institute of Health Guide forthe Care and Use of Laboratory Animal and approved by theScientific Investigation Board and TianjinMedical UniversityGeneral Hospital Tianjin China

22 Medium and Reagents The medium was RPMI1640(Nanjing Keygen Biotech Nanjing China) with 10 fetalbovine serum (FBS Sigma St Louis MO) CD4+CD25+Tregs isolation kits were from Miltenyi Biotec GmbH Ber-gisch Gladbach Germany Cell counting kit-8 (CCK-8) wasfrom Dojindo Kumamoto Japan Fluorescein isothiocya-nate- (FITC-) conjugated Annexin-V apoptotic kit puri-fied hamster anti-mouse CD3CD28 FITC-conjugated anti-mouse CTLA-4 FITC-conjugated anti-mouserat-Foxp-3

and allophycocyanin- (APC-) conjugated anti-mouserat-TGF-120573m+ were from eBioscience San Diego CA ELISA kitsfor interferon- (IFN-) 120574 IL-4 IL-10 and TGF-120573 were fromExcell Biol Shanghai China Alanine transaminase (ALT)aspartate transaminase (AST) creatinine (Cr) and arterialblood gas (ABG) kits were from Instrumentation LaboratoryCompanyMAUSAAll primers and SYBRGreenReal-TimePolymerase Chain Reaction (RT-PCR)MasterMix were fromApplied Biosystems Carlsbad CA

23 Isolation of Splenic CD4+CD25+ Tregs and CD4+CD25minusT Cells CD4+CD25+ Tregs and CD4+CD25minus T cells wereisolated usingmouse CD4+CD25+ Tregs isolation kits whichwe have recounted [23 24]

24 Sepsis Model The classical septic model that is CLPis used which we have recounted in our previous studies toinduce sepsis [23 24]

25 Experimental Design 180 mice were divided into controlgroup (30 mice) WT mice with CLP group [WT (CLP) 50mice] WT mice with CLP and subcutaneous injection of200mgKg parenteral Vit C group [WT (CLP + Vit C) 50mice] Gulominusminus mice with CLP group [Gulominusminus (CLP) 50mice] and Gulominusminus mice with CLP and subcutaneous injec-tion of 200mgKg parenteral Vit C group [Gulominusminus (CLP +Vit C) 50 mice] The first administration time of parenteralVit C was immediately after CLP and parenteral Vit C wasgiven again after 12 hours The survival time and rate wererecorded for 72 hours in various groups CD4+CD25+ Tregsand CD4+CD25minus T cells were isolated from every group atthe point of 24 hours after CLP The proliferative activityapoptotic rate and secretion ability (including IFN-120574 andIL-4) of CD4+CD25minus T cells as well as the expression ofFoxp-3 Foxp3-TSDR Helios CTLA-4 TGF-120573m+ apoptoticrate and secretion ability (including IL-10 and TGF-120573) ofCD4+CD25+ Tregs were determined The serum levels ofAST ALT and Cr as well as ABG were determined by TheCentral Laboratory of Tianjin Medical University GeneralHospital

26 Flow Cytometric Analysis The expression levels ofFoxp-3 CTLA-4 and TGF-120573m+ of CD4+CD25+ Tregsas well as the apoptotic rates of CD4+CD25+ Tregs andCD4+CD25minus T cells were determined by flow cytometer(Becton-Dickinson) which we have recounted [23 24]

27 CCK-8 Measurement The proliferative ability ofCD4+CD25minus T cells was determined by CCK-8 which wehave recounted [23 24]

28 SYBR Green and Methylation-Sensitive RT-PCR 1 times 106cellsgroup were used for distilling the total RNA via the sin-gle technique of acid guanidinium thiocyanate-chloroformextraction according to the manufacturerrsquos instruction ThemRNA expressions of Foxp-3 Helios CTLA-4 TGF-120573 andIL-10 were tested by SYBR Green RT-PCR The primer

Mediators of Inflammation 3

Table 1 Targeted genes and their primer sequences for SYBR Green and Methylation-Sensitive Real-Time Polymerase Chain Reaction

Gene Primer sequences

Mouse Foxp-3 Forward 51015840-CAGCTGCCTACAGTGCCCCTAG-3Reverse 51015840-CATTTGCCAGCAGTGGGTAG-31015840

Mouse Helios Forward 51015840-TAAGCTCAGCTTATTCTCAGGTCTATCA-31015840Reverse 51015840-ATGTTGTTTTCGTGACTATCAGATGTT-31015840

Mouse CTLA-4 Forward 51015840-CGCAGATTTATGTCATTGATCC-31015840Reverse 51015840-TTTTCACATAGACCCCTGTTGT-31015840

Mouse TGF-120573 Forward 51015840-AACAATTCCTGGCGTTACCTT-31015840Reverse 51015840-GAATCGAAAGCCCTGTATTCC-31015840

Mouse IL-10 Forward 51015840-ACAGCCGGGAAGACAATAAC-31015840Reverse 51015840-CAGCTGGTCCTTTGTTTGAAAG-31015840

Mouse Foxp3-TSDR Forward 51015840-CTTGCTTCCTGGCACGAGATTTGAATTGGATATGGTTTGT-31015840Reverse 51015840-CAGGAAACAGCTATGACAACCTTAAACCCCTCTAACATC-3

sequences were listed in Table 1 The amplification of PCRconsisted of 1min denaturation step at 95∘C followed by 40cycles of 15 s at 95∘C and 40 s at 60∘C using the SequenceDetection System (Applied Biosystems) The methylationlevel of Foxp3-TSDR was determined by Methylation-Sensitive RT-PCR which has been recounted by Tatura et al[11] The primers for Foxp3-TSDR were listed in Table 1

29 ELISA Measurement The levels of IFN-120574 IL-4 IL-10and TGF-120573were determined by ELISA kits strictly accordingto the protocols provided by manufacturer using microplatereader at OD 450

210 Statistical Analysis Data were represented as mean plusmnstandard deviation (SD) and analyzed by software SPSS 170with one-way ANOVA Unpaired Studentrsquos 119905-test was used toevaluate significant differences between groups A 119901 value of005 or 001 was considered statistically significant Survivalrate in septicmice was evaluated by Kaplan-Meier via the log-rank test

3 Results

31 Parenteral Vit C Improved the Outcome of Sepsis andSepsis-Induced MODS As shown in Figure 1(a) comparedwith WT (CLP) group the deficiency of Vit C reducedthe 72-hour survival rate of Gulominusminus mice (119901 lt 005)administration of parenteral Vit C significantly increasedthe survival rate of WT and Gulominusminus mice (119901 lt 001) insepsis Compared with WT (CLP) group at 24 hours afterCLP the deficiency of Vit C worsened the organs function ofGulominusminus (CLP) mice with sepsis acting as the serum levelsof AST (Figure 1(b)) ALT (Figure 1(c)) Cr (Figure 1(d))and Lac (Figure 1(g)) were elevated but the levels of PCO2(Figure 1(e)) and PO2 (Figure 1(f)) were decreased (119901 lt005 or 001) administration of parenteral Vit C after CLPsignificantly improved the appellate quotas of organ functionof WT mice and Gulominusminus mice (119901 lt 005 or 001)

32 Parenteral Vit C Weakened the Stability of CD4+CD25+Tregs with Sepsis CD4+CD25+ Tregs were isolated from

every group the deficiency of Vit C inhibited the expressionof Foxp-3 (Figure 2(a)) CTLA-4 (Figure 2(b)) and TGF-120573m+

(Figure 2(c)) on splenic CD4+CD25+ Tregs of Gulominusminus mice(119901 lt 005 or 001) but the expressions of Foxp-3 CTLA-4and TGF-120573m+ were significantly weakened when parenteralVit C was administered after CLP to WT mice and Gulominusminusmice (119901 lt 005 or 001) Figure 2(d) showed that therewere nodifferences between WT and Gulominusminus mice on the apoptoticlevel of CD4+CD25+ Tregs with or without administeredparenteral Vit Cwith sepsis (119901 gt 005)The supernatant levelsof TGF-120573 (Figure 2(e)) and IL-10 (Figure 2(f)) were signifi-cantly increased fromCD4+CD25+ Tregs whichwere isolatedfrom Gulominusminus (CLP) groups after being cultured for 24 hours(119901 lt 001) but significantly decreased from CD4+CD25+

Tregs which were isolated from WT and Gulominusminus mice withadministration of parenteral Vit C (119901 lt 005 or 001)

33 Parenteral Vit C Inhibited the Immunosuppressive Func-tion of CD4+CD25+ Tregs CD4+CD25+ Tregs were cocul-tured with conventional CD4+CD25minus T cells for another24 hours in a ratio of 1 1 with anti-CD3 (5120583gmL) andanti-CD28 (2 120583gmL) for polyclonal activation of T cellsrespectively CD4+CD25+ Tregs which were isolated fromGulominusminus (CLP) group significantly inhibited the proliferativeactivity (Figure 3(a)) and release of IFN-120574 (Figure 3(c)) butenhanced the apoptotic rate (Figure 3(b)) and release ofIL-4 (Figure 3(d)) of CD4+CD25minus T cells (119901 lt 005 or001) however treatment with parenteral Vit C of WT andGulominusminus mice significantly inhibited the immunosuppressivefunction of CD4+CD25+ Tregs to CD4+CD25minus T cells whichincreased the proliferative response and release of IFN-120574 butdecreased the apoptotic rate and release of IL-4 of conven-tional CD4+CD25minus T cells (119901 lt 005 or 001)

34 The Impact of Parenteral Vit C on the mRNA Expres-sion of Foxp-3 Helios CTLA-4 TGF-120573 IL-10 and theMethylation Level of Foxp3-TSDR The deficiency of VitC significantly promoted the mRNA expression of Foxp-3(Figure 4(a)) Helios (Figure 4(c)) CTLA-4 (Figure 4(d))TGF-120573 (Figure 4(e)) and IL-10 (Figure 4(f)) but decreasedthe methylation level of Foxp3-TSDR (Figure 4(b)) in

4 Mediators of Inflammation

Frac

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Time after CLP (hours)0 20 40 60 80

WT (CLP + Vit C) WT (CLP)Gulominusminus (CLP + Vit C) Gulominusminus (CLP)

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f PCO

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Figure 1 Continued

Mediators of Inflammation 5

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f PO

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lowast

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The A

BG o

f Lac

(mm

olL

)

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(g)

Figure 1 The impact of parenteral Vit C treatment on the 72-hour survival rate of septic mice and sepsis-induced MODS The 72-hoursurvival rate of WT mice and Gulominusminus mice was improved on administration of 200mgKg parenteral Vit C twice after CLP (a) ParenteralVit C improved the biomarkers of liver (b and c) renal (d) respiratory (e and f) and circulatory (g) function in CLP-exposed WT mice andVit C deficient Gulominusminus mice The survival rate was analyzed by Kaplan-Meier via the log-rank test 119899 = 50 per group WT (CLP) versus WT(CLP + Vit C) 119901 lt 001 Gulominusminus (CLP) versus Gulominusminus (CLP + Vit C) 119901 lt 001 WT (CLP) versus Gulominusminus (CLP) 119901 lt 005 Data wererepresented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 orlowastlowast119901 lt 001

CD4+CD25+ Tregs of Gulominusminus (CLP) group in comparison toWT (CLP) group (119901 lt 001) Treatment with parenteral Vit Cafter CLP of WT and Gulominusminus mice inhibited the expressionof Foxp-3 Helios CTLA-4 TGF-120573 and IL-10 but enhancedthe methylation level of Foxp3-TSDR in CD4+CD25+ Tregs(119901 lt 005 or 001)

35 Parenteral Vit C ImprovedCD4+ TCells-MediatedCellularImmunosuppression CD4+CD25minus T cells were isolated fromevery group and cultured for another 24 hours The defi-ciency of Vit C significantly worsened the proliferative ability(Figure 5(a)) and IFN-120574 level (Figure 5(c)) but increasedthe release of IL-4 (Figure 5(d)) of splenic CD4+CD25minus Tcells of Gulominusminus mice (119901 lt 001) however treatment withparenteral Vit C afterCLP significantly enhanced the prolifer-ative response and IFN-120574 level but inhibited the release of IL-4 of CD4+CD25minus T cells ofWT and Gulominusminusmice (119901 lt 001)As shown in Figure 5(b) the deficiency of Vit C significantlyincreased the apoptotic rate of splenic CD4+CD25minus T cells ofGulominusminus mice (119901 lt 001) however treatment with parenteralVit C after CLP decreased the apoptotic rate of CD4+CD25minus

T cells of WT mice and Gulominusminus mice with sepsis (119901 lt 005or 001)

4 Discussion

MODS is the principal cause of death in critically ill patientswith sepsis [3] Fisher BJ and his colleagues showed that

low circulatory level of Vit C was significantly susceptible tosepsis-inducedMODS [16] In the current study we reportedthat administration of parenteral Vit C per se markedlyimproved sepsis and sepsis-induced MODS in WT andGulominusminus mice which was in accordance with the findingsof Fisher BJ Meanwhile we first reported the mechanismsof parenteral Vit C in improving the outcome of sepsis andsepsis-induced MODS via regulating cellular immunosup-pression

The immune dysfunction of CD4+ T cells is the primarycellular mechanisms with sepsis and sepsis-induced MODSImmediate specimens from liver kidney and lung even thecellular number of circulatory system in septic patients whodied in intensive care units showed a progressive profoundapoptosis-induced loss of adaptive immunocytes especiallythe early decrease of T-lymphocytes thereby resulting inan attenuated ability of clearing life-threatening pathogens[2 5 7 8]The activated CD4+ T cells canmainly differentiateinto Th1 and Th2 which mainly produced IFN-120574 and IL-4respectively A shift to Th2 response was noted to be corrob-orated with sepsis-inducedMODS and the outcome of sepsis[8 20 25 26] We showed that administration of parenteralVit C had the ability to improve the immune dysfunction ofCD4+ T cells Sepsis significantly weakened the proliferationand IFN-120574 secretion but enhanced the apoptotic level and IL-4 secretion of CD4+CD25minus T cells of WT mice and Gulominusminusmice however treatment with 200mgKg parenteral Vit Cfor two times after CLP significantly took a turn for the

6 Mediators of Inflammation

lowast lowastlowast

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)

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(pg

mL)

400

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(f)

Figure 2 Continued

Mediators of Inflammation 7

M1 M1 M1 M1

M1M1M1M1

M1 M1 M1 M1

Cou

nts

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nts

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PI

Annexin-V100 101 102 103 104

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Annexin-V

WT (CLP + Vit C)WT (CLP) Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

(g)

Figure 2 Parenteral Vit Cmarkedlyweakened the stability of CD4+CD25+ Tregs in sepsisThe expressions of Foxp-3 CTLA-4 andTGF-120573m+as well as the apoptotic ability of splenic CD4+CD25+ Tregs were subjected to flow cytometric analysis by flow cytometer (g) Parenteral VitC downregulated the expression of Foxp-3 (a) CTLA-4 (b) and TGF-120573m+ (c) of CD4+CD25+ Tregs in sepsis Parenteral Vit C did not alterthe apoptotic ability of splenic CD4+CD25+ Tregs in sepsis (d) Parenteral Vit C inhibited the secretion of TGF-120573 (e) and IL-10 (f) fromCD4+CD25+ Tregs in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

better and acted on upregulating the proliferative responseand IFN-120574 secretion as well as downregulating the apoptoticrate and IL-4 secretion The immunological mechanisms ofVit C on CD4+ T cells have not been elucidated reactiveoxygen species (ROS) was formed during CD4+ T cellsactivation which acted as the second messenger [19ndash21 27]We conjectured that parenteral Vit C affects T cell behaviorsduring sepsis via ROS

We have reported that the percentage and stability ofTregs were higher in septic patients than those without sepsis

a reduction in the stability of Tregs was accompanied by animprovement in survival rate and immune dysfunction ofT-lymphocytes with sepsis [12ndash15 23 24] Foxp- 3 whichis still the mainly intracellular marker for identification ofTregs was also critical for their function differentiation andmaintenance [10 11] Our previous study demonstrated thatan increased expression of Foxp-3 in Tregs was positivelycorrelated to themortality of septicmice [12] Parenteral Vit Cweakened the expression of Foxp-3 at protein and gene levelsbut the deficiency of Vit C promoted the expression of Foxp-3

8 Mediators of Inflammation

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

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ominusminus

(CLP

+ V

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ominusminus

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lowastlowast

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15

10

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(CLP

+ V

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(CLP

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ominusminus

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+ V

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lowastlowast

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ate o

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(CLP

+ V

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(CLP

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ominusminus

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+ V

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(CLP

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lowastlowast

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-120574(p

gm

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(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

lowast

IL-4

(pg

mL)

400

300

200

100

0

(d)

Figure 3 The impact of parenteral Vit C on the immunosuppressive function of CD4+CD25+ Tregs The parenteral Vit C inhibited theimmunosuppressive function of CD4+CD25+ Tregs which upregulated the proliferative response (a) and release of IFN-120574 (c) but significantlydownregulated the apoptotic rate (b) and release of IL-4 (d) of splenic conventional CD4+CD25minus T cells Data were represented as mean plusmnstandard deviation (SD) and analyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

in Gulominusminus mice septic model The recent researches showedthat the expression of transcription factor Helios controlleda great deal of pivotal aspects of Tregsrsquo suppressive functiondifferentiation and survival [28]We first reported that sepsisper se promoted the mRNA expression of Helios especiallyin the absence of Vit C but treatment with parenteral VitC after CLP of WT and Gulominusminus mice inhibited the mRNAexpression of Helios These suggested that parenteral Vit Chad significant ability to decrease the stability of Tregs withsepsis

During the development of sepsis Tregs can mainlyinhibit the activation of CD4+ T cells through variousmecha-nisms mainly including the pathway of cellular contact (egCTLA-4 and TGF-120573m+) and inhibitory cytokines (eg IL-10 and TGF-120573) as well as upregulating the antiapoptoticability of Tregs [11ndash15 23 24] Accumulated evidence hasshown that a combination of Foxp-3 CTLA-4 TGF-120573m+and inhibitory cytokines (IL-10 and TGF-120573) might serve asactive markers for Tregs with sepsis Our previous studydemonstrated that sepsis obviously enhanced the negative

Mediators of Inflammation 9

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowastlowast

Relat

ive l

evel

of F

oxp-3

mRN

A 6

4

2

0

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive o

f Fox

p3-T

SDR

mRN

A 6

4

2

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

Relat

ive l

evel

of H

elio

s mRN

A 4

3

2

1

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowastlowastlowast

lowastlowast

Relat

ive l

evel

of C

TLA-4

mRN

A 5

4

3

2

1

0

(d)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowast

Rela

tive l

evel

of T

GF-120573

mRN

A 6

4

2

0

(e)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive l

evel

of I

L-10

mRN

A 4

3

2

1

0

(f)

Figure 4 The impact of parenteral Vit C on the mRNA expression of Foxp-3 Helios CTLA-4 TGF-120573 IL-10 and the methylation level ofFoxp3-TSDR mRNA in CD4+CD25+ Tregs CD4+CD25+ Tregs were harvested for SYBR Green and Methylation-Sensitive RT-PCRT fromevery group after CLP The parenteral Vit C inhibited the expression of Foxp-3 (a) Helios (c) CTLA-4 (d) TGF-120573 (e) and IL-10 (f) butincreased the methylation level of Foxp3-TSDR (b) in CD4+CD25+ Tregs Data were represented as mean plusmn standard deviation (SD) andanalyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

10 Mediators of Inflammation

OD450

15

10

05

00

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

lowastlowastlowastlowast

lowast

lowastlowast

Gul

ominusminus

(CLP

)

(a)

The r

ate o

f apo

ptos

is (

)

100

80

60

40

20

0

lowastlowast

lowastlowast

lowast

lowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(b)

