shedding of the tumor necrosis factor (tnf) receptor from ... · for 8 hours, their plasma...

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Shedding of the tumor necrosis factor (TNF)receptor from the surface of hepatocytes duringsepsis limits inflammation through cGMP signalingMeihong Deng,1 Patricia A. Loughran,1,2 Liyong Zhang,1 Melanie J. Scott,1 Timothy R. Billiar1*

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Proteolytic cleavage of the tumor necrosis factor (TNF) receptor (TNFR) from the cell surface contributes toanti-inflammatory responses andmay be beneficial in reducing the excessive inflammation associatedwithmultiple organ failure andmortality during sepsis. Using a clinically relevant mousemodel of polymicrobialabdominal sepsis, we found that the production of inducible nitric oxide synthase (iNOS) in hepatocytesled to the cyclic guanosine monophosphate (cGMP)–dependent activation of the protease TACE (TNF-converting enzyme) and the shedding of TNFR. Furthermore, treating mice with a cGMP analog after theinduction of sepsis increased TNFR shedding and decreased systemic inflammation. Similarly, increas-ing the abundance of cGMP with a clinically approved phosphodiesterase 5 inhibitor (sildenafil) alsodecreasedmarkers of systemic inflammation, protected against organ injury, and increased circulatingamounts of TNFR1 inmice with sepsis. We further confirmed that a similar iNOS-cGMP-TACE pathway wasrequired for TNFR1 shedding by human hepatocytes in response to the bacterial product lipopolysac-charide. Our data suggest that increasing the bioavailability of cGMP might be beneficial in amelioratingthe inflammation associated with sepsis.

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INTRODUCTION
Tumor necrosis factor (TNF), a proinflammatory cytokine secreted mostlyby immune cells, plays an important role in the pathophysiology of sepsis.Appropriate amounts of TNF most likely exert a beneficial effect on hostsurvival to infection, but exaggerated or sustained increases in TNF abun-dance can lead to toxicity (1, 2), which, at a cellular level, manifests as celldeath (3, 4). Plasma TNF concentrations correlatewith the severity of sepsis(5), and a meta-analysis of anti-TNF therapies in human sepsis trials indi-cated that inhibiting TNF or TNF-dependent signaling was on the “side ofbenefit” (6). Thus, TNF remains a target of interest in studies on the patho-biology of sepsis, as well as a target in new human sepsis trials (7).

TNF initiates cellular inflammatory responses through engagement withtwo cell surface receptors: TNF receptor 1 (TNFR1) and TNFR2. TNFR1 isfound on immune cells, such as macrophages (8), and parenchymal cells,such as hepatocytes (9). Excessive activation of TNFR1 byTNF leads to theapoptosis of hepatocytes, and thus leads to organ damage (9); however,membrane-bound TNFR1 can be cleaved through proteolytic shedding ofits ectodomain, which is dependent on activation of TNF-converting en-zyme (TACE, also known as ADAM17) (10). Receptor shedding is thoughtto be protective by reducing cellular responses to TNFand by binding to andsequestering extracellular TNF.

Endotoxemia (10) and sepsis (9, 11) are associated with marked in-creases in soluble TNFR1 (sTNFR1) concentrations in the circulation. Stu-dies showed that neutralizing TNFwith sTNFR1 lessens organ damage (12)and mortality (13) in micewith sepsis. We previously demonstrated that theToll-like receptor 4 (TLR4)–dependent expression of the gene encodinginducible nitric oxide synthase (iNOS) in hepatocytes leads to nitric oxide(NO) production and activation of the cyclic guanosine monophosphate(cGMP)– and protein kinase G (PKG)–dependent activation of TACE,which cleaves TNFR1 (10). However, the signaling pathways that mediate

1Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213,USA. 2Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA15213, USA.*Corresponding author. E-mail: [email protected]

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shedding of hepatocyte TNFR1 (HC-TNFR1) during sepsis are unclear. Inpolymicrobial sepsis, multiple pathogen-derived ligands of TLRs and cyto-kines, such as interleukin-1 (IL-1), are potent inducers of iNOS productionby hepatocytes (14) and may also be potent inducers of HC-TNFR1shedding. Therefore, we aimed to elucidate the stimuli and signaling path-ways that mediate HC-TNFR1 shedding in polymicrobial sepsis.We provideevidence that multiple TLR ligands and cytokines stimulate HC-TNFR1shedding in sepsis. Furthermore,we found thatmyeloiddifferentiationmarker88 (MyD88)–dependent iNOS production in hepatocytes led to the cGMP-dependent activation of TACE and shedding of TNFR1. Finally, strategiesthat increased cGMP bioavailability enhanced receptor shedding, reducedsystemic inflammation, andprotected organs from injury in sepsis. These dataindicate a link between MyD88, the iNOS-NO-cGMP pathway, and TNFsignaling during sepsis. This mechanism for TNFR1 shedding could beexploited therapeutically to limit excessive inflammation during sepsis.

RESULTS

Multiple TLR ligands and cytokines stimulate HC-TNFR1shedding during polymicrobial sepsisWepreviously showed that HC-TNFR1 shedding is stimulated by the TLR4ligand lipopolysaccharide (LPS) in vitro and in vivo (10); however, duringpolymicrobial sepsis, multiple TLR ligands and cytokines are involved inpropagating the inflammatory response. To determine the mechanismsinvolved in a clinically relevant polymicrobial sepsis model, we assessedTNFR1 shedding in mice during sepsis induced by cecal ligation and punc-ture (CLP). The control mice underwent laparotomy and manipulation ofthe cecum without puncture. Concentrations of sTNFR1 in the circulationincreased over time in both the sublethal and lethalmodels ofCLP (fig. S1A).In the sublethal model, all of the mice survived until 24 hours after CLP. Theamounts of sTNFR1 were greatest at 18 hours after CLP and returned tobaseline by 24 hours. In the lethal model, all of the mice survived until18 hours after CLP; however, mortality reached 66% by 24 hours. Theamounts of plasma sTNFR1were substantially greater in the lethal model thanin the sublethal model at 18 hours (fig. S1A). Plasma TNF concentrations

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increased early in both models, with the greatest amounts at 6 hours (fig.S1B). That the amount of sTNFR1 peaked much later than did that ofTNF suggests that direct TNF-TNFR1 interactions do not play a majorrole in sTNFR1 release in sepsis.