IFN

-120574(p

gm

L)

500

400

300

200

100

0

lowast

lowastlowastlowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(c)

IL-4

(pg

mL)

500

400

300

200

100

0

lowastlowastlowastlowast

lowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(d)

WT (CLP + Vit C)WT (CLP)Control group

Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

PI

104

103

102

101

100

104103102101100

Annexin-V

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

104103102101100

Annexin-V

104103102101100

Annexin-V104103102101100

Annexin-V104103102101100

Annexin-V

(e)

Figure 5 The impact of parenteral Vit C on CD4+ T cells-mediated cellular immunosuppression in sepsis Treatment with 200mgKg par-enteral Vit C upregulated the proliferative response of CD4+CD25minus T cells of WT mice and Gulominusminus mice in sepsis (a) The apoptotic rate ofsplenic CD4+CD25minusT cells was analyzed with Annexin-V-FITCPI by flow cytometry at 24 hours after CLP-inducedmidgrade sepsis (e)Theapoptotic level of splenic CD4+CD25minusT cells was decreased when 200mgKg parenteral Vit C was administered after CLP to WT mice andGulominusminus mice in sepsis (b) Parenteral Vit C increased the serum level of IFN-120574 (c) as well as decreasing the serum levels of IL-4 (d) of WTmice and Gulominusminus mice in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

Mediators of Inflammation 11

immunoregulation of Nrp-1highCD4+CD25+ Tregs whichcorrelated to the expression of Foxp-3 and CTLA-4 as wellas the secretion of IL-10 and TGF-120573 [23] We showed thatparenteral Vit C could inhibit the expression of exteriorCTLA-4 and TGF-120573m+ and the secretion of TGF-120573 and IL-10at protein and gene levels but did not alter the apoptotic levelof CD4+CD25+ Tregs These findings were in accordancewith the results of our previous studies [12 13] The stabilityof Tregs includes the expression of Foxp-3 and negativeimmunoregulation with sepsis which is crucially dependenton the demethylation status of the Foxp3-TSDR [11] Ourprevious study suggested that recombinant Nrp-1 polyclonalantibody could decrease the demethylation of Foxp3-TSDRin the presence of LPS which is associated with inhibition ofthe negative immunoregulation of Tregs [23] In the currentstudy parenteral Vit C had the ability to decrease thedemethylation level of Foxp3-TSDR but the deficiency ofVit C increased the demethylation level of Foxp3-TSDR inGulominusminusmice septicmodel whichwas in accordancewith ourprevious findings

5 Conclusion

In conclusion administration of parenteral Vit C improvedthe outcome of sepsis and sepsis-induced MODS of WT andGulominusminus mice The negative immunoregulation of Tregs wasinhibitedmainly including inhibiting the expression of Foxp-3 CTLA-4 and TGF-120573m+ and the secretion of inhibitorycytokines (TGF-120573 and IL-10) CD4+ T cells-mediated cellularimmunosuppression was improved by parenteral Vit C inWT and Gulominusminus septic mice These results suggested thatparenteral Vit C has the ability to improve the outcomeof sepsis and sepsis-induced MODS and is associated withimprovement in cellular immunosuppression

Competing Interests

The authors declare that they have no competing financialinterests This work is attributed to Tianjin Medical Univer-sity General Hospital Tianjin China

Authorsrsquo Contributions

Yu-Lei Gao Bin Lu and Jian-Hua Zhai contributed equally tothis work

Acknowledgments

This work was supported by grants from the TMUGHfunding (nos ZYYFY2015020 and ZYYFY2014014) and theRui-E funding (no 2016020) The authors thank ProfessorShu-Zhang Cui of Emergency Department of TianjinMedicalUniversity General Hospital for the guidance of experimentaldesign

References

[1] M Singer C S Deutschman C W Seymour et al ldquoThe thirdinternational consensus definitions for sepsis and septic shock

(Sepsis-3)rdquoThe Journal of theAmericanMedical Association vol315 no 8 pp 801ndash810 2016

[2] K-M Kaukonen M Bailey D Pilcher D J Cooper and RBellomo ldquoSystemic inflammatory response syndrome criteriain defining severe sepsisrdquoTheNew England Journal of Medicinevol 372 no 17 pp 1629ndash1638 2015

[3] D F Gaieski J M Edwards M J Kallan and B G CarrldquoBenchmarking the incidence and mortality of severe sepsis inthe united statesrdquo Critical Care Medicine vol 41 no 5 pp 1167ndash1174 2013

[4] B D Winters M Eberlein J Leung D M Needham P JPronovost and J E Sevransky ldquoLong-termmortality and qual-ity of life in sepsis a systematic reviewrdquo Critical Care Medicinevol 38 no 5 pp 1276ndash1283 2010

[5] S Inoue K Suzuki-Utsunomiya Y Okada et al ldquoReduction ofimmunocompetent T cells followed by prolonged lymphopeniain severe sepsis in the elderlyrdquo Critical Care Medicine vol 41no 3 pp 810ndash819 2013

[6] L P Skrupky P W Kerby and R S Hotchkiss ldquoAdvancesin the management of sepsis and the understanding of keyimmunologic defectsrdquo Anesthesiology vol 115 no 6 pp 1349ndash1362 2011

[7] J S Boomer K To K C Chang et al ldquoImmunosuppressionin patients who die of sepsis and multiple organ failurerdquo TheJournal of the American Medical Association vol 306 no 23pp 2594ndash2605 2011

[8] C J Darcy G Minigo K A Piera et al ldquoNeutrophils withmyeloid derived suppressor function deplete arginine andconstrain T cell function in septic shock patientsrdquoCritical Carevol 18 no 4 article R163 2014

[9] K Chang C Svabek C Vazquez-Guillamet et al ldquoTargetingthe programmed cell death 1 programmed cell death ligand 1pathway reverses T cell exhaustion in patients with sepsisrdquoCrit-ical Care vol 18 no 1 article R3 2014

[10] D C Nascimento J C Alves-Filho F Sonego et al ldquoRole ofregulatory T cells in long-term immune dysfunction associatedwith severe sepsisrdquo Critical Care Medicine vol 38 no 8 pp1718ndash1725 2010

[11] R TaturaM ZeschnigkWHansen et al ldquoRelevance of Foxp3+regulatory T cells for early and late phases of murine sepsisrdquoImmunology vol 146 no 1 pp 144ndash156 2015

[12] Q Y Liu Y M Yao Y Yu et al ldquoAstragalus polysaccharidesattenuate postburn sepsisvia inhibiting negative immunoreg-ulation of CD4+CD25highT cellsrdquo PLoS ONE vol 6 no 6Article ID e19811 2011

[13] X-M Zhu Y-M Yao H-P Liang et al ldquoHigh mobility groupbox-1 protein regulate immunosuppression of regulatory T cellsthrough toll-like receptor 4rdquo Cytokine vol 54 no 3 pp 296ndash304 2011

[14] Y Zhang Y-M Yao L-F Huang N Dong Y Yu and Z-YSheng ldquoThe potential effect and mechanism of high-mobilitygroup box 1 protein on regulatory T cell-mediated immunosup-pressionrdquo Journal of Interferon and Cytokine Research vol 31no 2 pp 249ndash257 2011

[15] Y-Y Luan C-F Yin Q-H Qin et al ldquoEffect of regulatory Tcells on promoting apoptosis of T lymphocyte and its regula-tory mechanism in sepsisrdquo Journal of Interferon and CytokineResearch vol 35 no 12 pp 969ndash980 2015

[16] B J Fisher D Kraskauskas E J Martin et al ldquoAttenuation ofsepsis-induced organ injury in mice by vitamin Crdquo Journal ofParenteral andEnteralNutrition vol 38 no 7 pp 825ndash839 2014

12 Mediators of Inflammation

[17] P E Marik ldquolsquoVitamin Srsquo (Steroids) and Vitamin C for the treat-ment of severe sepsis and septic shockrdquo Critical Care Medicinevol 44 no 6 pp 1228ndash1229 2016

[18] J-M Hong J-H Kim J S Kang W J Lee and Y-I HwangldquoVitamin C is taken up by human T cells via sodium-dependentvitamin C transporter 2 (SVCT2) and exerts inhibitory effectson the activation of these cells in vitrordquo Anatomy and CellBiology vol 49 no 2 pp 88ndash98 2016

[19] J Manning B Mitchell D A Appadurai et al ldquoVitamin C pro-motes maturation of T-cellsrdquo Antioxidants and Redox Signalingvol 19 no 17 pp 2054ndash2067 2013

[20] S Mburu J L Marnewick A Abayomi and H Ipp ldquoMod-ulation of LPS-induced CD4+ T-cell activation and apopto-sis by antioxidants in untreated asymptomatic HIV infectedparticipants An In Vitro Studyrdquo Clinical and DevelopmentalImmunology vol 2013 Article ID 631063 9 pages 2013

[21] P H Tan P Sagoo C Chan et al ldquoInhibition of NF-120581B andoxidative pathways in human dendritic cells by antioxidativevitamins generates regulatory T cellsrdquo Journal of Immunologyvol 174 no 12 pp 7633ndash7644 2005

[22] V S Nair M H Song and K I Oh ldquoVitamin C facilitatesdemethylation of the Foxp3 enhancer in a Tet-dependentmannerrdquo Journal of Immunology vol 196 no 5 pp 2119ndash21312016

[23] Y-L Gao Y-F Chai A-L Qi et al ldquoNeuropilin-1ℎ119894119892ℎCD4+CD25+ regulatory T cells exhibit primary negativeimmunoregulation in sepsisrdquo Mediators of Inflammation vol2016 Article ID 7132158 11 pages 2016

[24] Y L Gao Y F Chai N Dong et al ldquoTuftsin-derived T-peptideprevents cellular immunosuppression and improves survivalrate in septic micerdquo Scientific Reports vol 5 Article ID 167252015

[25] Z Yang Y Zhang L Dong et al ldquoThe reduction of peripheralblood CD4+ T cell indicates persistent organ failure in acutepancreatitisrdquo PLoSONE vol 10 no 5 Article ID e0125529 2015

[26] R Sharma P R Sharma Y C Kim et al ldquoIL-2-controlledexpression ofmultiple T cell trafficking genes andTh2 cytokinesin the regulatory T cell-deficient scurfy mice implication tomultiorgan inflammation and control of skin and lung inflam-mationrdquo The Journal of Immunology vol 186 no 2 pp 1268ndash1278 2011

[27] M J A J Huijskens M Walczak N Koller et al ldquoTechnicaladvance ascorbic acid induces development of double-positiveT cells from human hematopoietic stem cells in the absence ofStromal cellsrdquo Journal of Leukocyte Biology vol 96 no 6 pp1165ndash1175 2014

[28] H-J Kim R A Barnitz T Kreslavsky et al ldquoStable inhibitoryactivity of regulatory T cells requires the transcription factorHeliosrdquo Science vol 350 no 6258 pp 334ndash339 2015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Mediators of Inflammation

Frac

tion

surv

ival

15

10

05

00

Time after CLP (hours)0 20 40 60 80

WT (CLP + Vit C) WT (CLP)Gulominusminus (CLP + Vit C) Gulominusminus (CLP)

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

The c

once

ntra

tion

of A

ST (U

L)

250

200

150

100

50

0

lowast

lowast

lowastlowast

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

The c

once

ntra

tion

of A

LT (U

L)

150

100

50

0

lowast

lowastlowast lowastlowast

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowast

lowastlowastlowastlowast100

80

60

40

20

0

The c

once

ntra

tion

of C

r (120583

mol

L)

(d)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowast lowastlowast

lowastlowast

The A

BG o

f PCO

2(m

mH

g)

40

30

20

10

0

(e)

Figure 1 Continued

Mediators of Inflammation 5

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

The A

BG o

f PO

2(m

mH

g)150

100

50

0

(f)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowast

lowast

lowastlowast

The A

BG o

f Lac

(mm

olL

)

6

4

2

0

(g)

Figure 1 The impact of parenteral Vit C treatment on the 72-hour survival rate of septic mice and sepsis-induced MODS The 72-hoursurvival rate of WT mice and Gulominusminus mice was improved on administration of 200mgKg parenteral Vit C twice after CLP (a) ParenteralVit C improved the biomarkers of liver (b and c) renal (d) respiratory (e and f) and circulatory (g) function in CLP-exposed WT mice andVit C deficient Gulominusminus mice The survival rate was analyzed by Kaplan-Meier via the log-rank test 119899 = 50 per group WT (CLP) versus WT(CLP + Vit C) 119901 lt 001 Gulominusminus (CLP) versus Gulominusminus (CLP + Vit C) 119901 lt 001 WT (CLP) versus Gulominusminus (CLP) 119901 lt 005 Data wererepresented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 orlowastlowast119901 lt 001

CD4+CD25+ Tregs of Gulominusminus (CLP) group in comparison toWT (CLP) group (119901 lt 001) Treatment with parenteral Vit Cafter CLP of WT and Gulominusminus mice inhibited the expressionof Foxp-3 Helios CTLA-4 TGF-120573 and IL-10 but enhancedthe methylation level of Foxp3-TSDR in CD4+CD25+ Tregs(119901 lt 005 or 001)

35 Parenteral Vit C ImprovedCD4+ TCells-MediatedCellularImmunosuppression CD4+CD25minus T cells were isolated fromevery group and cultured for another 24 hours The defi-ciency of Vit C significantly worsened the proliferative ability(Figure 5(a)) and IFN-120574 level (Figure 5(c)) but increasedthe release of IL-4 (Figure 5(d)) of splenic CD4+CD25minus Tcells of Gulominusminus mice (119901 lt 001) however treatment withparenteral Vit C afterCLP significantly enhanced the prolifer-ative response and IFN-120574 level but inhibited the release of IL-4 of CD4+CD25minus T cells ofWT and Gulominusminusmice (119901 lt 001)As shown in Figure 5(b) the deficiency of Vit C significantlyincreased the apoptotic rate of splenic CD4+CD25minus T cells ofGulominusminus mice (119901 lt 001) however treatment with parenteralVit C after CLP decreased the apoptotic rate of CD4+CD25minus

T cells of WT mice and Gulominusminus mice with sepsis (119901 lt 005or 001)

4 Discussion

MODS is the principal cause of death in critically ill patientswith sepsis [3] Fisher BJ and his colleagues showed that

low circulatory level of Vit C was significantly susceptible tosepsis-inducedMODS [16] In the current study we reportedthat administration of parenteral Vit C per se markedlyimproved sepsis and sepsis-induced MODS in WT andGulominusminus mice which was in accordance with the findingsof Fisher BJ Meanwhile we first reported the mechanismsof parenteral Vit C in improving the outcome of sepsis andsepsis-induced MODS via regulating cellular immunosup-pression

The immune dysfunction of CD4+ T cells is the primarycellular mechanisms with sepsis and sepsis-induced MODSImmediate specimens from liver kidney and lung even thecellular number of circulatory system in septic patients whodied in intensive care units showed a progressive profoundapoptosis-induced loss of adaptive immunocytes especiallythe early decrease of T-lymphocytes thereby resulting inan attenuated ability of clearing life-threatening pathogens[2 5 7 8]The activated CD4+ T cells canmainly differentiateinto Th1 and Th2 which mainly produced IFN-120574 and IL-4respectively A shift to Th2 response was noted to be corrob-orated with sepsis-inducedMODS and the outcome of sepsis[8 20 25 26] We showed that administration of parenteralVit C had the ability to improve the immune dysfunction ofCD4+ T cells Sepsis significantly weakened the proliferationand IFN-120574 secretion but enhanced the apoptotic level and IL-4 secretion of CD4+CD25minus T cells of WT mice and Gulominusminusmice however treatment with 200mgKg parenteral Vit Cfor two times after CLP significantly took a turn for the

6 Mediators of Inflammation

lowast lowastlowast

lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

Foxp

-3(

)

100

80

60

40

20

0

(a)

lowast

lowastlowast lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

CTLA

-4(

)

100

80

60

40

20

0

(b)

lowastlowastlowastlowast

lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

100

80

60

40

20

0

TGF-120573

m+

()

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

The r

ate o

f apo

ptos

is (

)40

30

20

10

0

(d)

lowastlowastlowastlowast

lowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

TGF-120573

(pg

mL)

600

400

200

0

(e)

lowastlowast

lowastlowast

lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

IL-10

(pg

mL)

400

300

200

100

0

(f)

Figure 2 Continued

Mediators of Inflammation 7

M1 M1 M1 M1

M1M1M1M1

M1 M1 M1 M1

Cou

nts

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

100 101 102 103 104

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

PI

Annexin-V100 101 102 103 104

Annexin-V100 101 102 103 104

Annexin-V100

100

101

101

102

102

103

103

104

104

PI

100

101

102

103

104

PI

100

101

102

103

104

PI

100

101

102

103

104

Annexin-V

WT (CLP + Vit C)WT (CLP) Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

(g)

Figure 2 Parenteral Vit Cmarkedlyweakened the stability of CD4+CD25+ Tregs in sepsisThe expressions of Foxp-3 CTLA-4 andTGF-120573m+as well as the apoptotic ability of splenic CD4+CD25+ Tregs were subjected to flow cytometric analysis by flow cytometer (g) Parenteral VitC downregulated the expression of Foxp-3 (a) CTLA-4 (b) and TGF-120573m+ (c) of CD4+CD25+ Tregs in sepsis Parenteral Vit C did not alterthe apoptotic ability of splenic CD4+CD25+ Tregs in sepsis (d) Parenteral Vit C inhibited the secretion of TGF-120573 (e) and IL-10 (f) fromCD4+CD25+ Tregs in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

better and acted on upregulating the proliferative responseand IFN-120574 secretion as well as downregulating the apoptoticrate and IL-4 secretion The immunological mechanisms ofVit C on CD4+ T cells have not been elucidated reactiveoxygen species (ROS) was formed during CD4+ T cellsactivation which acted as the second messenger [19ndash21 27]We conjectured that parenteral Vit C affects T cell behaviorsduring sepsis via ROS

We have reported that the percentage and stability ofTregs were higher in septic patients than those without sepsis

a reduction in the stability of Tregs was accompanied by animprovement in survival rate and immune dysfunction ofT-lymphocytes with sepsis [12ndash15 23 24] Foxp- 3 whichis still the mainly intracellular marker for identification ofTregs was also critical for their function differentiation andmaintenance [10 11] Our previous study demonstrated thatan increased expression of Foxp-3 in Tregs was positivelycorrelated to themortality of septicmice [12] Parenteral Vit Cweakened the expression of Foxp-3 at protein and gene levelsbut the deficiency of Vit C promoted the expression of Foxp-3

8 Mediators of Inflammation

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD450

15

10

05

00

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowast

lowastlowast

The r

ate o

f apo

ptos

is (

)

80

60

40

20

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

IFN

-120574(p

gm

L)

150

100

50

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

lowast

IL-4

(pg

mL)

400

300

200

100

0

(d)

Figure 3 The impact of parenteral Vit C on the immunosuppressive function of CD4+CD25+ Tregs The parenteral Vit C inhibited theimmunosuppressive function of CD4+CD25+ Tregs which upregulated the proliferative response (a) and release of IFN-120574 (c) but significantlydownregulated the apoptotic rate (b) and release of IL-4 (d) of splenic conventional CD4+CD25minus T cells Data were represented as mean plusmnstandard deviation (SD) and analyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

in Gulominusminus mice septic model The recent researches showedthat the expression of transcription factor Helios controlleda great deal of pivotal aspects of Tregsrsquo suppressive functiondifferentiation and survival [28]We first reported that sepsisper se promoted the mRNA expression of Helios especiallyin the absence of Vit C but treatment with parenteral VitC after CLP of WT and Gulominusminus mice inhibited the mRNAexpression of Helios These suggested that parenteral Vit Chad significant ability to decrease the stability of Tregs withsepsis

During the development of sepsis Tregs can mainlyinhibit the activation of CD4+ T cells through variousmecha-nisms mainly including the pathway of cellular contact (egCTLA-4 and TGF-120573m+) and inhibitory cytokines (eg IL-10 and TGF-120573) as well as upregulating the antiapoptoticability of Tregs [11ndash15 23 24] Accumulated evidence hasshown that a combination of Foxp-3 CTLA-4 TGF-120573m+and inhibitory cytokines (IL-10 and TGF-120573) might serve asactive markers for Tregs with sepsis Our previous studydemonstrated that sepsis obviously enhanced the negative

Mediators of Inflammation 9

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowastlowast

Relat

ive l

evel

of F

oxp-3

mRN

A 6

4

2

0

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive o

f Fox

p3-T

SDR

mRN

A 6

4

2

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

Relat

ive l

evel

of H

elio

s mRN

A 4

3

2

1

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowastlowastlowast

lowastlowast

Relat

ive l

evel

of C

TLA-4

mRN

A 5

4

3

2

1

0

(d)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowast

Rela

tive l

evel

of T

GF-120573

mRN

A 6

4

2

0

(e)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive l

evel

of I

L-10

mRN

A 4

3

2

1

0

(f)

Figure 4 The impact of parenteral Vit C on the mRNA expression of Foxp-3 Helios CTLA-4 TGF-120573 IL-10 and the methylation level ofFoxp3-TSDR mRNA in CD4+CD25+ Tregs CD4+CD25+ Tregs were harvested for SYBR Green and Methylation-Sensitive RT-PCRT fromevery group after CLP The parenteral Vit C inhibited the expression of Foxp-3 (a) Helios (c) CTLA-4 (d) TGF-120573 (e) and IL-10 (f) butincreased the methylation level of Foxp3-TSDR (b) in CD4+CD25+ Tregs Data were represented as mean plusmn standard deviation (SD) andanalyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

10 Mediators of Inflammation

OD450

15

10

05

00

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

lowastlowastlowastlowast

lowast

lowastlowast

Gul

ominusminus

(CLP

)

(a)

The r

ate o

f apo

ptos

is (

)

100

80

60

40

20

0

lowastlowast

lowastlowast

lowast

lowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(b)

IFN

-120574(p

gm

L)

500

400

300

200

100

0

lowast

lowastlowastlowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(c)

IL-4

(pg

mL)

500

400

300

200

100

0

lowastlowastlowastlowast

lowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(d)

WT (CLP + Vit C)WT (CLP)Control group

Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

PI

104

103

102

101

100

104103102101100

Annexin-V

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

104103102101100

Annexin-V

104103102101100

Annexin-V104103102101100

Annexin-V104103102101100

Annexin-V

(e)

Figure 5 The impact of parenteral Vit C on CD4+ T cells-mediated cellular immunosuppression in sepsis Treatment with 200mgKg par-enteral Vit C upregulated the proliferative response of CD4+CD25minus T cells of WT mice and Gulominusminus mice in sepsis (a) The apoptotic rate ofsplenic CD4+CD25minusT cells was analyzed with Annexin-V-FITCPI by flow cytometry at 24 hours after CLP-inducedmidgrade sepsis (e)Theapoptotic level of splenic CD4+CD25minusT cells was decreased when 200mgKg parenteral Vit C was administered after CLP to WT mice andGulominusminus mice in sepsis (b) Parenteral Vit C increased the serum level of IFN-120574 (c) as well as decreasing the serum levels of IL-4 (d) of WTmice and Gulominusminus mice in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

Mediators of Inflammation 11

immunoregulation of Nrp-1highCD4+CD25+ Tregs whichcorrelated to the expression of Foxp-3 and CTLA-4 as wellas the secretion of IL-10 and TGF-120573 [23] We showed thatparenteral Vit C could inhibit the expression of exteriorCTLA-4 and TGF-120573m+ and the secretion of TGF-120573 and IL-10at protein and gene levels but did not alter the apoptotic levelof CD4+CD25+ Tregs These findings were in accordancewith the results of our previous studies [12 13] The stabilityof Tregs includes the expression of Foxp-3 and negativeimmunoregulation with sepsis which is crucially dependenton the demethylation status of the Foxp3-TSDR [11] Ourprevious study suggested that recombinant Nrp-1 polyclonalantibody could decrease the demethylation of Foxp3-TSDRin the presence of LPS which is associated with inhibition ofthe negative immunoregulation of Tregs [23] In the currentstudy parenteral Vit C had the ability to decrease thedemethylation level of Foxp3-TSDR but the deficiency ofVit C increased the demethylation level of Foxp3-TSDR inGulominusminusmice septicmodel whichwas in accordancewith ourprevious findings

5 Conclusion

In conclusion administration of parenteral Vit C improvedthe outcome of sepsis and sepsis-induced MODS of WT andGulominusminus mice The negative immunoregulation of Tregs wasinhibitedmainly including inhibiting the expression of Foxp-3 CTLA-4 and TGF-120573m+ and the secretion of inhibitorycytokines (TGF-120573 and IL-10) CD4+ T cells-mediated cellularimmunosuppression was improved by parenteral Vit C inWT and Gulominusminus septic mice These results suggested thatparenteral Vit C has the ability to improve the outcomeof sepsis and sepsis-induced MODS and is associated withimprovement in cellular immunosuppression

Competing Interests

The authors declare that they have no competing financialinterests This work is attributed to Tianjin Medical Univer-sity General Hospital Tianjin China

Authorsrsquo Contributions

Yu-Lei Gao Bin Lu and Jian-Hua Zhai contributed equally tothis work

Acknowledgments

This work was supported by grants from the TMUGHfunding (nos ZYYFY2015020 and ZYYFY2014014) and theRui-E funding (no 2016020) The authors thank ProfessorShu-Zhang Cui of Emergency Department of TianjinMedicalUniversity General Hospital for the guidance of experimentaldesign

References

[1] M Singer C S Deutschman C W Seymour et al ldquoThe thirdinternational consensus definitions for sepsis and septic shock

(Sepsis-3)rdquoThe Journal of theAmericanMedical Association vol315 no 8 pp 801ndash810 2016

[2] K-M Kaukonen M Bailey D Pilcher D J Cooper and RBellomo ldquoSystemic inflammatory response syndrome criteriain defining severe sepsisrdquoTheNew England Journal of Medicinevol 372 no 17 pp 1629ndash1638 2015

[3] D F Gaieski J M Edwards M J Kallan and B G CarrldquoBenchmarking the incidence and mortality of severe sepsis inthe united statesrdquo Critical Care Medicine vol 41 no 5 pp 1167ndash1174 2013

[4] B D Winters M Eberlein J Leung D M Needham P JPronovost and J E Sevransky ldquoLong-termmortality and qual-ity of life in sepsis a systematic reviewrdquo Critical Care Medicinevol 38 no 5 pp 1276ndash1283 2010

[5] S Inoue K Suzuki-Utsunomiya Y Okada et al ldquoReduction ofimmunocompetent T cells followed by prolonged lymphopeniain severe sepsis in the elderlyrdquo Critical Care Medicine vol 41no 3 pp 810ndash819 2013

[6] L P Skrupky P W Kerby and R S Hotchkiss ldquoAdvancesin the management of sepsis and the understanding of keyimmunologic defectsrdquo Anesthesiology vol 115 no 6 pp 1349ndash1362 2011

[7] J S Boomer K To K C Chang et al ldquoImmunosuppressionin patients who die of sepsis and multiple organ failurerdquo TheJournal of the American Medical Association vol 306 no 23pp 2594ndash2605 2011

[8] C J Darcy G Minigo K A Piera et al ldquoNeutrophils withmyeloid derived suppressor function deplete arginine andconstrain T cell function in septic shock patientsrdquoCritical Carevol 18 no 4 article R163 2014

[9] K Chang C Svabek C Vazquez-Guillamet et al ldquoTargetingthe programmed cell death 1 programmed cell death ligand 1pathway reverses T cell exhaustion in patients with sepsisrdquoCrit-ical Care vol 18 no 1 article R3 2014

[10] D C Nascimento J C Alves-Filho F Sonego et al ldquoRole ofregulatory T cells in long-term immune dysfunction associatedwith severe sepsisrdquo Critical Care Medicine vol 38 no 8 pp1718ndash1725 2010

[11] R TaturaM ZeschnigkWHansen et al ldquoRelevance of Foxp3+regulatory T cells for early and late phases of murine sepsisrdquoImmunology vol 146 no 1 pp 144ndash156 2015

[12] Q Y Liu Y M Yao Y Yu et al ldquoAstragalus polysaccharidesattenuate postburn sepsisvia inhibiting negative immunoreg-ulation of CD4+CD25highT cellsrdquo PLoS ONE vol 6 no 6Article ID e19811 2011

[13] X-M Zhu Y-M Yao H-P Liang et al ldquoHigh mobility groupbox-1 protein regulate immunosuppression of regulatory T cellsthrough toll-like receptor 4rdquo Cytokine vol 54 no 3 pp 296ndash304 2011

[14] Y Zhang Y-M Yao L-F Huang N Dong Y Yu and Z-YSheng ldquoThe potential effect and mechanism of high-mobilitygroup box 1 protein on regulatory T cell-mediated immunosup-pressionrdquo Journal of Interferon and Cytokine Research vol 31no 2 pp 249ndash257 2011

[15] Y-Y Luan C-F Yin Q-H Qin et al ldquoEffect of regulatory Tcells on promoting apoptosis of T lymphocyte and its regula-tory mechanism in sepsisrdquo Journal of Interferon and CytokineResearch vol 35 no 12 pp 969ndash980 2015

[16] B J Fisher D Kraskauskas E J Martin et al ldquoAttenuation ofsepsis-induced organ injury in mice by vitamin Crdquo Journal ofParenteral andEnteralNutrition vol 38 no 7 pp 825ndash839 2014

12 Mediators of Inflammation

[17] P E Marik ldquolsquoVitamin Srsquo (Steroids) and Vitamin C for the treat-ment of severe sepsis and septic shockrdquo Critical Care Medicinevol 44 no 6 pp 1228ndash1229 2016

[18] J-M Hong J-H Kim J S Kang W J Lee and Y-I HwangldquoVitamin C is taken up by human T cells via sodium-dependentvitamin C transporter 2 (SVCT2) and exerts inhibitory effectson the activation of these cells in vitrordquo Anatomy and CellBiology vol 49 no 2 pp 88ndash98 2016

[19] J Manning B Mitchell D A Appadurai et al ldquoVitamin C pro-motes maturation of T-cellsrdquo Antioxidants and Redox Signalingvol 19 no 17 pp 2054ndash2067 2013

[20] S Mburu J L Marnewick A Abayomi and H Ipp ldquoMod-ulation of LPS-induced CD4+ T-cell activation and apopto-sis by antioxidants in untreated asymptomatic HIV infectedparticipants An In Vitro Studyrdquo Clinical and DevelopmentalImmunology vol 2013 Article ID 631063 9 pages 2013

[21] P H Tan P Sagoo C Chan et al ldquoInhibition of NF-120581B andoxidative pathways in human dendritic cells by antioxidativevitamins generates regulatory T cellsrdquo Journal of Immunologyvol 174 no 12 pp 7633ndash7644 2005

[22] V S Nair M H Song and K I Oh ldquoVitamin C facilitatesdemethylation of the Foxp3 enhancer in a Tet-dependentmannerrdquo Journal of Immunology vol 196 no 5 pp 2119ndash21312016

[23] Y-L Gao Y-F Chai A-L Qi et al ldquoNeuropilin-1ℎ119894119892ℎCD4+CD25+ regulatory T cells exhibit primary negativeimmunoregulation in sepsisrdquo Mediators of Inflammation vol2016 Article ID 7132158 11 pages 2016

[24] Y L Gao Y F Chai N Dong et al ldquoTuftsin-derived T-peptideprevents cellular immunosuppression and improves survivalrate in septic micerdquo Scientific Reports vol 5 Article ID 167252015

[25] Z Yang Y Zhang L Dong et al ldquoThe reduction of peripheralblood CD4+ T cell indicates persistent organ failure in acutepancreatitisrdquo PLoSONE vol 10 no 5 Article ID e0125529 2015

[26] R Sharma P R Sharma Y C Kim et al ldquoIL-2-controlledexpression ofmultiple T cell trafficking genes andTh2 cytokinesin the regulatory T cell-deficient scurfy mice implication tomultiorgan inflammation and control of skin and lung inflam-mationrdquo The Journal of Immunology vol 186 no 2 pp 1268ndash1278 2011

[27] M J A J Huijskens M Walczak N Koller et al ldquoTechnicaladvance ascorbic acid induces development of double-positiveT cells from human hematopoietic stem cells in the absence ofStromal cellsrdquo Journal of Leukocyte Biology vol 96 no 6 pp1165ndash1175 2014

[28] H-J Kim R A Barnitz T Kreslavsky et al ldquoStable inhibitoryactivity of regulatory T cells requires the transcription factorHeliosrdquo Science vol 350 no 6258 pp 334ndash339 2015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 5

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

The A

BG o

f PO

2(m

mH

g)150

100

50

0

(f)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowast

lowast

lowastlowast

The A

BG o

f Lac

(mm

olL

)

6

4

2

0

(g)

Figure 1 The impact of parenteral Vit C treatment on the 72-hour survival rate of septic mice and sepsis-induced MODS The 72-hoursurvival rate of WT mice and Gulominusminus mice was improved on administration of 200mgKg parenteral Vit C twice after CLP (a) ParenteralVit C improved the biomarkers of liver (b and c) renal (d) respiratory (e and f) and circulatory (g) function in CLP-exposed WT mice andVit C deficient Gulominusminus mice The survival rate was analyzed by Kaplan-Meier via the log-rank test 119899 = 50 per group WT (CLP) versus WT(CLP + Vit C) 119901 lt 001 Gulominusminus (CLP) versus Gulominusminus (CLP + Vit C) 119901 lt 001 WT (CLP) versus Gulominusminus (CLP) 119901 lt 005 Data wererepresented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 orlowastlowast119901 lt 001

CD4+CD25+ Tregs of Gulominusminus (CLP) group in comparison toWT (CLP) group (119901 lt 001) Treatment with parenteral Vit Cafter CLP of WT and Gulominusminus mice inhibited the expressionof Foxp-3 Helios CTLA-4 TGF-120573 and IL-10 but enhancedthe methylation level of Foxp3-TSDR in CD4+CD25+ Tregs(119901 lt 005 or 001)

35 Parenteral Vit C ImprovedCD4+ TCells-MediatedCellularImmunosuppression CD4+CD25minus T cells were isolated fromevery group and cultured for another 24 hours The defi-ciency of Vit C significantly worsened the proliferative ability(Figure 5(a)) and IFN-120574 level (Figure 5(c)) but increasedthe release of IL-4 (Figure 5(d)) of splenic CD4+CD25minus Tcells of Gulominusminus mice (119901 lt 001) however treatment withparenteral Vit C afterCLP significantly enhanced the prolifer-ative response and IFN-120574 level but inhibited the release of IL-4 of CD4+CD25minus T cells ofWT and Gulominusminusmice (119901 lt 001)As shown in Figure 5(b) the deficiency of Vit C significantlyincreased the apoptotic rate of splenic CD4+CD25minus T cells ofGulominusminus mice (119901 lt 001) however treatment with parenteralVit C after CLP decreased the apoptotic rate of CD4+CD25minus

T cells of WT mice and Gulominusminus mice with sepsis (119901 lt 005or 001)

4 Discussion

MODS is the principal cause of death in critically ill patientswith sepsis [3] Fisher BJ and his colleagues showed that

low circulatory level of Vit C was significantly susceptible tosepsis-inducedMODS [16] In the current study we reportedthat administration of parenteral Vit C per se markedlyimproved sepsis and sepsis-induced MODS in WT andGulominusminus mice which was in accordance with the findingsof Fisher BJ Meanwhile we first reported the mechanismsof parenteral Vit C in improving the outcome of sepsis andsepsis-induced MODS via regulating cellular immunosup-pression

The immune dysfunction of CD4+ T cells is the primarycellular mechanisms with sepsis and sepsis-induced MODSImmediate specimens from liver kidney and lung even thecellular number of circulatory system in septic patients whodied in intensive care units showed a progressive profoundapoptosis-induced loss of adaptive immunocytes especiallythe early decrease of T-lymphocytes thereby resulting inan attenuated ability of clearing life-threatening pathogens[2 5 7 8]The activated CD4+ T cells canmainly differentiateinto Th1 and Th2 which mainly produced IFN-120574 and IL-4respectively A shift to Th2 response was noted to be corrob-orated with sepsis-inducedMODS and the outcome of sepsis[8 20 25 26] We showed that administration of parenteralVit C had the ability to improve the immune dysfunction ofCD4+ T cells Sepsis significantly weakened the proliferationand IFN-120574 secretion but enhanced the apoptotic level and IL-4 secretion of CD4+CD25minus T cells of WT mice and Gulominusminusmice however treatment with 200mgKg parenteral Vit Cfor two times after CLP significantly took a turn for the

6 Mediators of Inflammation

lowast lowastlowast

lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

Foxp

-3(

)

100

80

60

40

20

0

(a)

lowast

lowastlowast lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

CTLA

-4(

)

100

80

60

40

20

0

(b)

lowastlowastlowastlowast

lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

100

80

60

40

20

0

TGF-120573

m+

()

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

The r

ate o

f apo

ptos

is (

)40

30

20

10

0

(d)

lowastlowastlowastlowast

lowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

TGF-120573

(pg

mL)

600

400

200

0

(e)

lowastlowast

lowastlowast

lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

IL-10

(pg

mL)

400

300

200

100

0

(f)

Figure 2 Continued

Mediators of Inflammation 7

M1 M1 M1 M1

M1M1M1M1

M1 M1 M1 M1

Cou

nts

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

100 101 102 103 104

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

PI

Annexin-V100 101 102 103 104

Annexin-V100 101 102 103 104

Annexin-V100

100

101

101

102

102

103

103

104

104

PI

100

101

102

103

104

PI

100

101

102

103

104

PI

100

101

102

103

104

Annexin-V

WT (CLP + Vit C)WT (CLP) Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

(g)

Figure 2 Parenteral Vit Cmarkedlyweakened the stability of CD4+CD25+ Tregs in sepsisThe expressions of Foxp-3 CTLA-4 andTGF-120573m+as well as the apoptotic ability of splenic CD4+CD25+ Tregs were subjected to flow cytometric analysis by flow cytometer (g) Parenteral VitC downregulated the expression of Foxp-3 (a) CTLA-4 (b) and TGF-120573m+ (c) of CD4+CD25+ Tregs in sepsis Parenteral Vit C did not alterthe apoptotic ability of splenic CD4+CD25+ Tregs in sepsis (d) Parenteral Vit C inhibited the secretion of TGF-120573 (e) and IL-10 (f) fromCD4+CD25+ Tregs in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

better and acted on upregulating the proliferative responseand IFN-120574 secretion as well as downregulating the apoptoticrate and IL-4 secretion The immunological mechanisms ofVit C on CD4+ T cells have not been elucidated reactiveoxygen species (ROS) was formed during CD4+ T cellsactivation which acted as the second messenger [19ndash21 27]We conjectured that parenteral Vit C affects T cell behaviorsduring sepsis via ROS

We have reported that the percentage and stability ofTregs were higher in septic patients than those without sepsis

a reduction in the stability of Tregs was accompanied by animprovement in survival rate and immune dysfunction ofT-lymphocytes with sepsis [12ndash15 23 24] Foxp- 3 whichis still the mainly intracellular marker for identification ofTregs was also critical for their function differentiation andmaintenance [10 11] Our previous study demonstrated thatan increased expression of Foxp-3 in Tregs was positivelycorrelated to themortality of septicmice [12] Parenteral Vit Cweakened the expression of Foxp-3 at protein and gene levelsbut the deficiency of Vit C promoted the expression of Foxp-3