We used the lethal model of CLP to assess the mechanisms of TNFR1shedding in polymicrobial sepsis. We first investigated the extent to whichincreases in plasma sTNFR1 abundance were TLR4-dependent in CLP. Wesubjected wild-type and TLR4-deficient (Tlr4−/−) mice to CLP and assessedcirculating amounts of sTNFR1 after 8 hours by enzyme-linked immuno-sorbent assay (ELISA). We found that plasma sTNFR1 amounts were sub-stantially increased in wild-type and Tlr4−/− mice after CLP, with nostatistically significant difference between both groups (Fig. 1A). Thus,TNFR1 shedding does not appear to be TLR4-dependent in this poly-microbial sepsis model. We next assessed the capacity of TLR ligands, inaddition to LPS, to induce HC-TNFR1 shedding in vitro. Compared to theamount of sTNFR1 shed by untreated control hepatocytes, the amounts of

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sTNFR1 in the culture media of hepatocytestreated for 24 hours with HKLM (heat-killedListeria monocytogenes, a TLR2 ligand),LPS (a TLR4 ligand), disulfide HMGB1(high-mobility group protein 1, a TLR4ligand), FLA-ST (flagellin from Salmonellatyphimurium, a TLR5 ligand), and OD1669(CpGoligonucleotide 1669, a TLR9 ligand)weremarkedly increased (Fig. 1B).Thisobser-vation suggests that multiple TLR ligandsstimulate HC-TNFR1 shedding in vitro.

Cytokines, such as TNF and IL-1b, areimportant early inflammatory mediatorsduring sepsis. To investigate whether thesecytokines also stimulated HC-TNFR1shedding, we treated primary hepatocyteswith TNF and IL-1b in culture. Treatmentwith either cytokine did not result in sub-stantial cell death (Fig. 1C). We found thathepatocytes spontaneously released lowamounts of sTNFR1 over time (Fig. 1D).The amounts of sTNFR1 in the mediaof cells treated with IL-1b increased in atime-dependent manner compared to thatin the media of untreated cells (Fig. 1D),whereas sTNFR1 amounts in the mediawere only slightly increased after 12 hoursof treatment with TNF. These results sug-gest that IL-1b stimulates HC-TNFR1shedding in vitro. Consistent with theseresults, knockout of caspase-1 or the inflam-masone component NLRP3 (NACHT,LRR, and PYD domains–containing pro-tein 3), both of which result in abrogation ofIL-1b processing (fig. S2), was associatedwith lower circulating concentrations ofsTNFR1 at 8 hours after CLP (Fig. 1E). Lossof IL-1 signaling using Il1r−/−mice was alsoassociated with substantially decreased cir-culating concentrations of sTNFR1 afterCLP compared to those of wild-type mice(Fig. 1E).These results suggest that IL-1b alsostimulates TNFR1 shedding in vivo.

TNFR1 shedding fromhepatocytes andmyeloid cells duringsepsis is dependent on MyD88Because TLR ligands and IL-1b initiate signaling mediated by theadaptor proteins MyD88 and TRIF (TIR domain–containing adaptor–inducing interferon-b), we hypothesized that TNFR1 shedding wasmediated by signaling pathways requiring one or both molecules.When wild-type, Myd88−/−, or Trif −/− mice were subjected to CLPfor 8 hours, their plasma concentrations of sTNFR1 increased substan-tially (Fig. 2A); however, the amounts of plasma sTNFR1 in Myd88−/−

mice subjected to CLP were markedly less than those in similarly treatedwild-type or Trif −/− mice (Fig. 2A). The abundance of Tnfr1 mRNAincreased twofold in the livers of all mice after CLP, with no statisti-cally significant difference between the strains (Fig. 2B). These datasuggest that MyD88 does not regulate Tnfr1 expression in mouse liversafter CLP but that it contributes to the signaling that leads to TNFR1shedding.

Fig. 1. Multiple TLR ligands and cytokines stimulateHC-TNFR1shedding in vitro and in vivo. (A)Wild-type(WT) and Tlr4−/− mice were subjected to CLP. In thecontrol mice, laparotomy and cecal manipulationwere performed without ligation and puncture. Eighthours later, plasma sTNFR1 concentrations weremeasured by ELISA. Data are means ± SD from 8to 10mice per group from two separate experiments.(B) Mouse hepatocytes were left untreated (Control)or were stimulated for 24 hours with the TLR ligandsHKLM (1 × 108 cells/ml), LPS (100 ng/ml), HMGB1

(5 mg/ml), FLA-ST (1 mg/ml), and ODN1669 (5 mg/ml). The concentrations of TNFR1 in the culture media

were measured by ELISA. Data are means ± SD from three experiments. *P < 0.05 versus control by two-tailed unpaired t test. (C andD) Mouse hepatocytes were left untreated or were incubatedwith TNF (500U/ml)or IL-1b (100 U/ml) for the indicated times. At each time point, (C) cell viability and (D) the concentration ofTNFR1 in the culture media were determined. Data are means ± SD from three experiments. *P < 0.05 versuscontrol by two-tailed unpaired t test. (E) Mice of the indicated genotypes were subjected to CLP. Eight hourslater, the plasma concentrations of TNFR1were determined by ELISA. Data aremeans ± SD from sixmice pergroup of each genotype from two experiments. *P < 0.05 versus control by two-tailed unpaired t test.

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Weand others previously reported that hepatocytes (10, 15) and immunecells (8, 16) shed TNFR1. To investigate the role of MyD88 and TRIF inTNFR1 shedding in different liver cell populations, we assessed the LPS-stimulated shedding of TNFR1 fromhepatocytes and nonparenchymal cells(NPCs), including endothelial cells, Kupffer cells, and stellate cells isolatedfrom wild-type,Myd88−/−, or Trif −/−mice. The amounts of sTNFR1 in theculture media of hepatocytes (Fig. 2C) and NPCs (Fig. 2D) from all strainsof mice increased in a time-dependent manner. TNFR1 abundance in themedia of hepatocytes was three- to fourfold greater than that in the mediaof NPCs for both wild-type and Trif −/−mice (Fig. 2, C and D), suggesting


that hepatocytes are a major source ofsTNFR1. Furthermore, the concentrationof sTNFR1 in the culture media ofMyd88−/−

hepatocytes was substantially less than thatin the media of wild-type and Trif −/− cells(Fig. 2C), suggesting that HC-TNFR1shedding is MyD88-dependent. Therewas no statistically significant differencein the concentration of sTNFR1 in the cul-turemedia ofwild-type,Myd88−/−, orTrif −/−

in NPCs (Fig. 2D), suggesting that NPC-TNFR1 shedding is not dependent on eitherMyD88 or TRIF alone.

To further investigate the cell-specificrole of MyD88 in TNFR1 shedding in vivoduring sepsis, we used the Cre-loxP systemto generate mouse strains with hepatocyte-specific (HC-Myd88−/−) or myeloid cell–specific (LysM-Myd88−/−) loss of MyD88.To test the effect of the Cre recombinaseon cellular toxicity and the response toLPS in mice, we isolated hepatocytes andmacrophages from Alb-Cre mice (overex-pression of Cre specifically in hepatocytes)and Lyz-Cre mice (overexpression of Crespecifically in myeloid cells), respectively.We did not observe any alterations in cel-lular toxicity or the responses to LPS of hepa-tocytes from Alb-Cre mice or macrophagesfrom Lyz-Cre mice, including TNFR1 shed-ding and IL-6 production (figs. S3 and S4).These data suggest that overexpression ofthe Cre recombinase specifically in thesecell types did not alter their responses to aseptic stimulus.