8 Mediators of Inflammation

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD450

15

10

05

00

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowast

lowastlowast

The r

ate o

f apo

ptos

is (

)

80

60

40

20

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

IFN

-120574(p

gm

L)

150

100

50

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

lowast

IL-4

(pg

mL)

400

300

200

100

0

(d)

Figure 3 The impact of parenteral Vit C on the immunosuppressive function of CD4+CD25+ Tregs The parenteral Vit C inhibited theimmunosuppressive function of CD4+CD25+ Tregs which upregulated the proliferative response (a) and release of IFN-120574 (c) but significantlydownregulated the apoptotic rate (b) and release of IL-4 (d) of splenic conventional CD4+CD25minus T cells Data were represented as mean plusmnstandard deviation (SD) and analyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

in Gulominusminus mice septic model The recent researches showedthat the expression of transcription factor Helios controlleda great deal of pivotal aspects of Tregsrsquo suppressive functiondifferentiation and survival [28]We first reported that sepsisper se promoted the mRNA expression of Helios especiallyin the absence of Vit C but treatment with parenteral VitC after CLP of WT and Gulominusminus mice inhibited the mRNAexpression of Helios These suggested that parenteral Vit Chad significant ability to decrease the stability of Tregs withsepsis

During the development of sepsis Tregs can mainlyinhibit the activation of CD4+ T cells through variousmecha-nisms mainly including the pathway of cellular contact (egCTLA-4 and TGF-120573m+) and inhibitory cytokines (eg IL-10 and TGF-120573) as well as upregulating the antiapoptoticability of Tregs [11ndash15 23 24] Accumulated evidence hasshown that a combination of Foxp-3 CTLA-4 TGF-120573m+and inhibitory cytokines (IL-10 and TGF-120573) might serve asactive markers for Tregs with sepsis Our previous studydemonstrated that sepsis obviously enhanced the negative

Mediators of Inflammation 9

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowastlowast

Relat

ive l

evel

of F

oxp-3

mRN

A 6

4

2

0

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive o

f Fox

p3-T

SDR

mRN

A 6

4

2

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

Relat

ive l

evel

of H

elio

s mRN

A 4

3

2

1

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowastlowastlowast

lowastlowast

Relat

ive l

evel

of C

TLA-4

mRN

A 5

4

3

2

1

0

(d)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowast

Rela

tive l

evel

of T

GF-120573

mRN

A 6

4

2

0

(e)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive l

evel

of I

L-10

mRN

A 4

3

2

1

0

(f)

Figure 4 The impact of parenteral Vit C on the mRNA expression of Foxp-3 Helios CTLA-4 TGF-120573 IL-10 and the methylation level ofFoxp3-TSDR mRNA in CD4+CD25+ Tregs CD4+CD25+ Tregs were harvested for SYBR Green and Methylation-Sensitive RT-PCRT fromevery group after CLP The parenteral Vit C inhibited the expression of Foxp-3 (a) Helios (c) CTLA-4 (d) TGF-120573 (e) and IL-10 (f) butincreased the methylation level of Foxp3-TSDR (b) in CD4+CD25+ Tregs Data were represented as mean plusmn standard deviation (SD) andanalyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

10 Mediators of Inflammation

OD450

15

10

05

00

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

lowastlowastlowastlowast

lowast

lowastlowast

Gul

ominusminus

(CLP

)

(a)

The r

ate o

f apo

ptos

is (

)

100

80

60

40

20

0

lowastlowast

lowastlowast

lowast

lowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(b)

IFN

-120574(p

gm

L)

500

400

300

200

100

0

lowast

lowastlowastlowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(c)

IL-4

(pg

mL)

500

400

300

200

100

0

lowastlowastlowastlowast

lowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(d)

WT (CLP + Vit C)WT (CLP)Control group

Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

PI

104

103

102

101

100

104103102101100

Annexin-V

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

104103102101100

Annexin-V

104103102101100

Annexin-V104103102101100

Annexin-V104103102101100

Annexin-V

(e)

Figure 5 The impact of parenteral Vit C on CD4+ T cells-mediated cellular immunosuppression in sepsis Treatment with 200mgKg par-enteral Vit C upregulated the proliferative response of CD4+CD25minus T cells of WT mice and Gulominusminus mice in sepsis (a) The apoptotic rate ofsplenic CD4+CD25minusT cells was analyzed with Annexin-V-FITCPI by flow cytometry at 24 hours after CLP-inducedmidgrade sepsis (e)Theapoptotic level of splenic CD4+CD25minusT cells was decreased when 200mgKg parenteral Vit C was administered after CLP to WT mice andGulominusminus mice in sepsis (b) Parenteral Vit C increased the serum level of IFN-120574 (c) as well as decreasing the serum levels of IL-4 (d) of WTmice and Gulominusminus mice in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

Mediators of Inflammation 11

immunoregulation of Nrp-1highCD4+CD25+ Tregs whichcorrelated to the expression of Foxp-3 and CTLA-4 as wellas the secretion of IL-10 and TGF-120573 [23] We showed thatparenteral Vit C could inhibit the expression of exteriorCTLA-4 and TGF-120573m+ and the secretion of TGF-120573 and IL-10at protein and gene levels but did not alter the apoptotic levelof CD4+CD25+ Tregs These findings were in accordancewith the results of our previous studies [12 13] The stabilityof Tregs includes the expression of Foxp-3 and negativeimmunoregulation with sepsis which is crucially dependenton the demethylation status of the Foxp3-TSDR [11] Ourprevious study suggested that recombinant Nrp-1 polyclonalantibody could decrease the demethylation of Foxp3-TSDRin the presence of LPS which is associated with inhibition ofthe negative immunoregulation of Tregs [23] In the currentstudy parenteral Vit C had the ability to decrease thedemethylation level of Foxp3-TSDR but the deficiency ofVit C increased the demethylation level of Foxp3-TSDR inGulominusminusmice septicmodel whichwas in accordancewith ourprevious findings

5 Conclusion

In conclusion administration of parenteral Vit C improvedthe outcome of sepsis and sepsis-induced MODS of WT andGulominusminus mice The negative immunoregulation of Tregs wasinhibitedmainly including inhibiting the expression of Foxp-3 CTLA-4 and TGF-120573m+ and the secretion of inhibitorycytokines (TGF-120573 and IL-10) CD4+ T cells-mediated cellularimmunosuppression was improved by parenteral Vit C inWT and Gulominusminus septic mice These results suggested thatparenteral Vit C has the ability to improve the outcomeof sepsis and sepsis-induced MODS and is associated withimprovement in cellular immunosuppression

Competing Interests

The authors declare that they have no competing financialinterests This work is attributed to Tianjin Medical Univer-sity General Hospital Tianjin China

Authorsrsquo Contributions

Yu-Lei Gao Bin Lu and Jian-Hua Zhai contributed equally tothis work

Acknowledgments

This work was supported by grants from the TMUGHfunding (nos ZYYFY2015020 and ZYYFY2014014) and theRui-E funding (no 2016020) The authors thank ProfessorShu-Zhang Cui of Emergency Department of TianjinMedicalUniversity General Hospital for the guidance of experimentaldesign

References

[1] M Singer C S Deutschman C W Seymour et al ldquoThe thirdinternational consensus definitions for sepsis and septic shock

(Sepsis-3)rdquoThe Journal of theAmericanMedical Association vol315 no 8 pp 801ndash810 2016

[2] K-M Kaukonen M Bailey D Pilcher D J Cooper and RBellomo ldquoSystemic inflammatory response syndrome criteriain defining severe sepsisrdquoTheNew England Journal of Medicinevol 372 no 17 pp 1629ndash1638 2015

[3] D F Gaieski J M Edwards M J Kallan and B G CarrldquoBenchmarking the incidence and mortality of severe sepsis inthe united statesrdquo Critical Care Medicine vol 41 no 5 pp 1167ndash1174 2013

[4] B D Winters M Eberlein J Leung D M Needham P JPronovost and J E Sevransky ldquoLong-termmortality and qual-ity of life in sepsis a systematic reviewrdquo Critical Care Medicinevol 38 no 5 pp 1276ndash1283 2010

[5] S Inoue K Suzuki-Utsunomiya Y Okada et al ldquoReduction ofimmunocompetent T cells followed by prolonged lymphopeniain severe sepsis in the elderlyrdquo Critical Care Medicine vol 41no 3 pp 810ndash819 2013

[6] L P Skrupky P W Kerby and R S Hotchkiss ldquoAdvancesin the management of sepsis and the understanding of keyimmunologic defectsrdquo Anesthesiology vol 115 no 6 pp 1349ndash1362 2011

[7] J S Boomer K To K C Chang et al ldquoImmunosuppressionin patients who die of sepsis and multiple organ failurerdquo TheJournal of the American Medical Association vol 306 no 23pp 2594ndash2605 2011

[8] C J Darcy G Minigo K A Piera et al ldquoNeutrophils withmyeloid derived suppressor function deplete arginine andconstrain T cell function in septic shock patientsrdquoCritical Carevol 18 no 4 article R163 2014

[9] K Chang C Svabek C Vazquez-Guillamet et al ldquoTargetingthe programmed cell death 1 programmed cell death ligand 1pathway reverses T cell exhaustion in patients with sepsisrdquoCrit-ical Care vol 18 no 1 article R3 2014

[10] D C Nascimento J C Alves-Filho F Sonego et al ldquoRole ofregulatory T cells in long-term immune dysfunction associatedwith severe sepsisrdquo Critical Care Medicine vol 38 no 8 pp1718ndash1725 2010

[11] R TaturaM ZeschnigkWHansen et al ldquoRelevance of Foxp3+regulatory T cells for early and late phases of murine sepsisrdquoImmunology vol 146 no 1 pp 144ndash156 2015

[12] Q Y Liu Y M Yao Y Yu et al ldquoAstragalus polysaccharidesattenuate postburn sepsisvia inhibiting negative immunoreg-ulation of CD4+CD25highT cellsrdquo PLoS ONE vol 6 no 6Article ID e19811 2011

[13] X-M Zhu Y-M Yao H-P Liang et al ldquoHigh mobility groupbox-1 protein regulate immunosuppression of regulatory T cellsthrough toll-like receptor 4rdquo Cytokine vol 54 no 3 pp 296ndash304 2011

[14] Y Zhang Y-M Yao L-F Huang N Dong Y Yu and Z-YSheng ldquoThe potential effect and mechanism of high-mobilitygroup box 1 protein on regulatory T cell-mediated immunosup-pressionrdquo Journal of Interferon and Cytokine Research vol 31no 2 pp 249ndash257 2011

[15] Y-Y Luan C-F Yin Q-H Qin et al ldquoEffect of regulatory Tcells on promoting apoptosis of T lymphocyte and its regula-tory mechanism in sepsisrdquo Journal of Interferon and CytokineResearch vol 35 no 12 pp 969ndash980 2015

[16] B J Fisher D Kraskauskas E J Martin et al ldquoAttenuation ofsepsis-induced organ injury in mice by vitamin Crdquo Journal ofParenteral andEnteralNutrition vol 38 no 7 pp 825ndash839 2014

12 Mediators of Inflammation

[17] P E Marik ldquolsquoVitamin Srsquo (Steroids) and Vitamin C for the treat-ment of severe sepsis and septic shockrdquo Critical Care Medicinevol 44 no 6 pp 1228ndash1229 2016

[18] J-M Hong J-H Kim J S Kang W J Lee and Y-I HwangldquoVitamin C is taken up by human T cells via sodium-dependentvitamin C transporter 2 (SVCT2) and exerts inhibitory effectson the activation of these cells in vitrordquo Anatomy and CellBiology vol 49 no 2 pp 88ndash98 2016

[19] J Manning B Mitchell D A Appadurai et al ldquoVitamin C pro-motes maturation of T-cellsrdquo Antioxidants and Redox Signalingvol 19 no 17 pp 2054ndash2067 2013

[20] S Mburu J L Marnewick A Abayomi and H Ipp ldquoMod-ulation of LPS-induced CD4+ T-cell activation and apopto-sis by antioxidants in untreated asymptomatic HIV infectedparticipants An In Vitro Studyrdquo Clinical and DevelopmentalImmunology vol 2013 Article ID 631063 9 pages 2013

[21] P H Tan P Sagoo C Chan et al ldquoInhibition of NF-120581B andoxidative pathways in human dendritic cells by antioxidativevitamins generates regulatory T cellsrdquo Journal of Immunologyvol 174 no 12 pp 7633ndash7644 2005

[22] V S Nair M H Song and K I Oh ldquoVitamin C facilitatesdemethylation of the Foxp3 enhancer in a Tet-dependentmannerrdquo Journal of Immunology vol 196 no 5 pp 2119ndash21312016

[23] Y-L Gao Y-F Chai A-L Qi et al ldquoNeuropilin-1ℎ119894119892ℎCD4+CD25+ regulatory T cells exhibit primary negativeimmunoregulation in sepsisrdquo Mediators of Inflammation vol2016 Article ID 7132158 11 pages 2016

[24] Y L Gao Y F Chai N Dong et al ldquoTuftsin-derived T-peptideprevents cellular immunosuppression and improves survivalrate in septic micerdquo Scientific Reports vol 5 Article ID 167252015

[25] Z Yang Y Zhang L Dong et al ldquoThe reduction of peripheralblood CD4+ T cell indicates persistent organ failure in acutepancreatitisrdquo PLoSONE vol 10 no 5 Article ID e0125529 2015

[26] R Sharma P R Sharma Y C Kim et al ldquoIL-2-controlledexpression ofmultiple T cell trafficking genes andTh2 cytokinesin the regulatory T cell-deficient scurfy mice implication tomultiorgan inflammation and control of skin and lung inflam-mationrdquo The Journal of Immunology vol 186 no 2 pp 1268ndash1278 2011

[27] M J A J Huijskens M Walczak N Koller et al ldquoTechnicaladvance ascorbic acid induces development of double-positiveT cells from human hematopoietic stem cells in the absence ofStromal cellsrdquo Journal of Leukocyte Biology vol 96 no 6 pp1165ndash1175 2014

[28] H-J Kim R A Barnitz T Kreslavsky et al ldquoStable inhibitoryactivity of regulatory T cells requires the transcription factorHeliosrdquo Science vol 350 no 6258 pp 334ndash339 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Mediators of Inflammation

lowast lowastlowast

lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

Foxp

-3(

)

100

80

60

40

20

0

(a)

lowast

lowastlowast lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

CTLA

-4(

)

100

80

60

40

20

0

(b)

lowastlowastlowastlowast

lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

100

80

60

40

20

0

TGF-120573

m+

()

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

The r

ate o

f apo

ptos

is (

)40

30

20

10

0

(d)

lowastlowastlowastlowast

lowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

TGF-120573

(pg

mL)

600

400

200

0

(e)

lowastlowast

lowastlowast

lowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

IL-10

(pg

mL)

400

300

200

100

0

(f)

Figure 2 Continued

Mediators of Inflammation 7

M1 M1 M1 M1

M1M1M1M1

M1 M1 M1 M1

Cou

nts

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

100 101 102 103 104

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

PI

Annexin-V100 101 102 103 104

Annexin-V100 101 102 103 104

Annexin-V100

100

101

101

102

102

103

103

104

104

PI

100

101

102

103

104

PI

100

101

102

103

104

PI

100

101

102

103

104

Annexin-V

WT (CLP + Vit C)WT (CLP) Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

(g)

Figure 2 Parenteral Vit Cmarkedlyweakened the stability of CD4+CD25+ Tregs in sepsisThe expressions of Foxp-3 CTLA-4 andTGF-120573m+as well as the apoptotic ability of splenic CD4+CD25+ Tregs were subjected to flow cytometric analysis by flow cytometer (g) Parenteral VitC downregulated the expression of Foxp-3 (a) CTLA-4 (b) and TGF-120573m+ (c) of CD4+CD25+ Tregs in sepsis Parenteral Vit C did not alterthe apoptotic ability of splenic CD4+CD25+ Tregs in sepsis (d) Parenteral Vit C inhibited the secretion of TGF-120573 (e) and IL-10 (f) fromCD4+CD25+ Tregs in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

better and acted on upregulating the proliferative responseand IFN-120574 secretion as well as downregulating the apoptoticrate and IL-4 secretion The immunological mechanisms ofVit C on CD4+ T cells have not been elucidated reactiveoxygen species (ROS) was formed during CD4+ T cellsactivation which acted as the second messenger [19ndash21 27]We conjectured that parenteral Vit C affects T cell behaviorsduring sepsis via ROS

We have reported that the percentage and stability ofTregs were higher in septic patients than those without sepsis

a reduction in the stability of Tregs was accompanied by animprovement in survival rate and immune dysfunction ofT-lymphocytes with sepsis [12ndash15 23 24] Foxp- 3 whichis still the mainly intracellular marker for identification ofTregs was also critical for their function differentiation andmaintenance [10 11] Our previous study demonstrated thatan increased expression of Foxp-3 in Tregs was positivelycorrelated to themortality of septicmice [12] Parenteral Vit Cweakened the expression of Foxp-3 at protein and gene levelsbut the deficiency of Vit C promoted the expression of Foxp-3

8 Mediators of Inflammation

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD450

15

10

05

00

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowast

lowastlowast

The r

ate o

f apo

ptos

is (

)

80

60

40

20

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

IFN

-120574(p

gm

L)

150

100

50

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

lowast

IL-4

(pg

mL)

400

300

200

100

0

(d)

Figure 3 The impact of parenteral Vit C on the immunosuppressive function of CD4+CD25+ Tregs The parenteral Vit C inhibited theimmunosuppressive function of CD4+CD25+ Tregs which upregulated the proliferative response (a) and release of IFN-120574 (c) but significantlydownregulated the apoptotic rate (b) and release of IL-4 (d) of splenic conventional CD4+CD25minus T cells Data were represented as mean plusmnstandard deviation (SD) and analyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

in Gulominusminus mice septic model The recent researches showedthat the expression of transcription factor Helios controlleda great deal of pivotal aspects of Tregsrsquo suppressive functiondifferentiation and survival [28]We first reported that sepsisper se promoted the mRNA expression of Helios especiallyin the absence of Vit C but treatment with parenteral VitC after CLP of WT and Gulominusminus mice inhibited the mRNAexpression of Helios These suggested that parenteral Vit Chad significant ability to decrease the stability of Tregs withsepsis

During the development of sepsis Tregs can mainlyinhibit the activation of CD4+ T cells through variousmecha-nisms mainly including the pathway of cellular contact (egCTLA-4 and TGF-120573m+) and inhibitory cytokines (eg IL-10 and TGF-120573) as well as upregulating the antiapoptoticability of Tregs [11ndash15 23 24] Accumulated evidence hasshown that a combination of Foxp-3 CTLA-4 TGF-120573m+and inhibitory cytokines (IL-10 and TGF-120573) might serve asactive markers for Tregs with sepsis Our previous studydemonstrated that sepsis obviously enhanced the negative

Mediators of Inflammation 9

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowastlowast

Relat

ive l

evel

of F

oxp-3

mRN

A 6

4

2

0

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive o

f Fox

p3-T

SDR

mRN

A 6

4

2

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

Relat

ive l

evel

of H

elio

s mRN

A 4

3

2

1

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowastlowastlowast

lowastlowast

Relat

ive l

evel

of C

TLA-4

mRN

A 5

4

3

2

1

0

(d)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowast

Rela

tive l

evel

of T

GF-120573

mRN

A 6

4

2

0

(e)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive l

evel

of I

L-10

mRN

A 4

3

2

1

0

(f)

Figure 4 The impact of parenteral Vit C on the mRNA expression of Foxp-3 Helios CTLA-4 TGF-120573 IL-10 and the methylation level ofFoxp3-TSDR mRNA in CD4+CD25+ Tregs CD4+CD25+ Tregs were harvested for SYBR Green and Methylation-Sensitive RT-PCRT fromevery group after CLP The parenteral Vit C inhibited the expression of Foxp-3 (a) Helios (c) CTLA-4 (d) TGF-120573 (e) and IL-10 (f) butincreased the methylation level of Foxp3-TSDR (b) in CD4+CD25+ Tregs Data were represented as mean plusmn standard deviation (SD) andanalyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

10 Mediators of Inflammation

OD450

15

10

05

00

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

lowastlowastlowastlowast

lowast

lowastlowast

Gul

ominusminus

(CLP

)

(a)

The r

ate o

f apo

ptos

is (

)

100

80

60

40

20

0

lowastlowast

lowastlowast

lowast

lowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(b)

IFN

-120574(p

gm

L)

500

400

300

200

100

0

lowast

lowastlowastlowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(c)

IL-4

(pg

mL)

500

400

300

200

100

0

lowastlowastlowastlowast

lowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(d)

WT (CLP + Vit C)WT (CLP)Control group

Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

PI

104

103

102

101

100

104103102101100

Annexin-V

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

104103102101100

Annexin-V

104103102101100

Annexin-V104103102101100

Annexin-V104103102101100

Annexin-V

(e)

Figure 5 The impact of parenteral Vit C on CD4+ T cells-mediated cellular immunosuppression in sepsis Treatment with 200mgKg par-enteral Vit C upregulated the proliferative response of CD4+CD25minus T cells of WT mice and Gulominusminus mice in sepsis (a) The apoptotic rate ofsplenic CD4+CD25minusT cells was analyzed with Annexin-V-FITCPI by flow cytometry at 24 hours after CLP-inducedmidgrade sepsis (e)Theapoptotic level of splenic CD4+CD25minusT cells was decreased when 200mgKg parenteral Vit C was administered after CLP to WT mice andGulominusminus mice in sepsis (b) Parenteral Vit C increased the serum level of IFN-120574 (c) as well as decreasing the serum levels of IL-4 (d) of WTmice and Gulominusminus mice in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

Mediators of Inflammation 11

immunoregulation of Nrp-1highCD4+CD25+ Tregs whichcorrelated to the expression of Foxp-3 and CTLA-4 as wellas the secretion of IL-10 and TGF-120573 [23] We showed thatparenteral Vit C could inhibit the expression of exteriorCTLA-4 and TGF-120573m+ and the secretion of TGF-120573 and IL-10at protein and gene levels but did not alter the apoptotic levelof CD4+CD25+ Tregs These findings were in accordancewith the results of our previous studies [12 13] The stabilityof Tregs includes the expression of Foxp-3 and negativeimmunoregulation with sepsis which is crucially dependenton the demethylation status of the Foxp3-TSDR [11] Ourprevious study suggested that recombinant Nrp-1 polyclonalantibody could decrease the demethylation of Foxp3-TSDRin the presence of LPS which is associated with inhibition ofthe negative immunoregulation of Tregs [23] In the currentstudy parenteral Vit C had the ability to decrease thedemethylation level of Foxp3-TSDR but the deficiency ofVit C increased the demethylation level of Foxp3-TSDR inGulominusminusmice septicmodel whichwas in accordancewith ourprevious findings

5 Conclusion

In conclusion administration of parenteral Vit C improvedthe outcome of sepsis and sepsis-induced MODS of WT andGulominusminus mice The negative immunoregulation of Tregs wasinhibitedmainly including inhibiting the expression of Foxp-3 CTLA-4 and TGF-120573m+ and the secretion of inhibitorycytokines (TGF-120573 and IL-10) CD4+ T cells-mediated cellularimmunosuppression was improved by parenteral Vit C inWT and Gulominusminus septic mice These results suggested thatparenteral Vit C has the ability to improve the outcomeof sepsis and sepsis-induced MODS and is associated withimprovement in cellular immunosuppression

Competing Interests

The authors declare that they have no competing financialinterests This work is attributed to Tianjin Medical Univer-sity General Hospital Tianjin China

Authorsrsquo Contributions

Yu-Lei Gao Bin Lu and Jian-Hua Zhai contributed equally tothis work

Acknowledgments

This work was supported by grants from the TMUGHfunding (nos ZYYFY2015020 and ZYYFY2014014) and theRui-E funding (no 2016020) The authors thank ProfessorShu-Zhang Cui of Emergency Department of TianjinMedicalUniversity General Hospital for the guidance of experimentaldesign

References

[1] M Singer C S Deutschman C W Seymour et al ldquoThe thirdinternational consensus definitions for sepsis and septic shock

(Sepsis-3)rdquoThe Journal of theAmericanMedical Association vol315 no 8 pp 801ndash810 2016

[2] K-M Kaukonen M Bailey D Pilcher D J Cooper and RBellomo ldquoSystemic inflammatory response syndrome criteriain defining severe sepsisrdquoTheNew England Journal of Medicinevol 372 no 17 pp 1629ndash1638 2015

[3] D F Gaieski J M Edwards M J Kallan and B G CarrldquoBenchmarking the incidence and mortality of severe sepsis inthe united statesrdquo Critical Care Medicine vol 41 no 5 pp 1167ndash1174 2013

[4] B D Winters M Eberlein J Leung D M Needham P JPronovost and J E Sevransky ldquoLong-termmortality and qual-ity of life in sepsis a systematic reviewrdquo Critical Care Medicinevol 38 no 5 pp 1276ndash1283 2010

[5] S Inoue K Suzuki-Utsunomiya Y Okada et al ldquoReduction ofimmunocompetent T cells followed by prolonged lymphopeniain severe sepsis in the elderlyrdquo Critical Care Medicine vol 41no 3 pp 810ndash819 2013

[6] L P Skrupky P W Kerby and R S Hotchkiss ldquoAdvancesin the management of sepsis and the understanding of keyimmunologic defectsrdquo Anesthesiology vol 115 no 6 pp 1349ndash1362 2011

[7] J S Boomer K To K C Chang et al ldquoImmunosuppressionin patients who die of sepsis and multiple organ failurerdquo TheJournal of the American Medical Association vol 306 no 23pp 2594ndash2605 2011

[8] C J Darcy G Minigo K A Piera et al ldquoNeutrophils withmyeloid derived suppressor function deplete arginine andconstrain T cell function in septic shock patientsrdquoCritical Carevol 18 no 4 article R163 2014

[9] K Chang C Svabek C Vazquez-Guillamet et al ldquoTargetingthe programmed cell death 1 programmed cell death ligand 1pathway reverses T cell exhaustion in patients with sepsisrdquoCrit-ical Care vol 18 no 1 article R3 2014

[10] D C Nascimento J C Alves-Filho F Sonego et al ldquoRole ofregulatory T cells in long-term immune dysfunction associatedwith severe sepsisrdquo Critical Care Medicine vol 38 no 8 pp1718ndash1725 2010

[11] R TaturaM ZeschnigkWHansen et al ldquoRelevance of Foxp3+regulatory T cells for early and late phases of murine sepsisrdquoImmunology vol 146 no 1 pp 144ndash156 2015

[12] Q Y Liu Y M Yao Y Yu et al ldquoAstragalus polysaccharidesattenuate postburn sepsisvia inhibiting negative immunoreg-ulation of CD4+CD25highT cellsrdquo PLoS ONE vol 6 no 6Article ID e19811 2011

[13] X-M Zhu Y-M Yao H-P Liang et al ldquoHigh mobility groupbox-1 protein regulate immunosuppression of regulatory T cellsthrough toll-like receptor 4rdquo Cytokine vol 54 no 3 pp 296ndash304 2011

[14] Y Zhang Y-M Yao L-F Huang N Dong Y Yu and Z-YSheng ldquoThe potential effect and mechanism of high-mobilitygroup box 1 protein on regulatory T cell-mediated immunosup-pressionrdquo Journal of Interferon and Cytokine Research vol 31no 2 pp 249ndash257 2011

[15] Y-Y Luan C-F Yin Q-H Qin et al ldquoEffect of regulatory Tcells on promoting apoptosis of T lymphocyte and its regula-tory mechanism in sepsisrdquo Journal of Interferon and CytokineResearch vol 35 no 12 pp 969ndash980 2015

[16] B J Fisher D Kraskauskas E J Martin et al ldquoAttenuation ofsepsis-induced organ injury in mice by vitamin Crdquo Journal ofParenteral andEnteralNutrition vol 38 no 7 pp 825ndash839 2014

12 Mediators of Inflammation

[17] P E Marik ldquolsquoVitamin Srsquo (Steroids) and Vitamin C for the treat-ment of severe sepsis and septic shockrdquo Critical Care Medicinevol 44 no 6 pp 1228ndash1229 2016

[18] J-M Hong J-H Kim J S Kang W J Lee and Y-I HwangldquoVitamin C is taken up by human T cells via sodium-dependentvitamin C transporter 2 (SVCT2) and exerts inhibitory effectson the activation of these cells in vitrordquo Anatomy and CellBiology vol 49 no 2 pp 88ndash98 2016

[19] J Manning B Mitchell D A Appadurai et al ldquoVitamin C pro-motes maturation of T-cellsrdquo Antioxidants and Redox Signalingvol 19 no 17 pp 2054ndash2067 2013

[20] S Mburu J L Marnewick A Abayomi and H Ipp ldquoMod-ulation of LPS-induced CD4+ T-cell activation and apopto-sis by antioxidants in untreated asymptomatic HIV infectedparticipants An In Vitro Studyrdquo Clinical and DevelopmentalImmunology vol 2013 Article ID 631063 9 pages 2013

[21] P H Tan P Sagoo C Chan et al ldquoInhibition of NF-120581B andoxidative pathways in human dendritic cells by antioxidativevitamins generates regulatory T cellsrdquo Journal of Immunologyvol 174 no 12 pp 7633ndash7644 2005

[22] V S Nair M H Song and K I Oh ldquoVitamin C facilitatesdemethylation of the Foxp3 enhancer in a Tet-dependentmannerrdquo Journal of Immunology vol 196 no 5 pp 2119ndash21312016

[23] Y-L Gao Y-F Chai A-L Qi et al ldquoNeuropilin-1ℎ119894119892ℎCD4+CD25+ regulatory T cells exhibit primary negativeimmunoregulation in sepsisrdquo Mediators of Inflammation vol2016 Article ID 7132158 11 pages 2016

[24] Y L Gao Y F Chai N Dong et al ldquoTuftsin-derived T-peptideprevents cellular immunosuppression and improves survivalrate in septic micerdquo Scientific Reports vol 5 Article ID 167252015

[25] Z Yang Y Zhang L Dong et al ldquoThe reduction of peripheralblood CD4+ T cell indicates persistent organ failure in acutepancreatitisrdquo PLoSONE vol 10 no 5 Article ID e0125529 2015

[26] R Sharma P R Sharma Y C Kim et al ldquoIL-2-controlledexpression ofmultiple T cell trafficking genes andTh2 cytokinesin the regulatory T cell-deficient scurfy mice implication tomultiorgan inflammation and control of skin and lung inflam-mationrdquo The Journal of Immunology vol 186 no 2 pp 1268ndash1278 2011

[27] M J A J Huijskens M Walczak N Koller et al ldquoTechnicaladvance ascorbic acid induces development of double-positiveT cells from human hematopoietic stem cells in the absence ofStromal cellsrdquo Journal of Leukocyte Biology vol 96 no 6 pp1165ndash1175 2014

[28] H-J Kim R A Barnitz T Kreslavsky et al ldquoStable inhibitoryactivity of regulatory T cells requires the transcription factorHeliosrdquo Science vol 350 no 6258 pp 334ndash339 2015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 7

M1 M1 M1 M1

M1M1M1M1

M1 M1 M1 M1

Cou

nts

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

Foxp-3100 101 102 103 104

100 101 102 103 104

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

Cou

nts

200

160

120

80

40

0

100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100 101 102 103 104

CTLA-4100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

Cou

nts

100

80

60

40

20

0

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

TGF-120573m+

100 101 102 103 104

PI

Annexin-V100 101 102 103 104

Annexin-V100 101 102 103 104

Annexin-V100

100

101

101

102

102

103

103

104

104

PI

100

101

102

103

104

PI

100

101

102

103

104

PI

100

101

102

103

104

Annexin-V

WT (CLP + Vit C)WT (CLP) Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

(g)

Figure 2 Parenteral Vit Cmarkedlyweakened the stability of CD4+CD25+ Tregs in sepsisThe expressions of Foxp-3 CTLA-4 andTGF-120573m+as well as the apoptotic ability of splenic CD4+CD25+ Tregs were subjected to flow cytometric analysis by flow cytometer (g) Parenteral VitC downregulated the expression of Foxp-3 (a) CTLA-4 (b) and TGF-120573m+ (c) of CD4+CD25+ Tregs in sepsis Parenteral Vit C did not alterthe apoptotic ability of splenic CD4+CD25+ Tregs in sepsis (d) Parenteral Vit C inhibited the secretion of TGF-120573 (e) and IL-10 (f) fromCD4+CD25+ Tregs in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

better and acted on upregulating the proliferative responseand IFN-120574 secretion as well as downregulating the apoptoticrate and IL-4 secretion The immunological mechanisms ofVit C on CD4+ T cells have not been elucidated reactiveoxygen species (ROS) was formed during CD4+ T cellsactivation which acted as the second messenger [19ndash21 27]We conjectured that parenteral Vit C affects T cell behaviorsduring sepsis via ROS

We have reported that the percentage and stability ofTregs were higher in septic patients than those without sepsis

a reduction in the stability of Tregs was accompanied by animprovement in survival rate and immune dysfunction ofT-lymphocytes with sepsis [12ndash15 23 24] Foxp- 3 whichis still the mainly intracellular marker for identification ofTregs was also critical for their function differentiation andmaintenance [10 11] Our previous study demonstrated thatan increased expression of Foxp-3 in Tregs was positivelycorrelated to themortality of septicmice [12] Parenteral Vit Cweakened the expression of Foxp-3 at protein and gene levelsbut the deficiency of Vit C promoted the expression of Foxp-3

8 Mediators of Inflammation

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD450

15

10

05

00

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowast

lowastlowast

The r

ate o

f apo

ptos

is (

)

80

60

40

20

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

IFN

-120574(p

gm

L)

150

100

50

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

lowast

IL-4

(pg

mL)

400

300

200

100

0

(d)

Figure 3 The impact of parenteral Vit C on the immunosuppressive function of CD4+CD25+ Tregs The parenteral Vit C inhibited theimmunosuppressive function of CD4+CD25+ Tregs which upregulated the proliferative response (a) and release of IFN-120574 (c) but significantlydownregulated the apoptotic rate (b) and release of IL-4 (d) of splenic conventional CD4+CD25minus T cells Data were represented as mean plusmnstandard deviation (SD) and analyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

in Gulominusminus mice septic model The recent researches showedthat the expression of transcription factor Helios controlleda great deal of pivotal aspects of Tregsrsquo suppressive functiondifferentiation and survival [28]We first reported that sepsisper se promoted the mRNA expression of Helios especiallyin the absence of Vit C but treatment with parenteral VitC after CLP of WT and Gulominusminus mice inhibited the mRNAexpression of Helios These suggested that parenteral Vit Chad significant ability to decrease the stability of Tregs withsepsis

During the development of sepsis Tregs can mainlyinhibit the activation of CD4+ T cells through variousmecha-nisms mainly including the pathway of cellular contact (egCTLA-4 and TGF-120573m+) and inhibitory cytokines (eg IL-10 and TGF-120573) as well as upregulating the antiapoptoticability of Tregs [11ndash15 23 24] Accumulated evidence hasshown that a combination of Foxp-3 CTLA-4 TGF-120573m+and inhibitory cytokines (IL-10 and TGF-120573) might serve asactive markers for Tregs with sepsis Our previous studydemonstrated that sepsis obviously enhanced the negative

Mediators of Inflammation 9

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowastlowast

Relat

ive l

evel

of F

oxp-3

mRN

A 6

4

2

0

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive o

f Fox

p3-T

SDR

mRN

A 6

4

2

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

Relat

ive l

evel

of H

elio

s mRN

A 4

3

2

1

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowastlowastlowast

lowastlowast

Relat

ive l

evel

of C

TLA-4

mRN

A 5

4

3

2

1

0

(d)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowast

Rela

tive l

evel

of T

GF-120573

mRN

A 6

4

2

0

(e)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive l

evel

of I

L-10

mRN

A 4

3

2

1

0

(f)

Figure 4 The impact of parenteral Vit C on the mRNA expression of Foxp-3 Helios CTLA-4 TGF-120573 IL-10 and the methylation level ofFoxp3-TSDR mRNA in CD4+CD25+ Tregs CD4+CD25+ Tregs were harvested for SYBR Green and Methylation-Sensitive RT-PCRT fromevery group after CLP The parenteral Vit C inhibited the expression of Foxp-3 (a) Helios (c) CTLA-4 (d) TGF-120573 (e) and IL-10 (f) butincreased the methylation level of Foxp3-TSDR (b) in CD4+CD25+ Tregs Data were represented as mean plusmn standard deviation (SD) andanalyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

10 Mediators of Inflammation

OD450

15

10

05

00

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

lowastlowastlowastlowast

lowast

lowastlowast

Gul

ominusminus

(CLP

)

(a)

The r

ate o

f apo

ptos

is (

)

100

80

60

40

20

0

lowastlowast

lowastlowast

lowast

lowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(b)

IFN

-120574(p

gm

L)

500

400

300

200

100

0

lowast

lowastlowastlowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(c)

IL-4

(pg

mL)

500

400

300

200

100

0

lowastlowastlowastlowast

lowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(d)

WT (CLP + Vit C)WT (CLP)Control group

Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

PI

104

103

102

101

100

104103102101100

Annexin-V

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

104103102101100

Annexin-V

104103102101100

Annexin-V104103102101100

Annexin-V104103102101100

Annexin-V

(e)

Figure 5 The impact of parenteral Vit C on CD4+ T cells-mediated cellular immunosuppression in sepsis Treatment with 200mgKg par-enteral Vit C upregulated the proliferative response of CD4+CD25minus T cells of WT mice and Gulominusminus mice in sepsis (a) The apoptotic rate ofsplenic CD4+CD25minusT cells was analyzed with Annexin-V-FITCPI by flow cytometry at 24 hours after CLP-inducedmidgrade sepsis (e)Theapoptotic level of splenic CD4+CD25minusT cells was decreased when 200mgKg parenteral Vit C was administered after CLP to WT mice andGulominusminus mice in sepsis (b) Parenteral Vit C increased the serum level of IFN-120574 (c) as well as decreasing the serum levels of IL-4 (d) of WTmice and Gulominusminus mice in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

Mediators of Inflammation 11

immunoregulation of Nrp-1highCD4+CD25+ Tregs whichcorrelated to the expression of Foxp-3 and CTLA-4 as wellas the secretion of IL-10 and TGF-120573 [23] We showed thatparenteral Vit C could inhibit the expression of exteriorCTLA-4 and TGF-120573m+ and the secretion of TGF-120573 and IL-10at protein and gene levels but did not alter the apoptotic levelof CD4+CD25+ Tregs These findings were in accordancewith the results of our previous studies [12 13] The stabilityof Tregs includes the expression of Foxp-3 and negativeimmunoregulation with sepsis which is crucially dependenton the demethylation status of the Foxp3-TSDR [11] Ourprevious study suggested that recombinant Nrp-1 polyclonalantibody could decrease the demethylation of Foxp3-TSDRin the presence of LPS which is associated with inhibition ofthe negative immunoregulation of Tregs [23] In the currentstudy parenteral Vit C had the ability to decrease thedemethylation level of Foxp3-TSDR but the deficiency ofVit C increased the demethylation level of Foxp3-TSDR inGulominusminusmice septicmodel whichwas in accordancewith ourprevious findings