We then subjected HC-Myd88−/−,LysM-Myd88−/−, and Myd88flox mice(cell-specific knockout controls) to CLP.The plasma concentrations of sTNFR1 inHC-Myd88−/− mice and LysM-Myd88−/−

mice were substantially lower than that inMyd88flox mice at 8 hours after CLP(Fig. 2E). The circulating amounts of IL-1b were similar in Myd88flox and HC-Myd88−/− mice (Fig. 2F), supporting ourhypothesis thatHC-TNFR1 shedding invivoduring sepsis is MyD88-dependent. How-ever, the circulating concentration of IL-1bin LysM-Myd88−/− after CLPwas markedlyless than that in Myd88flox mice (Fig. 2F).

IL-1b is a strong stimulator of HC-TNFR1 shedding (Fig. 2, C and D),which suggests that MyD88 in myeloid cells might indirectly regulateHC-TNFR1 shedding by stimulating the production of IL-1b. Together,these data suggest that MyD88 plays a dominant role in TNFR1 sheddingduring sepsis.

HC-TNFR1 shedding during sepsis is mediated throughthe MyD88-dependent production of iNOSWepreviously showed thatHC-TNFR1 shedding ismediated by iNOS (10).Therefore, we postulated that iNOSmight be downstream ofMyD88 in the

Fig. 2. TNFR1 shedding from hepatocytes and myeloid cells during sepsis depends on Myd88. (A and B) WT,Myd88−/−, and Trif −/− mice were subjected to CLP. Eight hours later, (A) plasma sTNFR1 concentrations were

determined by ELISA, and (B) Tnfr1 mRNA abundance in the liver was determined by real-time reversetranscription polymerase chain reaction (RT-PCR) analysis. Data are means ± SD from 8 to 10 mice per groupfrom two experiments. *P < 0.05 versus control by two-tailed unpaired t test. (C andD) Hepatocytes (C) andNPCs (D) from the indicated mice were left untreated (zero-hour time point) or were treated with LPS for theindicated times. At each time point, the concentration of TNFR1 in the culture media was determined byELISA. Data are means ± SD from three experiments. *P < 0.05 by one-way analysis of variance (ANOVA).(EandF)Myd88flox,HC-Myd88−/−, andLysM-Myd88−/−miceweresubjected toCLP.Eight hours later, plasmaconcentrations of (E) TNFR1 and (F) IL-1b were determined by ELISA. Data are means ± SD from 8 to 10 miceper group from two experiments. *P< 0.05 versusMyd88flox by two-tailed unpaired t test. N.D., not detectable.




regulation of TNFR1 shedding during sepsis. To test this hypothesis, weassessed the expression of Nos2 (the gene that encodes iNOS) in the liver.The abundance ofNos2mRNAwas increased in the liver ofwild-typemice,but notMyd88−/−mice, after CLP (Fig. 3A). Furthermore, Nos2 expressionin the liver ofHC-Myd88−/−micewas substantially less than that inMyD88-Flox mice after CLP (Fig. 3A). The immunofluorescence signal based onantibody-dependent visualization of iNOS protein increased in the liver ofwild-typemice after CLP, but no obvious increase in signalwas observed inthe liver of Myd88−/− mice after CLP (Fig. 3B). In vitro, the abundance ofiNOS protein in LPS-treated hepatocytes from wild-type mice, but notMyd88−/−mice, increased in a time-dependentmanner (Fig. 3C).HepatocyteiNOS abundance (Fig. 3C) correlated with sTNFR1 concentration in culturemedia (Fig. 2C),whereasHC-TNFR1abundance (Fig. 3C) inversely correlatedwith the concentrations of sTNFR1 in the culture media (Fig. 2C). These datasuggest that TNFR1 shedding during sepsis requires MyD88-dependent in-creases in iNOS abundance in hepatocytes.

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To further confirm the hypothesis that TNFR1 shedding in sepsiswas iNOS-dependent, we treated wild-type mice with 1400W (aniNOS inhibitor) and subjected them and Nos2−/− mice to CLP. As ex-pected, the concentration of sTNFR1 in the circulation was substantial-ly less in Nos2−/− mice and 1400W-treated mice than in control mice(Fig. 3, D and E). Together, these data suggest that TNFR1 sheddingduring polymicrobial sepsis is partly mediated through the MyD88-iNOS pathway.

Activation of TACE by cGMP induces hepatic TNFR1shedding during sepsisOur previous study showed that cGMP was required for iNOS-inducedTACE activation and TNFR1 shedding in hepatocytes (10). Therefore,we hypothesized that iNOS-induced cGMP production might activateTACE to cause TNFR1 shedding from hepatocytes during sepsis. To testthis hypothesis, we determined the amounts of cGMP in liver homoge-

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nates from mice treated with saline or the iNOS inhibitor1400W. Hepatic cGMP concentrations in wild-type miceincreased substantially at 8 hours after CLP compared tothose in the saline-treated control mice, whereas cGMP con-centrations remained at baseline afterCLP in the 1400W-treatedmice (Fig. 4A).

Soluble guanylyl cyclase (sGC) is an enzyme that is activatedby NO to produce cGMP from GTP (guanosine triphosphate).We next used the cGMP analog 8-(4-chlorophenylthio)-cGMP(8-CPT-cGMP) and the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) to modify hepatic cGMP amountsafter CLP. The abundance of cGMP in the liver of 8-CPT-cGMP–treated mice increased fivefold after CLP comparedwith that in the untreated control mice (Fig. 4A). In addition,8-CPT-cGMP restored the hepatic cGMP abundance in micethat were simultaneously treated with the iNOS inhibitor1400W (Fig. 4A). The CLP-dependent increase in hepaticcGMP abundance was substantially suppressed by ODQcompared with that in saline-treated mice (Fig. 4A). Theseresults suggest that treatment with exogenous cGMP or aninhibitor of sGC efficiently increased or decreased, respec-tively, the abundance of hepatic cGMP in vivo. Furthermore,circulating sTNFR1 concentrations correlated with hepaticcGMP abundance, which suggests that TNFR1 shedding maybe dependent on hepatic cGMP (Fig. 4B). Furthermore, theimmunofluorescence signal for active TACE increased in theliver parenchyma of mice after CLP compared with that incontrol mice (Fig. 4C). Active TACE immunofluorescence in-creased further in the livers of mice treated with 8-CPT-cGMPand then subjected to CLP than in the livers of mice treated withsaline after CLP, whereas there was no obvious CLP-dependentincrease in the TACE signal in livers from mice treated with1400W or ODQ after CLP (Fig. 4C).

Western blotting analysis of TACE abundance in liverhomogenates confirmed that the abundance ofTACEcorrelatedwith hepatic cGMP amounts during sepsis (Fig. 4D). We nexttreated hepatocytes in vitro with or without the TACE inhibitorTAPI-2 before stimulating themwithLPS for various times.Wefound that the concentration of sTNFR1 in the media of cellsnot exposed to the inhibitor increased in a time-dependentman-ner in response toLPS; however, this increase in sTNFR1 abun-dance was suppressed by TAPI-2 (Fig. 4E). These resultssuggest that HC-TNFR1 shedding during sepsis may bemediated through an iNOS-cGMP-TACE pathway.