5 Conclusion

In conclusion administration of parenteral Vit C improvedthe outcome of sepsis and sepsis-induced MODS of WT andGulominusminus mice The negative immunoregulation of Tregs wasinhibitedmainly including inhibiting the expression of Foxp-3 CTLA-4 and TGF-120573m+ and the secretion of inhibitorycytokines (TGF-120573 and IL-10) CD4+ T cells-mediated cellularimmunosuppression was improved by parenteral Vit C inWT and Gulominusminus septic mice These results suggested thatparenteral Vit C has the ability to improve the outcomeof sepsis and sepsis-induced MODS and is associated withimprovement in cellular immunosuppression

Competing Interests

The authors declare that they have no competing financialinterests This work is attributed to Tianjin Medical Univer-sity General Hospital Tianjin China

Authorsrsquo Contributions

Yu-Lei Gao Bin Lu and Jian-Hua Zhai contributed equally tothis work

Acknowledgments

This work was supported by grants from the TMUGHfunding (nos ZYYFY2015020 and ZYYFY2014014) and theRui-E funding (no 2016020) The authors thank ProfessorShu-Zhang Cui of Emergency Department of TianjinMedicalUniversity General Hospital for the guidance of experimentaldesign

References

[1] M Singer C S Deutschman C W Seymour et al ldquoThe thirdinternational consensus definitions for sepsis and septic shock

(Sepsis-3)rdquoThe Journal of theAmericanMedical Association vol315 no 8 pp 801ndash810 2016

[2] K-M Kaukonen M Bailey D Pilcher D J Cooper and RBellomo ldquoSystemic inflammatory response syndrome criteriain defining severe sepsisrdquoTheNew England Journal of Medicinevol 372 no 17 pp 1629ndash1638 2015

[3] D F Gaieski J M Edwards M J Kallan and B G CarrldquoBenchmarking the incidence and mortality of severe sepsis inthe united statesrdquo Critical Care Medicine vol 41 no 5 pp 1167ndash1174 2013

[4] B D Winters M Eberlein J Leung D M Needham P JPronovost and J E Sevransky ldquoLong-termmortality and qual-ity of life in sepsis a systematic reviewrdquo Critical Care Medicinevol 38 no 5 pp 1276ndash1283 2010

[5] S Inoue K Suzuki-Utsunomiya Y Okada et al ldquoReduction ofimmunocompetent T cells followed by prolonged lymphopeniain severe sepsis in the elderlyrdquo Critical Care Medicine vol 41no 3 pp 810ndash819 2013

[6] L P Skrupky P W Kerby and R S Hotchkiss ldquoAdvancesin the management of sepsis and the understanding of keyimmunologic defectsrdquo Anesthesiology vol 115 no 6 pp 1349ndash1362 2011

[7] J S Boomer K To K C Chang et al ldquoImmunosuppressionin patients who die of sepsis and multiple organ failurerdquo TheJournal of the American Medical Association vol 306 no 23pp 2594ndash2605 2011

[8] C J Darcy G Minigo K A Piera et al ldquoNeutrophils withmyeloid derived suppressor function deplete arginine andconstrain T cell function in septic shock patientsrdquoCritical Carevol 18 no 4 article R163 2014

[9] K Chang C Svabek C Vazquez-Guillamet et al ldquoTargetingthe programmed cell death 1 programmed cell death ligand 1pathway reverses T cell exhaustion in patients with sepsisrdquoCrit-ical Care vol 18 no 1 article R3 2014

[10] D C Nascimento J C Alves-Filho F Sonego et al ldquoRole ofregulatory T cells in long-term immune dysfunction associatedwith severe sepsisrdquo Critical Care Medicine vol 38 no 8 pp1718ndash1725 2010

[11] R TaturaM ZeschnigkWHansen et al ldquoRelevance of Foxp3+regulatory T cells for early and late phases of murine sepsisrdquoImmunology vol 146 no 1 pp 144ndash156 2015

[12] Q Y Liu Y M Yao Y Yu et al ldquoAstragalus polysaccharidesattenuate postburn sepsisvia inhibiting negative immunoreg-ulation of CD4+CD25highT cellsrdquo PLoS ONE vol 6 no 6Article ID e19811 2011

[13] X-M Zhu Y-M Yao H-P Liang et al ldquoHigh mobility groupbox-1 protein regulate immunosuppression of regulatory T cellsthrough toll-like receptor 4rdquo Cytokine vol 54 no 3 pp 296ndash304 2011

[14] Y Zhang Y-M Yao L-F Huang N Dong Y Yu and Z-YSheng ldquoThe potential effect and mechanism of high-mobilitygroup box 1 protein on regulatory T cell-mediated immunosup-pressionrdquo Journal of Interferon and Cytokine Research vol 31no 2 pp 249ndash257 2011

[15] Y-Y Luan C-F Yin Q-H Qin et al ldquoEffect of regulatory Tcells on promoting apoptosis of T lymphocyte and its regula-tory mechanism in sepsisrdquo Journal of Interferon and CytokineResearch vol 35 no 12 pp 969ndash980 2015

[16] B J Fisher D Kraskauskas E J Martin et al ldquoAttenuation ofsepsis-induced organ injury in mice by vitamin Crdquo Journal ofParenteral andEnteralNutrition vol 38 no 7 pp 825ndash839 2014

12 Mediators of Inflammation

[17] P E Marik ldquolsquoVitamin Srsquo (Steroids) and Vitamin C for the treat-ment of severe sepsis and septic shockrdquo Critical Care Medicinevol 44 no 6 pp 1228ndash1229 2016

[18] J-M Hong J-H Kim J S Kang W J Lee and Y-I HwangldquoVitamin C is taken up by human T cells via sodium-dependentvitamin C transporter 2 (SVCT2) and exerts inhibitory effectson the activation of these cells in vitrordquo Anatomy and CellBiology vol 49 no 2 pp 88ndash98 2016

[19] J Manning B Mitchell D A Appadurai et al ldquoVitamin C pro-motes maturation of T-cellsrdquo Antioxidants and Redox Signalingvol 19 no 17 pp 2054ndash2067 2013

[20] S Mburu J L Marnewick A Abayomi and H Ipp ldquoMod-ulation of LPS-induced CD4+ T-cell activation and apopto-sis by antioxidants in untreated asymptomatic HIV infectedparticipants An In Vitro Studyrdquo Clinical and DevelopmentalImmunology vol 2013 Article ID 631063 9 pages 2013

[21] P H Tan P Sagoo C Chan et al ldquoInhibition of NF-120581B andoxidative pathways in human dendritic cells by antioxidativevitamins generates regulatory T cellsrdquo Journal of Immunologyvol 174 no 12 pp 7633ndash7644 2005

[22] V S Nair M H Song and K I Oh ldquoVitamin C facilitatesdemethylation of the Foxp3 enhancer in a Tet-dependentmannerrdquo Journal of Immunology vol 196 no 5 pp 2119ndash21312016

[23] Y-L Gao Y-F Chai A-L Qi et al ldquoNeuropilin-1ℎ119894119892ℎCD4+CD25+ regulatory T cells exhibit primary negativeimmunoregulation in sepsisrdquo Mediators of Inflammation vol2016 Article ID 7132158 11 pages 2016

[24] Y L Gao Y F Chai N Dong et al ldquoTuftsin-derived T-peptideprevents cellular immunosuppression and improves survivalrate in septic micerdquo Scientific Reports vol 5 Article ID 167252015

[25] Z Yang Y Zhang L Dong et al ldquoThe reduction of peripheralblood CD4+ T cell indicates persistent organ failure in acutepancreatitisrdquo PLoSONE vol 10 no 5 Article ID e0125529 2015

[26] R Sharma P R Sharma Y C Kim et al ldquoIL-2-controlledexpression ofmultiple T cell trafficking genes andTh2 cytokinesin the regulatory T cell-deficient scurfy mice implication tomultiorgan inflammation and control of skin and lung inflam-mationrdquo The Journal of Immunology vol 186 no 2 pp 1268ndash1278 2011

[27] M J A J Huijskens M Walczak N Koller et al ldquoTechnicaladvance ascorbic acid induces development of double-positiveT cells from human hematopoietic stem cells in the absence ofStromal cellsrdquo Journal of Leukocyte Biology vol 96 no 6 pp1165ndash1175 2014

[28] H-J Kim R A Barnitz T Kreslavsky et al ldquoStable inhibitoryactivity of regulatory T cells requires the transcription factorHeliosrdquo Science vol 350 no 6258 pp 334ndash339 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Mediators of Inflammation

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD450

15

10

05

00

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowast

lowastlowast

The r

ate o

f apo

ptos

is (

)

80

60

40

20

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

IFN

-120574(p

gm

L)

150

100

50

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

lowast

IL-4

(pg

mL)

400

300

200

100

0

(d)

Figure 3 The impact of parenteral Vit C on the immunosuppressive function of CD4+CD25+ Tregs The parenteral Vit C inhibited theimmunosuppressive function of CD4+CD25+ Tregs which upregulated the proliferative response (a) and release of IFN-120574 (c) but significantlydownregulated the apoptotic rate (b) and release of IL-4 (d) of splenic conventional CD4+CD25minus T cells Data were represented as mean plusmnstandard deviation (SD) and analyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

in Gulominusminus mice septic model The recent researches showedthat the expression of transcription factor Helios controlleda great deal of pivotal aspects of Tregsrsquo suppressive functiondifferentiation and survival [28]We first reported that sepsisper se promoted the mRNA expression of Helios especiallyin the absence of Vit C but treatment with parenteral VitC after CLP of WT and Gulominusminus mice inhibited the mRNAexpression of Helios These suggested that parenteral Vit Chad significant ability to decrease the stability of Tregs withsepsis

During the development of sepsis Tregs can mainlyinhibit the activation of CD4+ T cells through variousmecha-nisms mainly including the pathway of cellular contact (egCTLA-4 and TGF-120573m+) and inhibitory cytokines (eg IL-10 and TGF-120573) as well as upregulating the antiapoptoticability of Tregs [11ndash15 23 24] Accumulated evidence hasshown that a combination of Foxp-3 CTLA-4 TGF-120573m+and inhibitory cytokines (IL-10 and TGF-120573) might serve asactive markers for Tregs with sepsis Our previous studydemonstrated that sepsis obviously enhanced the negative

Mediators of Inflammation 9

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowastlowast

Relat

ive l

evel

of F

oxp-3

mRN

A 6

4

2

0

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive o

f Fox

p3-T

SDR

mRN

A 6

4

2

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

Relat

ive l

evel

of H

elio

s mRN

A 4

3

2

1

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowastlowastlowast

lowastlowast

Relat

ive l

evel

of C

TLA-4

mRN

A 5

4

3

2

1

0

(d)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowast

Rela

tive l

evel

of T

GF-120573

mRN

A 6

4

2

0

(e)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive l

evel

of I

L-10

mRN

A 4

3

2

1

0

(f)

Figure 4 The impact of parenteral Vit C on the mRNA expression of Foxp-3 Helios CTLA-4 TGF-120573 IL-10 and the methylation level ofFoxp3-TSDR mRNA in CD4+CD25+ Tregs CD4+CD25+ Tregs were harvested for SYBR Green and Methylation-Sensitive RT-PCRT fromevery group after CLP The parenteral Vit C inhibited the expression of Foxp-3 (a) Helios (c) CTLA-4 (d) TGF-120573 (e) and IL-10 (f) butincreased the methylation level of Foxp3-TSDR (b) in CD4+CD25+ Tregs Data were represented as mean plusmn standard deviation (SD) andanalyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

10 Mediators of Inflammation

OD450

15

10

05

00

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

lowastlowastlowastlowast

lowast

lowastlowast

Gul

ominusminus

(CLP

)

(a)

The r

ate o

f apo

ptos

is (

)

100

80

60

40

20

0

lowastlowast

lowastlowast

lowast

lowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(b)

IFN

-120574(p

gm

L)

500

400

300

200

100

0

lowast

lowastlowastlowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(c)

IL-4

(pg

mL)

500

400

300

200

100

0

lowastlowastlowastlowast

lowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(d)

WT (CLP + Vit C)WT (CLP)Control group

Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

PI

104

103

102

101

100

104103102101100

Annexin-V

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

104103102101100

Annexin-V

104103102101100

Annexin-V104103102101100

Annexin-V104103102101100

Annexin-V

(e)

Figure 5 The impact of parenteral Vit C on CD4+ T cells-mediated cellular immunosuppression in sepsis Treatment with 200mgKg par-enteral Vit C upregulated the proliferative response of CD4+CD25minus T cells of WT mice and Gulominusminus mice in sepsis (a) The apoptotic rate ofsplenic CD4+CD25minusT cells was analyzed with Annexin-V-FITCPI by flow cytometry at 24 hours after CLP-inducedmidgrade sepsis (e)Theapoptotic level of splenic CD4+CD25minusT cells was decreased when 200mgKg parenteral Vit C was administered after CLP to WT mice andGulominusminus mice in sepsis (b) Parenteral Vit C increased the serum level of IFN-120574 (c) as well as decreasing the serum levels of IL-4 (d) of WTmice and Gulominusminus mice in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

Mediators of Inflammation 11

immunoregulation of Nrp-1highCD4+CD25+ Tregs whichcorrelated to the expression of Foxp-3 and CTLA-4 as wellas the secretion of IL-10 and TGF-120573 [23] We showed thatparenteral Vit C could inhibit the expression of exteriorCTLA-4 and TGF-120573m+ and the secretion of TGF-120573 and IL-10at protein and gene levels but did not alter the apoptotic levelof CD4+CD25+ Tregs These findings were in accordancewith the results of our previous studies [12 13] The stabilityof Tregs includes the expression of Foxp-3 and negativeimmunoregulation with sepsis which is crucially dependenton the demethylation status of the Foxp3-TSDR [11] Ourprevious study suggested that recombinant Nrp-1 polyclonalantibody could decrease the demethylation of Foxp3-TSDRin the presence of LPS which is associated with inhibition ofthe negative immunoregulation of Tregs [23] In the currentstudy parenteral Vit C had the ability to decrease thedemethylation level of Foxp3-TSDR but the deficiency ofVit C increased the demethylation level of Foxp3-TSDR inGulominusminusmice septicmodel whichwas in accordancewith ourprevious findings

5 Conclusion

In conclusion administration of parenteral Vit C improvedthe outcome of sepsis and sepsis-induced MODS of WT andGulominusminus mice The negative immunoregulation of Tregs wasinhibitedmainly including inhibiting the expression of Foxp-3 CTLA-4 and TGF-120573m+ and the secretion of inhibitorycytokines (TGF-120573 and IL-10) CD4+ T cells-mediated cellularimmunosuppression was improved by parenteral Vit C inWT and Gulominusminus septic mice These results suggested thatparenteral Vit C has the ability to improve the outcomeof sepsis and sepsis-induced MODS and is associated withimprovement in cellular immunosuppression

Competing Interests

The authors declare that they have no competing financialinterests This work is attributed to Tianjin Medical Univer-sity General Hospital Tianjin China

Authorsrsquo Contributions

Yu-Lei Gao Bin Lu and Jian-Hua Zhai contributed equally tothis work

Acknowledgments

This work was supported by grants from the TMUGHfunding (nos ZYYFY2015020 and ZYYFY2014014) and theRui-E funding (no 2016020) The authors thank ProfessorShu-Zhang Cui of Emergency Department of TianjinMedicalUniversity General Hospital for the guidance of experimentaldesign

References

[1] M Singer C S Deutschman C W Seymour et al ldquoThe thirdinternational consensus definitions for sepsis and septic shock

(Sepsis-3)rdquoThe Journal of theAmericanMedical Association vol315 no 8 pp 801ndash810 2016

[2] K-M Kaukonen M Bailey D Pilcher D J Cooper and RBellomo ldquoSystemic inflammatory response syndrome criteriain defining severe sepsisrdquoTheNew England Journal of Medicinevol 372 no 17 pp 1629ndash1638 2015

[3] D F Gaieski J M Edwards M J Kallan and B G CarrldquoBenchmarking the incidence and mortality of severe sepsis inthe united statesrdquo Critical Care Medicine vol 41 no 5 pp 1167ndash1174 2013

[4] B D Winters M Eberlein J Leung D M Needham P JPronovost and J E Sevransky ldquoLong-termmortality and qual-ity of life in sepsis a systematic reviewrdquo Critical Care Medicinevol 38 no 5 pp 1276ndash1283 2010

[5] S Inoue K Suzuki-Utsunomiya Y Okada et al ldquoReduction ofimmunocompetent T cells followed by prolonged lymphopeniain severe sepsis in the elderlyrdquo Critical Care Medicine vol 41no 3 pp 810ndash819 2013

[6] L P Skrupky P W Kerby and R S Hotchkiss ldquoAdvancesin the management of sepsis and the understanding of keyimmunologic defectsrdquo Anesthesiology vol 115 no 6 pp 1349ndash1362 2011

[7] J S Boomer K To K C Chang et al ldquoImmunosuppressionin patients who die of sepsis and multiple organ failurerdquo TheJournal of the American Medical Association vol 306 no 23pp 2594ndash2605 2011

[8] C J Darcy G Minigo K A Piera et al ldquoNeutrophils withmyeloid derived suppressor function deplete arginine andconstrain T cell function in septic shock patientsrdquoCritical Carevol 18 no 4 article R163 2014

[9] K Chang C Svabek C Vazquez-Guillamet et al ldquoTargetingthe programmed cell death 1 programmed cell death ligand 1pathway reverses T cell exhaustion in patients with sepsisrdquoCrit-ical Care vol 18 no 1 article R3 2014

[10] D C Nascimento J C Alves-Filho F Sonego et al ldquoRole ofregulatory T cells in long-term immune dysfunction associatedwith severe sepsisrdquo Critical Care Medicine vol 38 no 8 pp1718ndash1725 2010

[11] R TaturaM ZeschnigkWHansen et al ldquoRelevance of Foxp3+regulatory T cells for early and late phases of murine sepsisrdquoImmunology vol 146 no 1 pp 144ndash156 2015

[12] Q Y Liu Y M Yao Y Yu et al ldquoAstragalus polysaccharidesattenuate postburn sepsisvia inhibiting negative immunoreg-ulation of CD4+CD25highT cellsrdquo PLoS ONE vol 6 no 6Article ID e19811 2011

[13] X-M Zhu Y-M Yao H-P Liang et al ldquoHigh mobility groupbox-1 protein regulate immunosuppression of regulatory T cellsthrough toll-like receptor 4rdquo Cytokine vol 54 no 3 pp 296ndash304 2011

[14] Y Zhang Y-M Yao L-F Huang N Dong Y Yu and Z-YSheng ldquoThe potential effect and mechanism of high-mobilitygroup box 1 protein on regulatory T cell-mediated immunosup-pressionrdquo Journal of Interferon and Cytokine Research vol 31no 2 pp 249ndash257 2011

[15] Y-Y Luan C-F Yin Q-H Qin et al ldquoEffect of regulatory Tcells on promoting apoptosis of T lymphocyte and its regula-tory mechanism in sepsisrdquo Journal of Interferon and CytokineResearch vol 35 no 12 pp 969ndash980 2015

[16] B J Fisher D Kraskauskas E J Martin et al ldquoAttenuation ofsepsis-induced organ injury in mice by vitamin Crdquo Journal ofParenteral andEnteralNutrition vol 38 no 7 pp 825ndash839 2014

12 Mediators of Inflammation

[17] P E Marik ldquolsquoVitamin Srsquo (Steroids) and Vitamin C for the treat-ment of severe sepsis and septic shockrdquo Critical Care Medicinevol 44 no 6 pp 1228ndash1229 2016

[18] J-M Hong J-H Kim J S Kang W J Lee and Y-I HwangldquoVitamin C is taken up by human T cells via sodium-dependentvitamin C transporter 2 (SVCT2) and exerts inhibitory effectson the activation of these cells in vitrordquo Anatomy and CellBiology vol 49 no 2 pp 88ndash98 2016

[19] J Manning B Mitchell D A Appadurai et al ldquoVitamin C pro-motes maturation of T-cellsrdquo Antioxidants and Redox Signalingvol 19 no 17 pp 2054ndash2067 2013

[20] S Mburu J L Marnewick A Abayomi and H Ipp ldquoMod-ulation of LPS-induced CD4+ T-cell activation and apopto-sis by antioxidants in untreated asymptomatic HIV infectedparticipants An In Vitro Studyrdquo Clinical and DevelopmentalImmunology vol 2013 Article ID 631063 9 pages 2013

[21] P H Tan P Sagoo C Chan et al ldquoInhibition of NF-120581B andoxidative pathways in human dendritic cells by antioxidativevitamins generates regulatory T cellsrdquo Journal of Immunologyvol 174 no 12 pp 7633ndash7644 2005

[22] V S Nair M H Song and K I Oh ldquoVitamin C facilitatesdemethylation of the Foxp3 enhancer in a Tet-dependentmannerrdquo Journal of Immunology vol 196 no 5 pp 2119ndash21312016

[23] Y-L Gao Y-F Chai A-L Qi et al ldquoNeuropilin-1ℎ119894119892ℎCD4+CD25+ regulatory T cells exhibit primary negativeimmunoregulation in sepsisrdquo Mediators of Inflammation vol2016 Article ID 7132158 11 pages 2016

[24] Y L Gao Y F Chai N Dong et al ldquoTuftsin-derived T-peptideprevents cellular immunosuppression and improves survivalrate in septic micerdquo Scientific Reports vol 5 Article ID 167252015

[25] Z Yang Y Zhang L Dong et al ldquoThe reduction of peripheralblood CD4+ T cell indicates persistent organ failure in acutepancreatitisrdquo PLoSONE vol 10 no 5 Article ID e0125529 2015

[26] R Sharma P R Sharma Y C Kim et al ldquoIL-2-controlledexpression ofmultiple T cell trafficking genes andTh2 cytokinesin the regulatory T cell-deficient scurfy mice implication tomultiorgan inflammation and control of skin and lung inflam-mationrdquo The Journal of Immunology vol 186 no 2 pp 1268ndash1278 2011

[27] M J A J Huijskens M Walczak N Koller et al ldquoTechnicaladvance ascorbic acid induces development of double-positiveT cells from human hematopoietic stem cells in the absence ofStromal cellsrdquo Journal of Leukocyte Biology vol 96 no 6 pp1165ndash1175 2014

[28] H-J Kim R A Barnitz T Kreslavsky et al ldquoStable inhibitoryactivity of regulatory T cells requires the transcription factorHeliosrdquo Science vol 350 no 6258 pp 334ndash339 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 9

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowastlowast

Relat

ive l

evel

of F

oxp-3

mRN

A 6

4

2

0

(a)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive o

f Fox

p3-T

SDR

mRN

A 6

4

2

0

(b)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast lowastlowast

Relat

ive l

evel

of H

elio

s mRN

A 4

3

2

1

0

(c)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowastlowastlowast

lowastlowast

Relat

ive l

evel

of C

TLA-4

mRN

A 5

4

3

2

1

0

(d)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowast

lowast

Rela

tive l

evel

of T

GF-120573

mRN

A 6

4

2

0

(e)

WT

(CLP

+ V

it C)

WT

(CLP

)

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

lowastlowast

lowastlowast

lowastlowast

Relat

ive l

evel

of I

L-10

mRN

A 4

3

2

1

0

(f)

Figure 4 The impact of parenteral Vit C on the mRNA expression of Foxp-3 Helios CTLA-4 TGF-120573 IL-10 and the methylation level ofFoxp3-TSDR mRNA in CD4+CD25+ Tregs CD4+CD25+ Tregs were harvested for SYBR Green and Methylation-Sensitive RT-PCRT fromevery group after CLP The parenteral Vit C inhibited the expression of Foxp-3 (a) Helios (c) CTLA-4 (d) TGF-120573 (e) and IL-10 (f) butincreased the methylation level of Foxp3-TSDR (b) in CD4+CD25+ Tregs Data were represented as mean plusmn standard deviation (SD) andanalyzed by software SPSS 170 with one-way ANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

10 Mediators of Inflammation

OD450

15

10

05

00

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

lowastlowastlowastlowast

lowast

lowastlowast

Gul

ominusminus

(CLP

)

(a)

The r

ate o

f apo

ptos

is (

)

100

80

60

40

20

0

lowastlowast

lowastlowast

lowast

lowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(b)

IFN

-120574(p

gm

L)

500

400

300

200

100

0

lowast

lowastlowastlowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(c)

IL-4

(pg

mL)

500

400

300

200

100

0

lowastlowastlowastlowast

lowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(d)

WT (CLP + Vit C)WT (CLP)Control group

Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

PI

104

103

102

101

100

104103102101100

Annexin-V

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

104103102101100

Annexin-V

104103102101100

Annexin-V104103102101100

Annexin-V104103102101100

Annexin-V

(e)

Figure 5 The impact of parenteral Vit C on CD4+ T cells-mediated cellular immunosuppression in sepsis Treatment with 200mgKg par-enteral Vit C upregulated the proliferative response of CD4+CD25minus T cells of WT mice and Gulominusminus mice in sepsis (a) The apoptotic rate ofsplenic CD4+CD25minusT cells was analyzed with Annexin-V-FITCPI by flow cytometry at 24 hours after CLP-inducedmidgrade sepsis (e)Theapoptotic level of splenic CD4+CD25minusT cells was decreased when 200mgKg parenteral Vit C was administered after CLP to WT mice andGulominusminus mice in sepsis (b) Parenteral Vit C increased the serum level of IFN-120574 (c) as well as decreasing the serum levels of IL-4 (d) of WTmice and Gulominusminus mice in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

Mediators of Inflammation 11

immunoregulation of Nrp-1highCD4+CD25+ Tregs whichcorrelated to the expression of Foxp-3 and CTLA-4 as wellas the secretion of IL-10 and TGF-120573 [23] We showed thatparenteral Vit C could inhibit the expression of exteriorCTLA-4 and TGF-120573m+ and the secretion of TGF-120573 and IL-10at protein and gene levels but did not alter the apoptotic levelof CD4+CD25+ Tregs These findings were in accordancewith the results of our previous studies [12 13] The stabilityof Tregs includes the expression of Foxp-3 and negativeimmunoregulation with sepsis which is crucially dependenton the demethylation status of the Foxp3-TSDR [11] Ourprevious study suggested that recombinant Nrp-1 polyclonalantibody could decrease the demethylation of Foxp3-TSDRin the presence of LPS which is associated with inhibition ofthe negative immunoregulation of Tregs [23] In the currentstudy parenteral Vit C had the ability to decrease thedemethylation level of Foxp3-TSDR but the deficiency ofVit C increased the demethylation level of Foxp3-TSDR inGulominusminusmice septicmodel whichwas in accordancewith ourprevious findings

5 Conclusion

In conclusion administration of parenteral Vit C improvedthe outcome of sepsis and sepsis-induced MODS of WT andGulominusminus mice The negative immunoregulation of Tregs wasinhibitedmainly including inhibiting the expression of Foxp-3 CTLA-4 and TGF-120573m+ and the secretion of inhibitorycytokines (TGF-120573 and IL-10) CD4+ T cells-mediated cellularimmunosuppression was improved by parenteral Vit C inWT and Gulominusminus septic mice These results suggested thatparenteral Vit C has the ability to improve the outcomeof sepsis and sepsis-induced MODS and is associated withimprovement in cellular immunosuppression

Competing Interests

The authors declare that they have no competing financialinterests This work is attributed to Tianjin Medical Univer-sity General Hospital Tianjin China

Authorsrsquo Contributions

Yu-Lei Gao Bin Lu and Jian-Hua Zhai contributed equally tothis work

Acknowledgments

This work was supported by grants from the TMUGHfunding (nos ZYYFY2015020 and ZYYFY2014014) and theRui-E funding (no 2016020) The authors thank ProfessorShu-Zhang Cui of Emergency Department of TianjinMedicalUniversity General Hospital for the guidance of experimentaldesign

References

[1] M Singer C S Deutschman C W Seymour et al ldquoThe thirdinternational consensus definitions for sepsis and septic shock

(Sepsis-3)rdquoThe Journal of theAmericanMedical Association vol315 no 8 pp 801ndash810 2016

[2] K-M Kaukonen M Bailey D Pilcher D J Cooper and RBellomo ldquoSystemic inflammatory response syndrome criteriain defining severe sepsisrdquoTheNew England Journal of Medicinevol 372 no 17 pp 1629ndash1638 2015

[3] D F Gaieski J M Edwards M J Kallan and B G CarrldquoBenchmarking the incidence and mortality of severe sepsis inthe united statesrdquo Critical Care Medicine vol 41 no 5 pp 1167ndash1174 2013

[4] B D Winters M Eberlein J Leung D M Needham P JPronovost and J E Sevransky ldquoLong-termmortality and qual-ity of life in sepsis a systematic reviewrdquo Critical Care Medicinevol 38 no 5 pp 1276ndash1283 2010

[5] S Inoue K Suzuki-Utsunomiya Y Okada et al ldquoReduction ofimmunocompetent T cells followed by prolonged lymphopeniain severe sepsis in the elderlyrdquo Critical Care Medicine vol 41no 3 pp 810ndash819 2013

[6] L P Skrupky P W Kerby and R S Hotchkiss ldquoAdvancesin the management of sepsis and the understanding of keyimmunologic defectsrdquo Anesthesiology vol 115 no 6 pp 1349ndash1362 2011

[7] J S Boomer K To K C Chang et al ldquoImmunosuppressionin patients who die of sepsis and multiple organ failurerdquo TheJournal of the American Medical Association vol 306 no 23pp 2594ndash2605 2011

[8] C J Darcy G Minigo K A Piera et al ldquoNeutrophils withmyeloid derived suppressor function deplete arginine andconstrain T cell function in septic shock patientsrdquoCritical Carevol 18 no 4 article R163 2014

[9] K Chang C Svabek C Vazquez-Guillamet et al ldquoTargetingthe programmed cell death 1 programmed cell death ligand 1pathway reverses T cell exhaustion in patients with sepsisrdquoCrit-ical Care vol 18 no 1 article R3 2014

[10] D C Nascimento J C Alves-Filho F Sonego et al ldquoRole ofregulatory T cells in long-term immune dysfunction associatedwith severe sepsisrdquo Critical Care Medicine vol 38 no 8 pp1718ndash1725 2010

[11] R TaturaM ZeschnigkWHansen et al ldquoRelevance of Foxp3+regulatory T cells for early and late phases of murine sepsisrdquoImmunology vol 146 no 1 pp 144ndash156 2015

[12] Q Y Liu Y M Yao Y Yu et al ldquoAstragalus polysaccharidesattenuate postburn sepsisvia inhibiting negative immunoreg-ulation of CD4+CD25highT cellsrdquo PLoS ONE vol 6 no 6Article ID e19811 2011

[13] X-M Zhu Y-M Yao H-P Liang et al ldquoHigh mobility groupbox-1 protein regulate immunosuppression of regulatory T cellsthrough toll-like receptor 4rdquo Cytokine vol 54 no 3 pp 296ndash304 2011

[14] Y Zhang Y-M Yao L-F Huang N Dong Y Yu and Z-YSheng ldquoThe potential effect and mechanism of high-mobilitygroup box 1 protein on regulatory T cell-mediated immunosup-pressionrdquo Journal of Interferon and Cytokine Research vol 31no 2 pp 249ndash257 2011

[15] Y-Y Luan C-F Yin Q-H Qin et al ldquoEffect of regulatory Tcells on promoting apoptosis of T lymphocyte and its regula-tory mechanism in sepsisrdquo Journal of Interferon and CytokineResearch vol 35 no 12 pp 969ndash980 2015

[16] B J Fisher D Kraskauskas E J Martin et al ldquoAttenuation ofsepsis-induced organ injury in mice by vitamin Crdquo Journal ofParenteral andEnteralNutrition vol 38 no 7 pp 825ndash839 2014

12 Mediators of Inflammation

[17] P E Marik ldquolsquoVitamin Srsquo (Steroids) and Vitamin C for the treat-ment of severe sepsis and septic shockrdquo Critical Care Medicinevol 44 no 6 pp 1228ndash1229 2016

[18] J-M Hong J-H Kim J S Kang W J Lee and Y-I HwangldquoVitamin C is taken up by human T cells via sodium-dependentvitamin C transporter 2 (SVCT2) and exerts inhibitory effectson the activation of these cells in vitrordquo Anatomy and CellBiology vol 49 no 2 pp 88ndash98 2016

[19] J Manning B Mitchell D A Appadurai et al ldquoVitamin C pro-motes maturation of T-cellsrdquo Antioxidants and Redox Signalingvol 19 no 17 pp 2054ndash2067 2013

[20] S Mburu J L Marnewick A Abayomi and H Ipp ldquoMod-ulation of LPS-induced CD4+ T-cell activation and apopto-sis by antioxidants in untreated asymptomatic HIV infectedparticipants An In Vitro Studyrdquo Clinical and DevelopmentalImmunology vol 2013 Article ID 631063 9 pages 2013

[21] P H Tan P Sagoo C Chan et al ldquoInhibition of NF-120581B andoxidative pathways in human dendritic cells by antioxidativevitamins generates regulatory T cellsrdquo Journal of Immunologyvol 174 no 12 pp 7633ndash7644 2005

[22] V S Nair M H Song and K I Oh ldquoVitamin C facilitatesdemethylation of the Foxp3 enhancer in a Tet-dependentmannerrdquo Journal of Immunology vol 196 no 5 pp 2119ndash21312016

[23] Y-L Gao Y-F Chai A-L Qi et al ldquoNeuropilin-1ℎ119894119892ℎCD4+CD25+ regulatory T cells exhibit primary negativeimmunoregulation in sepsisrdquo Mediators of Inflammation vol2016 Article ID 7132158 11 pages 2016

[24] Y L Gao Y F Chai N Dong et al ldquoTuftsin-derived T-peptideprevents cellular immunosuppression and improves survivalrate in septic micerdquo Scientific Reports vol 5 Article ID 167252015

[25] Z Yang Y Zhang L Dong et al ldquoThe reduction of peripheralblood CD4+ T cell indicates persistent organ failure in acutepancreatitisrdquo PLoSONE vol 10 no 5 Article ID e0125529 2015

[26] R Sharma P R Sharma Y C Kim et al ldquoIL-2-controlledexpression ofmultiple T cell trafficking genes andTh2 cytokinesin the regulatory T cell-deficient scurfy mice implication tomultiorgan inflammation and control of skin and lung inflam-mationrdquo The Journal of Immunology vol 186 no 2 pp 1268ndash1278 2011

[27] M J A J Huijskens M Walczak N Koller et al ldquoTechnicaladvance ascorbic acid induces development of double-positiveT cells from human hematopoietic stem cells in the absence ofStromal cellsrdquo Journal of Leukocyte Biology vol 96 no 6 pp1165ndash1175 2014

[28] H-J Kim R A Barnitz T Kreslavsky et al ldquoStable inhibitoryactivity of regulatory T cells requires the transcription factorHeliosrdquo Science vol 350 no 6258 pp 334ndash339 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Mediators of Inflammation

OD450

15

10

05

00

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

lowastlowastlowastlowast

lowast

lowastlowast

Gul

ominusminus

(CLP

)

(a)

The r

ate o

f apo

ptos

is (

)

100

80

60

40

20

0

lowastlowast

lowastlowast

lowast

lowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(b)

IFN

-120574(p

gm

L)

500

400

300

200

100

0

lowast

lowastlowastlowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(c)

IL-4

(pg

mL)

500

400

300

200

100

0

lowastlowastlowastlowast

lowastlowastlowastlowast

WT

(CLP

+ V

it C)

WT

(CLP

)

Con

trol g

roup

Gul

ominusminus

(CLP

+ V

it C)

Gul

ominusminus

(CLP

)

(d)

WT (CLP + Vit C)WT (CLP)Control group

Gulominusminus (CLP + Vit C)Gulominusminus (CLP)

PI

104

103

102

101

100

104103102101100

Annexin-V

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

PI

104

103

102

101

100

104103102101100

Annexin-V

104103102101100

Annexin-V104103102101100

Annexin-V104103102101100

Annexin-V

(e)

Figure 5 The impact of parenteral Vit C on CD4+ T cells-mediated cellular immunosuppression in sepsis Treatment with 200mgKg par-enteral Vit C upregulated the proliferative response of CD4+CD25minus T cells of WT mice and Gulominusminus mice in sepsis (a) The apoptotic rate ofsplenic CD4+CD25minusT cells was analyzed with Annexin-V-FITCPI by flow cytometry at 24 hours after CLP-inducedmidgrade sepsis (e)Theapoptotic level of splenic CD4+CD25minusT cells was decreased when 200mgKg parenteral Vit C was administered after CLP to WT mice andGulominusminus mice in sepsis (b) Parenteral Vit C increased the serum level of IFN-120574 (c) as well as decreasing the serum levels of IL-4 (d) of WTmice and Gulominusminus mice in sepsis Data were represented as mean plusmn standard deviation (SD) and analyzed by software SPSS 170 with one-wayANOVA 119899 = 4 per group lowast119901 lt 005 or lowastlowast119901 lt 001

Mediators of Inflammation 11

immunoregulation of Nrp-1highCD4+CD25+ Tregs whichcorrelated to the expression of Foxp-3 and CTLA-4 as wellas the secretion of IL-10 and TGF-120573 [23] We showed thatparenteral Vit C could inhibit the expression of exteriorCTLA-4 and TGF-120573m+ and the secretion of TGF-120573 and IL-10at protein and gene levels but did not alter the apoptotic levelof CD4+CD25+ Tregs These findings were in accordancewith the results of our previous studies [12 13] The stabilityof Tregs includes the expression of Foxp-3 and negativeimmunoregulation with sepsis which is crucially dependenton the demethylation status of the Foxp3-TSDR [11] Ourprevious study suggested that recombinant Nrp-1 polyclonalantibody could decrease the demethylation of Foxp3-TSDRin the presence of LPS which is associated with inhibition ofthe negative immunoregulation of Tregs [23] In the currentstudy parenteral Vit C had the ability to decrease thedemethylation level of Foxp3-TSDR but the deficiency ofVit C increased the demethylation level of Foxp3-TSDR inGulominusminusmice septicmodel whichwas in accordancewith ourprevious findings

5 Conclusion

In conclusion administration of parenteral Vit C improvedthe outcome of sepsis and sepsis-induced MODS of WT andGulominusminus mice The negative immunoregulation of Tregs wasinhibitedmainly including inhibiting the expression of Foxp-3 CTLA-4 and TGF-120573m+ and the secretion of inhibitorycytokines (TGF-120573 and IL-10) CD4+ T cells-mediated cellularimmunosuppression was improved by parenteral Vit C inWT and Gulominusminus septic mice These results suggested thatparenteral Vit C has the ability to improve the outcomeof sepsis and sepsis-induced MODS and is associated withimprovement in cellular immunosuppression

Competing Interests

The authors declare that they have no competing financialinterests This work is attributed to Tianjin Medical Univer-sity General Hospital Tianjin China

Authorsrsquo Contributions

Yu-Lei Gao Bin Lu and Jian-Hua Zhai contributed equally tothis work

Acknowledgments

This work was supported by grants from the TMUGHfunding (nos ZYYFY2015020 and ZYYFY2014014) and theRui-E funding (no 2016020) The authors thank ProfessorShu-Zhang Cui of Emergency Department of TianjinMedicalUniversity General Hospital for the guidance of experimentaldesign

References

[1] M Singer C S Deutschman C W Seymour et al ldquoThe thirdinternational consensus definitions for sepsis and septic shock