Fig. 3. Shedding of HC-TNFR1 during polymicrobial sepsis is mediated through a

MyD88- and iNOS-dependent pathway. (A and B) Mice of the indicated genotypeswere subjected to CLP for 8 hours. (A) The relative expression of Nos2 was de-termined by real-time RT-PCR analysis. Data are means ± SD from six to eight miceof each genotype from two experiments. *P < 0.05 by one-way ANOVA. (B) The produc-tion of iNOS in the liver of the indicated mice was analyzed by immunofluorescence mi-croscopy. Nuclear staining is in blue, whereas iNOS is shown in green. Scale bar, 50 mm.(C) Hepatocytes isolated from the indicated mice were left untreated (zero time point) orwere treated with LPS (100 ng/ml) for the indicated times. Whole-cell lysates were thenprepared and analyzed by Western blotting with antibodies specific for iNOS andTNFR1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) abundance was ana-lyzed to control for loading. Western blots are representative of three independentexperiments. (D) WT and Nos2−/− mice were subjected to CLP. Eight hours later, plasmaconcentrations of TNFR1 were determined by ELISA. Data are means ± SD from six miceper group from two experiments. *P < 0.05 by one-way ANOVA. (E) WT mice weresubjected to CLP and then were left untreated or were treated with 1400W (5 mg/kg,administered subcutaneously). Eight hours after CLP, plasma concentrations of TNFR1were determined by ELISA. Data are means ± SD from six mice per group from twoexperiments. *P < 0.05 by one-way ANOVA.

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TNFR1 shedding through the iNOS-cGMP pathwayattenuates the inflammatory response and liverinjury during sepsisWe and others showed that TNF signaling through TNFR1 stimulatescytokine production in immune cells and apoptosis in parenchymal cells,such as hepatocytes (9). Therefore, we postulated that regulation of HC-TNFR1 shedding through the iNOS-cGMP pathway might modulate thesystemic inflammatory response and organ damage during sepsis. We col-lected plasma from mice after they were subjected to CLP and then treatedwith either the cGMP analog 8-CPT-cGMP (to increase cGMP abun-

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dance) or the sGC inhibitor ODQ (to decrease cGMP abun-dance). The amounts of secreted cytokines were determinedby ELISA to assess the regulation of the systemic inflam-matory response. Plasma IL-6 was not detectable in thecontrol mice, but it was readily detectable in mice after CLP(Fig. 5A). In mice treated with 1400W to inhibit iNOS, plas-ma IL-6 concentrations were increased compared to thosein mice treated with saline (Fig. 5A). Administration of ex-ogenous cGMP (8-CPT-cGMP) substantially reduced circu-lating amounts of IL-6 after CLP (Fig. 5A). Administrationof 8-CPT-cGMP also reduced the amount of IL-6 in theplasma of mice treated with 1400W after CLP. Further-more, the amounts of plasma IL-6 in mice treated withODQ (which inhibits cGMP production) were increased sub-stantially compared to those in saline-treated mice (Fig. 5A).The plasma concentrations of early inflammatory factors,including TNF, KC, and MIP-1a, as well as of late inflamma-tory factors, such as HMGB1, showed the same trend asthat of IL-6 (Fig. 5, B to E). Increasing the bioavailabilityof cGMP in the liver by treatment with 8-CPT-cGMP pre-vented liver injury, as determined by measurement of alanineaminotransferase (ALT) concentrations, whereas inhibitionof cGMP production with ODQ exaggerated liver injurycompared with that in saline-treated mice (Fig. 5F). Thus,the inflammatory response and organ injury correlated withthe amount of hepatic cGMP (Fig. 4A) and were inverselyproportional to the plasma sTNFR1 concentration (Fig. 4B).This suggests that the regulation of HC-TNFR1 shedding bycGMP can modulate the inflammatory response and liverinjury during sepsis in mice.

We next tested whether sildenafil, an inhibitor of phos-phodiesterase 5 (PDE5) that is used in the clinic, could reg-ulate TNFR1 shedding during sepsis. Sildenafil blocks thedegradation of cGMP (17). Mice were subjected to CLPand then were treated with either sildenafil (50 mg/kg) orsaline, and plasma was collected after 8 hours. HepaticcGMP amounts increased slightly after exposure to sildena-fil (4.36 ± 2.0 pmol/10 mg protein) compared with those insaline-treated control mice (2.8 ± 0.4 pmol/10 mg protein).However, after CLP was performed, hepatic cGMPamounts were substantially greater in the sildenafil-treatedmice (13.1 ± 2.0 pmol/10 mg protein) than in the saline-treated mice (7.23 ± 2.9 pmol/10 mg protein) (Fig. 5G).As expected, plasma sTNFR1 amounts correlated with theabundance of hepatic cGMP (Fig. 5H). Furthermore, circu-lating IL-6 amounts in mice that received sildenafil afterCLP were substantially lower than those in mice that weretreated with saline after CLP (Fig. 5I). Together, these datasuggest that TNFR1 shedding during sepsis is mediated

through the iNOS-cGMP pathway. Increases in TNFR1 shedding throughthis pathway may reduce the inflammatory response and improve organdamage during sepsis.

Human HC-TNFR1 shedding stimulated by LPS and IL-1b isdependent on the iNOS-cGMP-TACE pathwayOur earlier results suggest that HC-TNFR1 shedding is mediated throughthe MyD88-iNOS-cGMP-TACE pathway in mice. To test whether TNFR1shedding in human hepatocytes was also mediated through this pathway,we isolated human hepatocytes and treated them with LPS or IL-1b. Theamounts of TNFR1 in the culture media increased in a time-dependent

Fig. 4. iNOS-stimulated HC-TNFR1 shedding during polymicrobial sepsis is mediatedby cGMP and TACE. (A to D) WT mice were subjected to CLP and then were imme-

diately treated subcutaneously with saline, 1400W (5 mg/kg), 8-CPT-cGMP (5 mg/kg),both 1400W and 8-CPT-cGMP, or ODQ (20 mg/kg). Eight hours after CLP, the concen-trations of (A) cGMP in liver homogenates and (B) TNFR1 in plasma were determined.Data are means ± SD from six to eight mice per group from two experiments. *P < 0.05by one-way ANOVA. (C) Some of the same mice were analyzed by immunofluores-cence microscopy to detect active TACE in the liver. TACE is shown in green, nucleiare in blue, and actin is shown in white. Scale bar, 50 mm. (D) Liver homogenates fromthe same mice were analyzed by Western blotting to detect TACE. GAPDH was usedas a loading control. Western blots are representative of two independent experiments.(E) Hepatocytes isolated from WT mice were left untreated (zero-hour time point) orwere treated in vitro with LPS (100 ng/ml) in the absence or presence of 400 nMTAPI-2 for the indicated times. The concentration of TNFR1 in the culture media wasdetermined by ELISA. Data are means ± SD from three independent experiments. *P <0.05 by one-way ANOVA.

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manner after treatment with LPS or IL-1b (Fig. 6A). The immuno-fluorescence signals for iNOS (red) and TACE (green) increased markedlyat 6 hours after treatment with LPS compared with those in untreatedcontrol cells (Fig. 6B). Furthermore, TACE colocalized with iNOS andtranslocated from the perinuclear area to the perimembrane area in the cy-toplasm (Fig. 6B).

The TACE inhibitor TAPI-2 reduced the sTNFR1 abundance in theculture media 6 and 12 hours after treatment with LPS (Fig. 6C). In re-sponse to stimulation with LPS, TACE abundance increased in humanhepatocytes in a time-dependent manner (Fig. 6, D and E), whereas TNFR1amounts in cell lysates decreased (Fig. 6, D and F). TAPI-2 inhibited theincrease in the abundance of TACE in LPS-treated cells (Fig. 6, D and E).Furthermore, TNFR1 amounts in cells did not decrease in the TAPI-2–


treated human hepatocytes after stimula-tion with LPS (Fig. 6, D and F). To con-firm whether HC-TNFR1 shedding wasmediated through an iNOS-TACE inter-action, we immunoprecipitated TACE fromwhole-cell lysates and then analyzed thesamples by Western blotting with antibodiesspecific for TACE and iNOS. The interac-tion between iNOS and TACE increased ina time-dependent manner in LPS-stimulatedcells (Fig. 6, G and H). Thus, human HC-TNFR1 shedding was dependent on theiNOS-TACE pathway, which may involve adirect interaction between iNOS and TACE.

Next, we testedwhether TNFR1 sheddingby human hepatocytes was dependent oniNOSand cGMP. Isolated human hepatocyteswere treated with vehicle [0.01% dimethylsulfoxide (DMSO)], the iNOS inhibitor1400W, the cGMP analog 8-CPT-cGMP, orthe sGC inhibitor ODQ, and thenwere stimu-latedwithLPS for 6 or 12hours. The amountsof sTNFR1 in the culturemedia increased in atime-dependent manner after LPS stimulation(Fig. 6I). Inhibition of iNOS with 1400Wsubstantially decreased sTNFR1 abundancein the culture media after 12 hours of stim-ulation with LPS. The amount of sTNFR1in the culture media was substantially in-creased in cells treated with 8-CPT-cGMP(Fig. 6I). Inhibition of cGMP productionwith ODQ resulted in a substantial reduc-tion in sTNFR1 abundance in the culturemedia after stimulation with LPS (Fig. 6I).These results suggest that, similar to mousehepatocytes, TNFR1 shedding from humanhepatocytes is also mediated through theiNOS-cGMP-TACE pathway in vitro.

DISCUSSION

TNFR1 shedding is an important protectivemechanism to limit the bioactivity of TNFin inflammatory processes. We previouslyshowed that TNFR1 shedding is mediatedthrough the iNOS-cGMP-TACE signalingpathway in hepatocytes (10). Therefore, we

extended our studies to determine the mechanism of HC-TNFR1 sheddingduring sepsis in a clinically relevant model of peritonitis. Our data indi-cate that circulating sTNFR1 amounts correlate with the severity of sepsis.Multiple TLR ligands and cytokines stimulated HC-TNFR1 sheddingin vitro, whereas one of themajor pathways that stimulated TNFR1 sheddingduring sepsiswas regulated by theMyD88-dependent production of iNOSand the cGMP-mediated activation of TACE. Furthermore, we showedthat regulation of HC-TNFR1 shedding through manipulation of theiNOS-cGMP pathway modulated systemic inflammatory responses andliver injury during sepsis. We also showed that human HC-TNFR1 sheddingwas similarly dependent on iNOS- and cGMP-mediated TACE activa-tion and its translocation to the cell surface. Together with our previousfinding (10), these data suggest that HC-TNFR1 shedding mediated

Fig. 5. Regulation of hepatic TNFR1 shedding through the iNOS-cGMP pathway modulates inflam-matory responses and liver injury during sepsis. (A to F) WT mice were subjected to CLP and then

were immediately treated subcutaneously with saline, 1400W (5 mg/kg), 8-CPT-cGMP (5 mg/kg), both1400W and 8-CPT-cGMP, or ODQ (20 mg/kg). Eight hours after CLP, plasma concentrations of (A) IL-6,(B) TNF, (C) KC, (D) MIP-1a, (E) HMGB1, and (F) ALT were measured by ELISA. Data are means ± SDfrom 8 to 10 mice per group from two experiments. *P < 0.05 by one-way ANOVA. (G to I) WT mice weresubjected to CLP and then were immediately treated subcutaneously with saline or sildenafil (50 mg/kg).Eight hours after CLP, the concentrations of (G) cGMP in liver homogenates and of (H) TNFR1 and (I)IL-6 in the plasma were determined. Data are means ± SD from six mice per group. *P < 0.05 by one-way ANOVA.




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through the MyD88-iNOS-cGMP-TACE signaling pathway limits in-flammatory responses and organ damage during sepsis. We also showedthat this pathway can be manipulated to enhance the amount of plasmasTNFR1 in vivo during sepsis.

TNF is a potent cytokine that exerts proinflammatory activities duringhost defense in infectious disease; however, excessive TNFactivity leads totissue toxicity. TNFR1 shedding is a self-protectivemechanism to buffer theeffects of excessive TNF amounts and block intracellular signaling by TNF(18, 19). TNFR1 shedding is thought to be an important host defensemech-anism during infections with Staphylococcus aureus (20) and L. monocyto-genes (19); however, these studies are based on models of infection withsingle pathogen. The CLP model is thought to be the model that is most rep-resentativeof the sepsis commonlyencounteredbypatientswithpolymicrobialintraperitoneal infection associated with fecal contamination (21). Therefore,

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we performed our study in a CLP model with antibiotics to investigate theinfluence of TNFR1 shedding on the inflammatory response in sepsis. Weshowed that the concentration of circulating sTNFR1 correlated with theseverity of sepsis. Circulating amounts of sTNFR1 inversely correlatedwiththe amounts of circulating inflammatory cytokines, suggesting that TNFR1shedding is regulated to modulate inflammatory response during sepsis.However, circulating amounts of sTNFR1 increasedmost substantially afterthe early increase in TNF concentration, which suggests that the mechanismsthat control TNFR1 shedding may be inadequate to counteract an early ex-cess of TNF encountered during severe sepsis.

TNFR1 shedding in a complex and systemic process such as sepsis islikely to involve several mechanisms in multiple cell types. Many studiesreported that during inflammation, TNFR1 is shed frommesenchymal cells,such as epithelial cells (22–24), lung endothelial cells (25), macrophages

Fig. 6. TNFR1 shedding by human hepatocytes in vitro ismediated through an iNOS-cGMP-TACE pathway. (A)Human hepatocytes were left untreated (Control) or treatedwith LPS (100 ng/ml) or 400 nM IL-1b for the indicated times.The concentration of TNFR1 in the culture media wasdetermined by ELISA. Data are means ± SD from threeindependent experiments. *P < 0.05 by one-way ANOVA.(B) Human hepatocytes were left untreated (Control) orwere treated with LPS (100 ng/ml). Cells were then ana-lyzed by immunofluorescence microscopy to detect iNOS(red) and TACE (green). Arrows indicate the colocalizationof iNOS and TACE. Scale bar, 50 mm. Images are represent-ative of two independent experiments. (C to F) Human hepa-tocytes were left untreated (zero-hour time point) or weretreated with LPS (100 ng/ml) in the absence (Cont) or pres-ence of 400 nM TAPI-2 for the indicated times. (C) The con-centration of TNFR1 in the culture media was determined byELISA. Data are means ± SD from three independentexperiments. *P < 0.05 by one-way ANOVA. (D) The hepa-tocytes were analyzed by Western blotting with antibodiesspecific for TACE and TNFR1. Densitometric analysis wasperformed to determine the relative abundances of (E)TACE and (F) TNFR1 proteins normalized to the abun-dance of actin. Data are means ± SD from three indepen-dent experiments. *P < 0.05 by two-tailed unpaired t test.(G and H) Human hepatocytes were left untreated (zero-hour time point) or were treated with LPS (100 mg/ml) inthe absence (Cont) or presence of 400 nM TAPI-2 for theindicated times. (G) Samples were then subjected to immu-noprecipitation (IP) with an anti-TACE antibody and wereanalyzed by Western blotting (IB) with antibodies specificfor TACE and iNOS. Bands corresponding to iNOS in theTAPI-2–treated samples were only detectable when West-ern blots were separately developed with SuperSignal WestPico Chemiluminescent Substrate (Thermo Scientific). (H)Densitometric analysis was performed to determine therelative abundance of iNOS protein normalized to that ofTACE. Data are means ± SD from three independent ex-periments. *P < 0.05 by two-tailed unpaired t test. (I) Hu-man hepatocytes were treated with vehicle (0.01% DMSO),20 mM 1400W, 100 mM 8-CPT-cGMP, or 20 mM ODQ, andthen were stimulated with LPS (100 ng/ml) for the indicated

times. The concentration of TNFR1 in the culture media was determined by ELISA. Data are means ± SD from three experiments. *P < 0.05 byone-way ANOVA.




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(8, 20, 26), and neutrophils (27). We and others showed that parenchymalcells, such as hepatocytes (10, 15), also shed TNFR1. Shedding mecha-nisms in these cells depend on TACE. TACE is an important enzyme duringinflammation and tissue regeneration through its activation and cleavage(and thus release) of multiple proteins, including cytokines (TNF andtransforming growth factor–b) and receptors (TNFR1, TNFR2, and IL-6R),aswell as activation of ligands that activate the epithelial growth factor receptor(28, 29). However, the exact mechanisms of TACE activation are unclear andmay be cell type–, receptor type–, or tissue-specific. McMahan et al. (30)showed that TACE was important for TNFR1 shedding from hepatocytesand myeloid cells in an endotoxemia model. For other cell types, TACE-dependent TNFR1 shedding requires the extracellular signal–regulated kinase(ERK) signaling pathway (20, 24) or cGMP-independent Ca2+ signaling (25).Okuyama et al. (31) showed that NO increased the abundance and shedding ofTNFR1 in a human umbilical vein cell line through metalloproteinases inde-pendently of cGMP.

Our previous (10) and current studies demonstrated that HC-TNFR1shedding is mediated, in part, through a MyD88-iNOS-cGMP-TACEpathway in vitro and in vivo, and that this pathway is dependent on a directinteraction between iNOS and TACE. Whether ERK or Ca2+ signaling isupstream of this pathway in hepatocytes was not addressed. The nitrosyla-tion of TACE is involved inTNFmaturation inmacrophages (32); therefore,it is reasonable to speculate that iNOS contributes to TACE activation notonly through cGMP but also through a direct interaction with TACEthrough targeted nitrosylation. That the extent of TNFR1 shedding is onlypartially suppressed by inhibition of the iNOS-cGMP-TACEpathway in he-patocytes during CLP suggests that other mechanisms for TNFR1 sheddingoperate during sepsis.

In polymicrobial sepsis, pathogen-associated molecular patterns(PAMPs), damage-associated molecular patterns (DAMPs), and cytokinesare involved in the induction of inflammatory responses (33). TNFR1shedding can be induced by PAMPs, such as LPS (10), CpG (8), anddouble-stranded RNA (dsRNA) (16), as well as by IL-1b (23). Our in vitrostudy showed that HC-TNFR1 shedding is induced by multiple TLR ligandsand IL-1b, suggesting that multiple stimuli trigger HC-TNFR1 shedding ina polymicrobial sepsis model. Our in vivo study showed that prevention ofIL-1b maturation in Casp-1−/− and Nalp3−/− mice or of IL-1b signaling inIl1r−/− mice reduced circulating sTNFR1 concentrations after CLP. Notethat inflammasomes activate iNOS in either an IL-1b–dependent (34) oran IL-1b–independent manner (35). Whether inflammasomes can increaseiNOS generation independently of IL-1b production to induce HC-TNFR1shedding needs to be further investigated.

MyD88 and TRIF are receptor-proximal signaling adaptor moleculesthat mediate signaling activated by various pattern recognition receptors.Yu et al. (16) showed that virus-derived dsRNA induced TNFR1 sheddingfrom human airway endothelial cells through a TLR3-TRIF-RIP pathway;however, Myddosome formation is the proximal event in the signaling ofall TLRs (with the exception of TLR3) and IL-1R (36). TLR ligands,such as LPS (10), and IL-1b (14) are potent inducers of iNOS productionin hepatocytes. Our in vitro and in vivo results suggest that MyD88 isrequired for increased iNOS production in hepatocytes, and they supporta model in which hepatocytes respond directly to TLR activators or toIL-1b generated from other cells (such as myeloid cells) to enhanceMyD88-dependent signaling. Activation of the Myddosome increasesiNOS production in hepatocytes, which leads to TACE activation andTNFR1 shedding during sepsis.

The amounts of cGMP in the circulation increase when NO activatessGC in the cytoplasm (37). Therefore, NO generated by iNOS is likely tobe one of the mechanisms responsible for the increase in cGMP concentra-tion observed in the circulation during sepsis (38, 39). Our previous study

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showed that iNOS- and NO-mediated increases in cGMP concentration re-sult in the activation of TACE and iRhom2 (a serine protease of the rhom-boid family) and the trafficking of the TACE-iRhom2 complex to the cellsurface in hepatocytes and the liver (10). We suggest that cGMP activatesPKG, which then participates in the phosphorylation of both TACE andiRhom2. Here, our data from experiments with both mouse and humanhepatocytes suggest that cGMP stimulates TACE activity and TNFR1release in hepatocytes and in the liver during CLP sepsis. Thus, the bio-availability of cGMP appears to be an important determinant of both thecell surface abundance of TNFR1 and the circulating concentration ofsTNFR1. Indeed, treatments that modulate cGMP abundance have thera-peutic benefit in sepsis models, although the mechanisms involved havenot always been clear. Yu et al. (38) showed that restoration of theNO-cGMPpathway with propofol improved endothelial dysfunction after CLP.Fernandes et al. (39) reported that cGMP was important for survival inthe early phase of sepsis. Buys et al. (40) showed that cGMP generatedby sGCa1b1 protected against cardiac dysfunction and mortality duringendotoxin- and TNF-induced shock.

Our data suggest that one mechanism by which cGMPmediates protec-tion from TNF toxicity in sepsis could be through the enhanced sheddingof TNFR1.We also showed that sildenafil, which is currently approved foruse in patients and has acceptable safety profiles, is an effective agent toincrease cGMP availability in experimental sepsis. Sildenafil is a PDE5inhibitor, which prevents cGMP degradation (17). Cadirci et al. (41)showed that sildenafil is protective against lung and kidney damagecaused byCLP-induced sepsis by preventing oxidant-induced injury. There-fore, maintaining or enhancing cGMP abundance during sepsis could havebeneficial effects through multiple interrelated mechanisms.

In summary, the integrated response to sepsis includes the increasediNOS production throughMyD88-dependent pathwayswithin hepatocytes,as well as through the MyD88-dependent production of IL-1b in myeloidcells. Within hepatocytes, increased iNOS production promotes the cGMP-dependent activation of TACE and TNFR1 shedding, which is associatedwith the suppression of inflammation and organ damage. Pharmacologicmanipulation of theNO-cGMP-TACE-TNFR1 pathway could be of benefitin the early clinical course of severe sepsis.

MATERIALS AND METHODS

ReagentsUltrapure LPS (from Escherichia coli 0111:B4) was from List Bio-logical Laboratories Inc. The mouse TLR1 to TLR9 agonist kit was fromInvivoGen. Sildenafil, ODQ, 1400W, and 8-CPT-cGMP were obtainedfrom Sigma.

MiceExperimental protocols were approved by the Institutional Animal Careand Use Committee of the University of Pittsburgh. Male wild-typeC57BL/6 mice, Il1r−/− (Il1r1tm1Imx) mice, Albumin-Cre (Alb-Cre) mice[Tg(Alb-Cre) 21MgN)], Lyz-Cre mice [Lyz2tm1(cre)Ifo], and MyD88-Flox (Myd88tm1Defr) mice were purchased from The Jackson Laboratory.Myd88−/− (Myd88tm1Aki) mice on a C57BL/6 background were a gift fromR. Medzhitov [Howard Hughes Medical Institute (HHMI), Yale Uni-versity, New Haven, CT]. Casp1−/− mice (Casptm1Flv) on a C57BL/6 back-ground were a gift from R. Flavell (HHMI, Yale University). Nalp3−/−

(Nlrp3tm1Flv) mice on a C56BL/6 background were a gift from R. Flavellthrough a material transfer agreement with Millennium Pharmaceuticals.Trif −/− mice (Ticam1Lps2) on a C57BL/6 background were a gift fromB. Beutler (Scripps Research Institute). Myd88−/− mice, Casp1−/− mice,




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Nalp3−/− mice, and Trif −/− mice were bred in our animal facility. Tlr4−/−

(Tlr4tm1Djh) and Nos2−/− (Nos2tm1Mrl) mice were generated in our laboratoryon a C57BL/6 background and backcrossed at least six times. Hepatocyte-specific Myd88−/− mice (HC-Myd88−/−) and myeloid cell–specific Myd88−/−

mice (LysM-Myd88−/−) were generated with the Cre-loxP technique andwere identified by PCR-based genotyping with multiple primer pairs, asdescribed previously (42). These animals were bred in our facility.

CLP procedureSepsis was induced by CLP. Mice that were 25 to 30 g in weight were used.The skin was disinfected with a 2% iodine tincture. Laparotomy was per-formed under 2% isoflurane (Piramal Critical Care) with oxygen. For thesublethal model, 50% of the cecumwas ligated and punctured twicewitha 22-gauge needle. For the lethal model, 75% of the cecum was ligatedand punctured twice with an 18-gauge needle. Saline (1 ml) was givensubcutaneously for resuscitation immediately after operation. Primaxin(25 mg/kg, Merck) was dissolved in 5% dextrose (Baxter) and injected intomice subcutaneously every 12 hours starting 2 hours after CLP was per-formed. In the control mice, laparotomy and cecal manipulation were per-formed without ligation and puncture.

Isolation and culture of hepatocytesCells were isolated from mice through an in situ collagenase (type VI,Sigma) perfusion technique, modified as described previously (13). Hepa-tocyte purity exceeded 99% by flow cytometric assay. Cell viability wastypically >90% by trypan blue exclusion. Hepatocytes (150,000 cells/ml)were plated on gelatin-coated culture plates or coverslips coated withcollagen I (BD Pharmingen) in William’s medium E with 10% calf serum,15mMHepes, 1 mM insulin, 2 mM L-glutamine, penicillin (100 U/ml), andstreptomycin (100 U/ml). Hepatocytes were allowed to attach to platesovernight, and the culture medium was replaced with fresh medium beforethe cells were treated for experiments. After treatment, 1 ml of culture me-diumwas collected and centrifuged at 400g for 5 min. The supernatant wasremoved to a new tube and stored at −80°C for further analysis.

Crystal violet stainingHepatocytes were plated in 12-well plates. After treatment, hepatocyteswere stained with 0.1% crystal violet (Sigma) for 15 min and then washedwith sterile distilled water. The rinsed plates were left to air-dry overnight.The bound dye was released by the addition of 1% SDS. The elution liquidof crystal violet was removed to a new microplate, and the absorbance(A) was measured at a wavelength of 575 nm. The percentage of viablecells (% viability) was calculated by dividing the A575 of the treated sam-ple by the A575 of the control sample and multiplying by 100.

Assessment of TNFR1 and cytokine abundancePlasma and cell culture media samples were analyzed with ELISA kitsspecific for IL-6, TNF, KC, MIP-1a, and TNFR1 (R&D Systems Inc.)to assess their concentrations.

cGMP assayThe abundance of cGMP in liver tissue was analyzed with a cGMP EIA kit(Cayman Chemical). Briefly, frozen liver tissue (50 mg) was homogenizedwith 5% trichloroacetic acid (TCA) on ice. After centrifugation, the TCAwas extracted from the supernatant with water-saturated ether. After TCAextraction, the residual ether was removed by heating the sample at 70°C for5 min. Samples (50 ml) were incubated with cGMPAchE Tracer at 4°Covernight and developed with Ellman’s reagent. The amount of cGMP ineach sample was assessed by measuring the absorbance of the samples at405 nm.

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Isolation of human hepatocytesHuman hepatocyteswere isolated from histologically normal liver and wereprovided by D. Geller (Department of Surgery, University of Pittsburgh,Pittsburgh, PA) according to a protocol approved by the InstitutionalReviewBoard. Human hepatocytes were prepared by a three-step collagenase per-fusion technique, modified as described previously (43). Isolated humanhepatocytes were cultured in William’s medium E supplemented with 5%calf serum (GEHealthcare Life Sciences), penicillin (100U/ml), streptomy-cin (100 U/ml), 2 mM L-glutamine, and 15 mM Hepes.

Cell lysis and Western blotting analysisFor in vitro experiments, hepatocytes were washed with cold phosphate-buffered saline (PBS), collected in lysis buffer (Cell Signaling Technol-ogy), sonicated, and centrifuged at 16,000g for 15 min, after which thesupernatant was collected. For in vivo experiments, snap-frozen liver(median lobe) was homogenized in lysis buffer and centrifuged at16,000g for 15 min, after which the supernatant was collected. Proteinconcentrations were determined with the BCA (bicinchoninic acid) proteinassay kit (Thermo Fisher Scientific). Loading buffer was added to thesamples, which were then resolved by 10 or 15% SDS–polyacrylamidegel electrophoresis. Samples were then transferred onto a polyvinylidenedifluoride membrane at 250 mA for 2 hours. The membrane was blockedin 5% milk for 1 hour and then incubated overnight with primary antibodyin 1% milk. Membranes were washed in tris-buffered saline containingTween (TBS-T) for 10 min, incubated with horseradish peroxidase–conjugated secondary antibody for 1 hour, and then washed for 1 hour inTBS-T, before being developed for chemiluminescence (Thermo FisherScientific). The primary antibodies used were anti-iNOS (1:1000, R&DSystems Inc.), anti-TNFR1, anti-TACE, and anti-GAPDH (1:1000 for allantibodies, Abcam). Protein band intensities were quantified with ImageJsoftware (National Institutes of Health).

Comparative PCR analysisThemedian lobe of the liverwas frozen in liquid nitrogen and stored at−80°Cfor comparative PCR analysis. Total RNAwas extracted with the RNeasyMini extraction kit (Qiagen) according to the manufacturer’s instructions.From 1 mg of RNA and with oligo-dT primers (Qiagen) and Omniscript re-verse transcriptase (Qiagen), complementary DNAwas generated and usedfor real-time PCR analysis. SYBRGreen PCRMasterMix (PEApplied Bio-systems) was used to prepare the PCR reactionmixes. Two-step real-timeRT-PCR was performed with prevalidated forward and reverse primer pairs thatwere specific for Tnfr1 andNos2 (Qiagen). All samples were assayed in trip-licate, and data were normalized to actin mRNA abundance.

Immunofluorescence microscopyThe left lateral lobe of the liver was perfused with PBS and fixed in 2%paraformaldehyde according to a standardized protocol for cryo-preservation (44). Liverswere sectioned in a cryostat and stained as follows.Liver sections (5 mm)were incubatedwith 2%bovine serumalbumin (BSA)in PBS for 1 hour, followed by fivewasheswith PBS containing 0.5%BSA(PBB). The samples were then incubated overnight with primary antibodiesspecific for iNOS (1:500, R&DSystems Inc.) and TACE (1:300, Abcam) inPBB, 0.1% Triton X-100. Samples were washed five times with PBB andthen were incubated with the appropriate Alexa Fluor 488–labeled (1:500,Invitrogen) and Cy3-labeled (1:1000, Jackson ImmunoResearch Laboratories)secondary antibodies diluted in PBB. Sampleswerewashed three timeswithPBB, followed by a single wash with PBS, before being incubated for 30 swith Hoechst nuclear stain. The nuclear stain was removed, and sampleswere washed with PBS before being placed on a coverslip using Gelvatol(EMDMillipore). Positively stained cells in six random fields were imaged




with a FluoView 1000 confocal scanning microscope (Olympus). Imagingconditionsweremaintained at identical settingswithin each antibody labelingexperiment, with original gating performed with the negative control.

Statistical analysisThe data are presented as means ± SD. The experimental results wereanalyzed for statistical significance with GraphPad Prism (GraphPadSoftware). Normal distribution of samples was tested with a two-tailedStudent’s t test or by one-way ANOVA for multiple comparisons asindicated in the figure legends. Statistical significance was establishedat the 95% confidence level (P < 0.05).

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SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/8/361/ra11/DC1Fig. S1. The kinetics of TNFR1 and TNF release after CLP.Fig. S2. Analysis of plasma IL-1b concentrations at 8 hours after CLP.Fig. S3. The responses to LPS of hepatocytes from Alb-Cre mice are similar to those ofhepatocytes from wild-type mice.Fig. S4. The responses to LPS of cells from Lyz-Cre mice are similar to those of cells fromwild-type mice.

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Acknowledgments: We thank H. Liao, M. Bo, A. Frank, and N. Martik-Hays for their ex-cellent technical support. Funding: This work was supported by NIH grants R37-GM-044100 and R01-GM-050441. Author contributions: M.D. performed most of theexperiments and contributed to the experimental design and writing of the manuscript;P.A.L. performed all of the immunofluorescence experiments; L.Z. performed all of theimmunoprecipitation experiments; M.J.S. contributed to the experimental design and editedthe manuscript; and T.R.B. designed most of the research project, provided oversight ofexperiments, and edited the manuscript. Competing interests: The authors declare that theyhave no competing interests.

Submitted 29 May 2014Accepted 8 January 2015Final Publication 27 January 201510.1126/scisignal.2005548Citation: M. Deng, P. A. Loughran, L. Zhang, M. J. Scott, T. R. Billiar, Shedding of thetumor necrosis factor (TNF) receptor from the surface of hepatocytes during sepsis limitsinflammation through cGMP signaling. Sci. Signal. 8, ra11 (2015).

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SCIENCESIGNALING.org 27 January 2015 Vol 8 Issue 361 ra11 11

on April 7, 2021


oaded from


sepsis limits inflammation through cGMP signalingShedding of the tumor necrosis factor (TNF) receptor from the surface of hepatocytes during

Meihong Deng, Patricia A. Loughran, Liyong Zhang, Melanie J. Scott and Timothy R. Billiar

DOI: 10.1126/scisignal.2005548 (361), ra11.8Sci. Signal.

humans.inflammation and increased survival, suggesting that a similar strategy might provide a therapy against sepsis in decreasedmessenger cGMP. Treating mice that had sepsis with drugs that increased the plasma concentration of cGMP

secondbacterial product LPS resulted in increased shedding of TNFR by the protease TACE, an event dependent on the . showed that treating mouse or human hepatocytes with theet aland reduces TNF concentrations in the plasma. Deng

such as occurs during sepsis. The proteolytic shedding of TNFR from the cell surface decreases TNF signaling in cells damage,invading pathogens; however, excessive TNF signaling through its receptor TNFR leads to cell death and tissue

The proinflammatory cytokine tumor necrosis factor (TNF) is required for an effective immune response toTaming TNF during sepsis

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