(Sepsis-3)rdquoThe Journal of theAmericanMedical Association vol315 no 8 pp 801ndash810 2016

[2] K-M Kaukonen M Bailey D Pilcher D J Cooper and RBellomo ldquoSystemic inflammatory response syndrome criteriain defining severe sepsisrdquoTheNew England Journal of Medicinevol 372 no 17 pp 1629ndash1638 2015

[3] D F Gaieski J M Edwards M J Kallan and B G CarrldquoBenchmarking the incidence and mortality of severe sepsis inthe united statesrdquo Critical Care Medicine vol 41 no 5 pp 1167ndash1174 2013

[4] B D Winters M Eberlein J Leung D M Needham P JPronovost and J E Sevransky ldquoLong-termmortality and qual-ity of life in sepsis a systematic reviewrdquo Critical Care Medicinevol 38 no 5 pp 1276ndash1283 2010

[5] S Inoue K Suzuki-Utsunomiya Y Okada et al ldquoReduction ofimmunocompetent T cells followed by prolonged lymphopeniain severe sepsis in the elderlyrdquo Critical Care Medicine vol 41no 3 pp 810ndash819 2013

[6] L P Skrupky P W Kerby and R S Hotchkiss ldquoAdvancesin the management of sepsis and the understanding of keyimmunologic defectsrdquo Anesthesiology vol 115 no 6 pp 1349ndash1362 2011

[7] J S Boomer K To K C Chang et al ldquoImmunosuppressionin patients who die of sepsis and multiple organ failurerdquo TheJournal of the American Medical Association vol 306 no 23pp 2594ndash2605 2011

[8] C J Darcy G Minigo K A Piera et al ldquoNeutrophils withmyeloid derived suppressor function deplete arginine andconstrain T cell function in septic shock patientsrdquoCritical Carevol 18 no 4 article R163 2014

[9] K Chang C Svabek C Vazquez-Guillamet et al ldquoTargetingthe programmed cell death 1 programmed cell death ligand 1pathway reverses T cell exhaustion in patients with sepsisrdquoCrit-ical Care vol 18 no 1 article R3 2014

[10] D C Nascimento J C Alves-Filho F Sonego et al ldquoRole ofregulatory T cells in long-term immune dysfunction associatedwith severe sepsisrdquo Critical Care Medicine vol 38 no 8 pp1718ndash1725 2010

[11] R TaturaM ZeschnigkWHansen et al ldquoRelevance of Foxp3+regulatory T cells for early and late phases of murine sepsisrdquoImmunology vol 146 no 1 pp 144ndash156 2015

[12] Q Y Liu Y M Yao Y Yu et al ldquoAstragalus polysaccharidesattenuate postburn sepsisvia inhibiting negative immunoreg-ulation of CD4+CD25highT cellsrdquo PLoS ONE vol 6 no 6Article ID e19811 2011

[13] X-M Zhu Y-M Yao H-P Liang et al ldquoHigh mobility groupbox-1 protein regulate immunosuppression of regulatory T cellsthrough toll-like receptor 4rdquo Cytokine vol 54 no 3 pp 296ndash304 2011

[14] Y Zhang Y-M Yao L-F Huang N Dong Y Yu and Z-YSheng ldquoThe potential effect and mechanism of high-mobilitygroup box 1 protein on regulatory T cell-mediated immunosup-pressionrdquo Journal of Interferon and Cytokine Research vol 31no 2 pp 249ndash257 2011

[15] Y-Y Luan C-F Yin Q-H Qin et al ldquoEffect of regulatory Tcells on promoting apoptosis of T lymphocyte and its regula-tory mechanism in sepsisrdquo Journal of Interferon and CytokineResearch vol 35 no 12 pp 969ndash980 2015

[16] B J Fisher D Kraskauskas E J Martin et al ldquoAttenuation ofsepsis-induced organ injury in mice by vitamin Crdquo Journal ofParenteral andEnteralNutrition vol 38 no 7 pp 825ndash839 2014

12 Mediators of Inflammation

[17] P E Marik ldquolsquoVitamin Srsquo (Steroids) and Vitamin C for the treat-ment of severe sepsis and septic shockrdquo Critical Care Medicinevol 44 no 6 pp 1228ndash1229 2016

[18] J-M Hong J-H Kim J S Kang W J Lee and Y-I HwangldquoVitamin C is taken up by human T cells via sodium-dependentvitamin C transporter 2 (SVCT2) and exerts inhibitory effectson the activation of these cells in vitrordquo Anatomy and CellBiology vol 49 no 2 pp 88ndash98 2016

[19] J Manning B Mitchell D A Appadurai et al ldquoVitamin C pro-motes maturation of T-cellsrdquo Antioxidants and Redox Signalingvol 19 no 17 pp 2054ndash2067 2013

[20] S Mburu J L Marnewick A Abayomi and H Ipp ldquoMod-ulation of LPS-induced CD4+ T-cell activation and apopto-sis by antioxidants in untreated asymptomatic HIV infectedparticipants An In Vitro Studyrdquo Clinical and DevelopmentalImmunology vol 2013 Article ID 631063 9 pages 2013

[21] P H Tan P Sagoo C Chan et al ldquoInhibition of NF-120581B andoxidative pathways in human dendritic cells by antioxidativevitamins generates regulatory T cellsrdquo Journal of Immunologyvol 174 no 12 pp 7633ndash7644 2005

[22] V S Nair M H Song and K I Oh ldquoVitamin C facilitatesdemethylation of the Foxp3 enhancer in a Tet-dependentmannerrdquo Journal of Immunology vol 196 no 5 pp 2119ndash21312016

[23] Y-L Gao Y-F Chai A-L Qi et al ldquoNeuropilin-1ℎ119894119892ℎCD4+CD25+ regulatory T cells exhibit primary negativeimmunoregulation in sepsisrdquo Mediators of Inflammation vol2016 Article ID 7132158 11 pages 2016

[24] Y L Gao Y F Chai N Dong et al ldquoTuftsin-derived T-peptideprevents cellular immunosuppression and improves survivalrate in septic micerdquo Scientific Reports vol 5 Article ID 167252015

[25] Z Yang Y Zhang L Dong et al ldquoThe reduction of peripheralblood CD4+ T cell indicates persistent organ failure in acutepancreatitisrdquo PLoSONE vol 10 no 5 Article ID e0125529 2015

[26] R Sharma P R Sharma Y C Kim et al ldquoIL-2-controlledexpression ofmultiple T cell trafficking genes andTh2 cytokinesin the regulatory T cell-deficient scurfy mice implication tomultiorgan inflammation and control of skin and lung inflam-mationrdquo The Journal of Immunology vol 186 no 2 pp 1268ndash1278 2011

[27] M J A J Huijskens M Walczak N Koller et al ldquoTechnicaladvance ascorbic acid induces development of double-positiveT cells from human hematopoietic stem cells in the absence ofStromal cellsrdquo Journal of Leukocyte Biology vol 96 no 6 pp1165ndash1175 2014

[28] H-J Kim R A Barnitz T Kreslavsky et al ldquoStable inhibitoryactivity of regulatory T cells requires the transcription factorHeliosrdquo Science vol 350 no 6258 pp 334ndash339 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 11

immunoregulation of Nrp-1highCD4+CD25+ Tregs whichcorrelated to the expression of Foxp-3 and CTLA-4 as wellas the secretion of IL-10 and TGF-120573 [23] We showed thatparenteral Vit C could inhibit the expression of exteriorCTLA-4 and TGF-120573m+ and the secretion of TGF-120573 and IL-10at protein and gene levels but did not alter the apoptotic levelof CD4+CD25+ Tregs These findings were in accordancewith the results of our previous studies [12 13] The stabilityof Tregs includes the expression of Foxp-3 and negativeimmunoregulation with sepsis which is crucially dependenton the demethylation status of the Foxp3-TSDR [11] Ourprevious study suggested that recombinant Nrp-1 polyclonalantibody could decrease the demethylation of Foxp3-TSDRin the presence of LPS which is associated with inhibition ofthe negative immunoregulation of Tregs [23] In the currentstudy parenteral Vit C had the ability to decrease thedemethylation level of Foxp3-TSDR but the deficiency ofVit C increased the demethylation level of Foxp3-TSDR inGulominusminusmice septicmodel whichwas in accordancewith ourprevious findings

5 Conclusion

In conclusion administration of parenteral Vit C improvedthe outcome of sepsis and sepsis-induced MODS of WT andGulominusminus mice The negative immunoregulation of Tregs wasinhibitedmainly including inhibiting the expression of Foxp-3 CTLA-4 and TGF-120573m+ and the secretion of inhibitorycytokines (TGF-120573 and IL-10) CD4+ T cells-mediated cellularimmunosuppression was improved by parenteral Vit C inWT and Gulominusminus septic mice These results suggested thatparenteral Vit C has the ability to improve the outcomeof sepsis and sepsis-induced MODS and is associated withimprovement in cellular immunosuppression

Competing Interests

The authors declare that they have no competing financialinterests This work is attributed to Tianjin Medical Univer-sity General Hospital Tianjin China

Authorsrsquo Contributions

Yu-Lei Gao Bin Lu and Jian-Hua Zhai contributed equally tothis work

Acknowledgments

This work was supported by grants from the TMUGHfunding (nos ZYYFY2015020 and ZYYFY2014014) and theRui-E funding (no 2016020) The authors thank ProfessorShu-Zhang Cui of Emergency Department of TianjinMedicalUniversity General Hospital for the guidance of experimentaldesign

References

[1] M Singer C S Deutschman C W Seymour et al ldquoThe thirdinternational consensus definitions for sepsis and septic shock

(Sepsis-3)rdquoThe Journal of theAmericanMedical Association vol315 no 8 pp 801ndash810 2016

[2] K-M Kaukonen M Bailey D Pilcher D J Cooper and RBellomo ldquoSystemic inflammatory response syndrome criteriain defining severe sepsisrdquoTheNew England Journal of Medicinevol 372 no 17 pp 1629ndash1638 2015

[3] D F Gaieski J M Edwards M J Kallan and B G CarrldquoBenchmarking the incidence and mortality of severe sepsis inthe united statesrdquo Critical Care Medicine vol 41 no 5 pp 1167ndash1174 2013

[4] B D Winters M Eberlein J Leung D M Needham P JPronovost and J E Sevransky ldquoLong-termmortality and qual-ity of life in sepsis a systematic reviewrdquo Critical Care Medicinevol 38 no 5 pp 1276ndash1283 2010

[5] S Inoue K Suzuki-Utsunomiya Y Okada et al ldquoReduction ofimmunocompetent T cells followed by prolonged lymphopeniain severe sepsis in the elderlyrdquo Critical Care Medicine vol 41no 3 pp 810ndash819 2013

[6] L P Skrupky P W Kerby and R S Hotchkiss ldquoAdvancesin the management of sepsis and the understanding of keyimmunologic defectsrdquo Anesthesiology vol 115 no 6 pp 1349ndash1362 2011

[7] J S Boomer K To K C Chang et al ldquoImmunosuppressionin patients who die of sepsis and multiple organ failurerdquo TheJournal of the American Medical Association vol 306 no 23pp 2594ndash2605 2011

[8] C J Darcy G Minigo K A Piera et al ldquoNeutrophils withmyeloid derived suppressor function deplete arginine andconstrain T cell function in septic shock patientsrdquoCritical Carevol 18 no 4 article R163 2014

[9] K Chang C Svabek C Vazquez-Guillamet et al ldquoTargetingthe programmed cell death 1 programmed cell death ligand 1pathway reverses T cell exhaustion in patients with sepsisrdquoCrit-ical Care vol 18 no 1 article R3 2014

[10] D C Nascimento J C Alves-Filho F Sonego et al ldquoRole ofregulatory T cells in long-term immune dysfunction associatedwith severe sepsisrdquo Critical Care Medicine vol 38 no 8 pp1718ndash1725 2010

[11] R TaturaM ZeschnigkWHansen et al ldquoRelevance of Foxp3+regulatory T cells for early and late phases of murine sepsisrdquoImmunology vol 146 no 1 pp 144ndash156 2015

[12] Q Y Liu Y M Yao Y Yu et al ldquoAstragalus polysaccharidesattenuate postburn sepsisvia inhibiting negative immunoreg-ulation of CD4+CD25highT cellsrdquo PLoS ONE vol 6 no 6Article ID e19811 2011

[13] X-M Zhu Y-M Yao H-P Liang et al ldquoHigh mobility groupbox-1 protein regulate immunosuppression of regulatory T cellsthrough toll-like receptor 4rdquo Cytokine vol 54 no 3 pp 296ndash304 2011

[14] Y Zhang Y-M Yao L-F Huang N Dong Y Yu and Z-YSheng ldquoThe potential effect and mechanism of high-mobilitygroup box 1 protein on regulatory T cell-mediated immunosup-pressionrdquo Journal of Interferon and Cytokine Research vol 31no 2 pp 249ndash257 2011

[15] Y-Y Luan C-F Yin Q-H Qin et al ldquoEffect of regulatory Tcells on promoting apoptosis of T lymphocyte and its regula-tory mechanism in sepsisrdquo Journal of Interferon and CytokineResearch vol 35 no 12 pp 969ndash980 2015

[16] B J Fisher D Kraskauskas E J Martin et al ldquoAttenuation ofsepsis-induced organ injury in mice by vitamin Crdquo Journal ofParenteral andEnteralNutrition vol 38 no 7 pp 825ndash839 2014

12 Mediators of Inflammation

[17] P E Marik ldquolsquoVitamin Srsquo (Steroids) and Vitamin C for the treat-ment of severe sepsis and septic shockrdquo Critical Care Medicinevol 44 no 6 pp 1228ndash1229 2016

[18] J-M Hong J-H Kim J S Kang W J Lee and Y-I HwangldquoVitamin C is taken up by human T cells via sodium-dependentvitamin C transporter 2 (SVCT2) and exerts inhibitory effectson the activation of these cells in vitrordquo Anatomy and CellBiology vol 49 no 2 pp 88ndash98 2016

[19] J Manning B Mitchell D A Appadurai et al ldquoVitamin C pro-motes maturation of T-cellsrdquo Antioxidants and Redox Signalingvol 19 no 17 pp 2054ndash2067 2013

[20] S Mburu J L Marnewick A Abayomi and H Ipp ldquoMod-ulation of LPS-induced CD4+ T-cell activation and apopto-sis by antioxidants in untreated asymptomatic HIV infectedparticipants An In Vitro Studyrdquo Clinical and DevelopmentalImmunology vol 2013 Article ID 631063 9 pages 2013

[21] P H Tan P Sagoo C Chan et al ldquoInhibition of NF-120581B andoxidative pathways in human dendritic cells by antioxidativevitamins generates regulatory T cellsrdquo Journal of Immunologyvol 174 no 12 pp 7633ndash7644 2005

[22] V S Nair M H Song and K I Oh ldquoVitamin C facilitatesdemethylation of the Foxp3 enhancer in a Tet-dependentmannerrdquo Journal of Immunology vol 196 no 5 pp 2119ndash21312016

[23] Y-L Gao Y-F Chai A-L Qi et al ldquoNeuropilin-1ℎ119894119892ℎCD4+CD25+ regulatory T cells exhibit primary negativeimmunoregulation in sepsisrdquo Mediators of Inflammation vol2016 Article ID 7132158 11 pages 2016

[24] Y L Gao Y F Chai N Dong et al ldquoTuftsin-derived T-peptideprevents cellular immunosuppression and improves survivalrate in septic micerdquo Scientific Reports vol 5 Article ID 167252015

[25] Z Yang Y Zhang L Dong et al ldquoThe reduction of peripheralblood CD4+ T cell indicates persistent organ failure in acutepancreatitisrdquo PLoSONE vol 10 no 5 Article ID e0125529 2015

[26] R Sharma P R Sharma Y C Kim et al ldquoIL-2-controlledexpression ofmultiple T cell trafficking genes andTh2 cytokinesin the regulatory T cell-deficient scurfy mice implication tomultiorgan inflammation and control of skin and lung inflam-mationrdquo The Journal of Immunology vol 186 no 2 pp 1268ndash1278 2011

[27] M J A J Huijskens M Walczak N Koller et al ldquoTechnicaladvance ascorbic acid induces development of double-positiveT cells from human hematopoietic stem cells in the absence ofStromal cellsrdquo Journal of Leukocyte Biology vol 96 no 6 pp1165ndash1175 2014

[28] H-J Kim R A Barnitz T Kreslavsky et al ldquoStable inhibitoryactivity of regulatory T cells requires the transcription factorHeliosrdquo Science vol 350 no 6258 pp 334ndash339 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Mediators of Inflammation

[17] P E Marik ldquolsquoVitamin Srsquo (Steroids) and Vitamin C for the treat-ment of severe sepsis and septic shockrdquo Critical Care Medicinevol 44 no 6 pp 1228ndash1229 2016

[18] J-M Hong J-H Kim J S Kang W J Lee and Y-I HwangldquoVitamin C is taken up by human T cells via sodium-dependentvitamin C transporter 2 (SVCT2) and exerts inhibitory effectson the activation of these cells in vitrordquo Anatomy and CellBiology vol 49 no 2 pp 88ndash98 2016

[19] J Manning B Mitchell D A Appadurai et al ldquoVitamin C pro-motes maturation of T-cellsrdquo Antioxidants and Redox Signalingvol 19 no 17 pp 2054ndash2067 2013

[20] S Mburu J L Marnewick A Abayomi and H Ipp ldquoMod-ulation of LPS-induced CD4+ T-cell activation and apopto-sis by antioxidants in untreated asymptomatic HIV infectedparticipants An In Vitro Studyrdquo Clinical and DevelopmentalImmunology vol 2013 Article ID 631063 9 pages 2013

[21] P H Tan P Sagoo C Chan et al ldquoInhibition of NF-120581B andoxidative pathways in human dendritic cells by antioxidativevitamins generates regulatory T cellsrdquo Journal of Immunologyvol 174 no 12 pp 7633ndash7644 2005

[22] V S Nair M H Song and K I Oh ldquoVitamin C facilitatesdemethylation of the Foxp3 enhancer in a Tet-dependentmannerrdquo Journal of Immunology vol 196 no 5 pp 2119ndash21312016

[23] Y-L Gao Y-F Chai A-L Qi et al ldquoNeuropilin-1ℎ119894119892ℎCD4+CD25+ regulatory T cells exhibit primary negativeimmunoregulation in sepsisrdquo Mediators of Inflammation vol2016 Article ID 7132158 11 pages 2016

[24] Y L Gao Y F Chai N Dong et al ldquoTuftsin-derived T-peptideprevents cellular immunosuppression and improves survivalrate in septic micerdquo Scientific Reports vol 5 Article ID 167252015

[25] Z Yang Y Zhang L Dong et al ldquoThe reduction of peripheralblood CD4+ T cell indicates persistent organ failure in acutepancreatitisrdquo PLoSONE vol 10 no 5 Article ID e0125529 2015

[26] R Sharma P R Sharma Y C Kim et al ldquoIL-2-controlledexpression ofmultiple T cell trafficking genes andTh2 cytokinesin the regulatory T cell-deficient scurfy mice implication tomultiorgan inflammation and control of skin and lung inflam-mationrdquo The Journal of Immunology vol 186 no 2 pp 1268ndash1278 2011

[27] M J A J Huijskens M Walczak N Koller et al ldquoTechnicaladvance ascorbic acid induces development of double-positiveT cells from human hematopoietic stem cells in the absence ofStromal cellsrdquo Journal of Leukocyte Biology vol 96 no 6 pp1165ndash1175 2014

[28] H-J Kim R A Barnitz T Kreslavsky et al ldquoStable inhibitoryactivity of regulatory T cells requires the transcription factorHeliosrdquo Science vol 350 no 6258 pp 334ndash339 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom