tristetraprolin limits inﬂammatory cytokine production in ...n-tam t-tam signal intensity g-tam...

Microenvironment and Immunology

Tristetraprolin Limits Inflammatory CytokineProduction in Tumor-Associated Macrophages inan mRNA Decay–Independent MannerFranz Kratochvill1,2, Nina Gratz1, Joseph E. Qualls1,2, Lee-Ann Van De Velde1,2,Hongbo Chi2, Pavel Kovarik3, and Peter J. Murray1,2

Abstract

Tristetraprolin (TTP) is an inducible zinc finger AU-richRNA-binding protein essential for enforcing degradation ofmRNAs encoding inflammatory chemokines and cytokines.Most studies on TTP center on the connection between mRNAhalf-life and inflammatory output, because loss of TTP ampli-fies inflammation by increasing the stability of AU-richmRNAs. Here, we focused on how TTP controls cytokine andchemokine production in the nonresolving inflammation ofcancer using tissue-specific approaches. In contrast with mod-el in vitro macrophage systems, we found constitutive TTPexpression in late-stage tumor-associated macrophages

(TAM). However, TTP's effects on AU-rich mRNA stabilitywere negligible and limited by constitutive p38a MAPK activ-ity, which was the main driver of proinflammatory cytokineproduction in TAMs at the posttranscriptional level. Instead,elimination of TTP caused excessive protein production ofinflammatory mediators, suggesting TTP-dependent transla-tional suppression of AU-rich mRNAs. Manipulation of thep38a–TTP axis in macrophages has significant effects on thegrowth of tumors and therefore represents a means to mani-pulate inflammation in the tumor microenvironment. CancerRes; 75(15); 3054–64. �2015 AACR.

IntroductionNonresolving inflammation (NRI) underpins most chronic dis-

eases, including obesity, autoinflammatory diseases, atherosclero-sis, chronic responses to implanted medical devices, chronic orlatent infections, and cancer (1). NRI is caused by aberrantresponses to a persisting entity. The persistence of malignant cellsin cancer, for example, drives the productionof hematopoietic cellsfrom the bone marrow to seed a growing tumor (2). Therefore, achallenge of biology andmedicine is to understand the cellular andmolecular pathways underpinning NRI and how to mitigate theireffects in chronic diseases.

In resolving inflammation triggered by TLR signaling, the TNFmRNA is rapidly produced from poised Pol II at the Tnf promoter(3–5). TNF protein is made and secreted, followed by a down-regulation phase where the tristetraprolin (TTP) zinc finger pro-tein, also induced in the early phase of TLR signaling, plays adecisive role in eliminating TNF mRNA (6). In model, in vitrosystems such as BMDMs the TNF–TTP pathway is controlled bytwo key factors, p38a MAPK and IL10 (7–9). p38a activated by

TLR signaling phosphorylates TTP thereby blocking its functionand sustaining TNF output (9, 10). In the resolution phase, DUSPproteins dephosphorylate p38a, which leads to dephosphoryla-tionof TTP anda rise in TTP activity to eliminate TNFmRNA. IL10,stimulated by TLR signaling to act in an autocrine way, furtherenhances removal of the TNF mRNA because it drives TTP andDUSP1 expression in a STAT3-dependent way (7, 11, 12).

To begin to understand the signaling pathways involved inregulating chronic inflammation in tumor-associated macro-phages (TAM), we used transplantable tumor models with pre-dictable temporal inflammatory responses that can be tracked byflow cytometry and that are amenable to the use of genetics toprobe signaling pathways andhow they becomedysregulated.Wefocused on how TTP regulates cytokine and chemokine produc-tion because inflammatory cytokines are the key drivers of chronicinflammation such as cancer. A key tactic was using a conditionalknockout allele of Zfp36 (encoding TTP) to avoid complicationsof systemic inflammation present in conventional Zfp36�/� miceearly in development (13–15). Our results show that TTP main-tains macrophages on a "knife-edge" between counterregulationof inflammation and death.Our results also suggest TTP primarilyregulates steps beyond mRNA stability such as mRNA translationin macrophages associated with cancer inflammation.

Materials and MethodsMice

C57BL/6, Tie2-Cre (B6.Cg-Tg(Tek-cre)1Ywa/J), and LysM-Cre(Lyz2tm1(cre)Ifo/J) mice were obtained from The Jackson Laboratory.TTPKO (Zfp36�/�) mice were a gift of P. Blackshear (NationalInstitute of Environmental Health Sciences, Research Triangle Park,NC). The TTPDM and Mapk14flox/flox mice have been describedelsewhere (15, 16). Th-MYCN transgenic (tg; ref. 17) andRbflox/flox;p53flox/flox;Osx-Creþ (mixed background) mice (18) wereobtained from M. Dyer, St. Jude Children's Research Hospital,

1Department of Infectious Diseases, St. Jude Children's Research Hospi-tal, Memphis, Tennessee. 2Department of Immunology, St. Jude Chil-dren's Research Hospital, Memphis, Tennessee. 3Max F. Perutz Labora-tories,Center forMolecularBiology,UniversityofVienna,Vienna,Austria.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for J.E. Qualls: Cincinnati Children's Hospital Medical Center,Cincinnati, OH.

Corresponding Author: Peter J. Murray, Infectious Diseases, MS320, Room E8078,St. JudeChildren'sResearchHospital, 262DannyThomasPlace,Memphis, TN38105-3678. Phone: 901-595-3219; Fax: 901-595-3099. E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-0205

�2015 American Association for Cancer Research.

CancerResearch

Cancer Res; 75(15) August 1, 20153054

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Published OnlineFirst July 16, 2015; DOI: 10.1158/0008-5472.CAN-15-0205

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Memphis, TN. For experiments, sex-matched mice were groupedand used between 6 and 10 weeks. All mice were on a C57BL/6backgroundunless indicatedotherwise.Micewereusedwithin theAnimal Resource Center according to protocols approved by theInstitutional Animal Care and Use Committee of St. Jude Chil-dren's Research Hospital.

Zymosan A induced peritonitisChronic peritonitis was induced by i.p. injection of 10 mg/

mouse of zymosan A (Sigma) as described (19) andCD11bþ cells

were purified by Miltenyi magnetic beads according to the man-ufacturer's protocol.

TAM collection from solid tumorsSolid tumors were minced and digested with 5 mL of fresh

Tumor Digestion Media as described in the Supplementary Mate-rials and Methods. The suspension was then passed through a70-mm strainer and cells were overlaid on a 35%/60% percollgradient. After centrifugation cells were collected from the 35%/60% interphase and TAMs were isolated by CD11b purification

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Figure 1.NRI-associated macrophages share a common mode of development and express proinflammatory cytokines. A and B, flow-cytometry plots of CD11bþLy6G�

myeloid cells isolated from EG7 thymomas (A) or zymosan induced peritonitis (B) at different time points as indicated. Plots are representative for atleast two experiments. Gates A to D indicate TAM or peritonitis-associated macrophage (PAM) populations used for sorting experiments or to measurecytokine production by intracellular flow cytometry. C, quantitative analysis of EG7 TAM populations A and C (gated as in A) over time. Values are from one oftwo independent experiments (n � 4); error bars, SEM. D, quantitative analysis of cytokine production by flow cytometry of EG7 TAM populations A to Cgated as shown in A. Values from individual mice are either depicted as the percentage of positive cells (left) or median fluorescence intensity (MFI, middle) ofTNF or IL1a-positive (TNFþ and IL1aþ) compared with negative (TNF� and IL1a�) macrophages within population A to C. Example plots are shown (right)indicating the gating strategy for cells considered positive and negative. Unstained control, gray. Data are mean � SEM of two independent experiments (n � 2).

TTP Limits Inflammation in Tumor Macrophages

www.aacrjournals.org Cancer Res; 75(15) August 1, 2015 3055




using Miltenyi magnetic beads according to the manufacturer'sprotocol.

Tumor models—in vivoEG7 lymphoma line (ATCC)originating from the thymus (EL4),

stably expressing OVA protein was tested for pathogen contami-nation before injection. A total of 3� 106 EG7 cells were injected s.c. into theflank of recipientmice and harvested 12days later unlessindicated otherwise. Orthotopically grown gliomas (20) and thy-momas were harvested from mice between 1.5 and 2 weeksposttransplant. Spontaneous neuroblastomas from Th-MYCNtransgenic (tg) and osteosarcomas collected from Rbflox/flox;p53flox/flox;Osx-Creþ mice (18) were detected by ultrasound orvisual inspection of tumors on the long bones or jaws, respectively.

Antibodies and immunoblottingFor flow cytometry: Anti-mouse MHC II (I-A/I-E; clone M5/

114.15.2, cat. no. 12-5321-83), anti-mouseCcl3 (cloneDNT3CC,cat. no. 12-7532-80), anti-mouse IL10 (clone Jess-16E3, cat. no.11-7101-81), anti-mouse IL1a (clone ALF 161, cat. no. 12-7011-81), anti-mouse IL1b (clone NJTEN3, cat. no. 12-7117-80)all eBiosience, anti-mouse TNFa (cat. no. 554419; PharMingen),

p-p38MAPK (T180/Y82; clone 3D7, cat. no. 9608S;Cell SignalingTechnology), anti-mouse/human CD11b (clone M1/70, cat. no.101224, BioLegend), anti-mouse Ly6C (clone HK1.4, cat. no.128016; BioLegend). For immunoblotting rabbit antibody to TTP(7). p-p38 MAPK and p38 MAPK were from Cell SignalingTechnology (T180/Y182; clone D3F9, cat. no. 4511S) and SantaCruz Biotechnology (clone A-12, cat. no. sc-7972), respectively.Whole-cell extracts were prepared and assayed by Western blot-ting as described (7) using 4% to 15% Tris–HCl gels (Criterion;Bio-Rad).Equal loading was controlled by anti-mouse GRB2antibody (cat. no. 610112; BD Biosciences).

Enzyme-linked immunosorbent assayMurine TNF production was measured from supernatants of

TAMs resting on tissue-culture plates for 6 hours using preopti-mized Capture and Detection antibodies (both eBioscience).

ResultsTAMs produce proinflammatory cytokines during all stages ofdevelopment

Blood-derived monocytes invade almost all types of solidtumors where they develop into macrophages. Macrophages are

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Figure 2.TTP is highly expressed in selected TAM populations. A, immunoblot analysis of TTP expression in neuroblastoma (N), osteosarcoma (O), and EG7 thymoma(T) TAMs compared with 1 hour LPS-stimulated BMDMs. Blot was reprobed with anti-GRB2 to test for equal loading and represents one of three experiments. B,TTPqRT-PCR of EG7 TAMs sorted for the indicated populations as in Fig. 1A. Data from individual mice are expression values from four experiments (n � 6).All values were first normalized to the corresponding GAPDH and then to the mean of TAM-C within each experiment. Data were analyzed by one-way ANOVAfollowed by the Bonferroni post-test. The mean per group is shown as a black line; error bars, SEM. C, qRT-PCR analysis of TTP in TAMs or BMDMs leftuntreated (ns) or stimulatedwith LPSþ IFNg over time. Datawere normalized to the corresponding GAPDH for each value and represent themean� SEM (n� 3). D,microarray analysis of TAMs isolated from neuroblastomas (N-TAM), EG7 Thymomas (T-TAM), and gliomas (G-TAM) showing average signal intensities ofselected AU-rich element binding proteins (ARE-BPs, n ¼ 3 per TAM type). Right, the rank of different ARE-BPs in N-TAMs together with the rank as thepercentage of all 45,038 probe sets, respectively. The full microarray dataset has been submitted (GEO accession number GSE59047).

Kratochvill et al.

Cancer Res; 75(15) August 1, 2015 Cancer Research3056




the most numerous myeloid cells in chronic inflammation, andthe source of many key proinflammatory cytokines and chemo-kines that drive inflammation forward (21). To dissect macro-

phage function in cancer inflammation,wedevelopedmethods toreliably distinguish macrophages at different points in theirdevelopment from neutrophils and eosinophils. Building on a

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Figure 3.Myeloid TTP counteracts inflammation and supports tumor growth. A, immunoblot analysis using anti-TTP antibody of whole-cell lysates of EG7 TAMs isolated fromWT, TTPDM, or TTP�/� tumor bearing animals. Blots were reprobed using anti-GRB2 antibody to confirm equal loading and represent one out of at least twoindependent experiments (Note: the brighter band detected in TTPKO TAMs is as it appears on the blot and has not been manipulated). B, tumor massof EG7 thymomas grown in WT (Zfp36flox/flox) and TTPDM mice for 13 days. Data from individual mice from three independent experiments (n � 23) areshown together with the mean and SEM. C, active caspase-3 staining of representative EG7 tumors isolated from WT and TTPDM animals; n ¼ 3 animals pergenotype. D, flow-cytomery analysis of Annexin Vþ cells in EG7 thymomas grown in WT and TTPDM mice. Values from individual mice are shown as the percentageof total cells from one of three experiments (n � 6). E and F, cytokine production in EG7 TAMs (gated on CD11bþ) measured by intracellular flow cytometry inWT (Zfp36flox/flox) or TTPDM animals. The percentage of positive cells of CD11bþ (E) or median fluorescence intensity (MFI) of cytokineþCD11bþ cells (F) fromindividual mice is from one of two to five experiments (n � 6). The mean is depicted as a black line; error bars, SEM. G, TNF cytokine production detected byELISA in the supernatants of WT and TTPDMTAMs after 6 hours (right) or resting or 3 hours LPS-stimulated BMDMs (left). The mean is depicted as a black line; errorbars, SEM. Values represent TAMs from individual mice (n � 2) from one of two experiments.






system developed by Movahedi and colleagues, we identifiedthree monocyte-derived populations of macrophages in solidtumors as well as in chronic zymosan peritonitis, a complemen-

tary model for chronic inflammation where monocytes infiltratethe inflammatory site (Fig. 1A and B; ref. 22). The "A" populationcomprises inflammatory monocytes (Ly6Chi and MHCII�).

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Figure 4.TTP-dependent mRNA decay is limited in TAMs. A,qRT-PCR analysis of EG7 TAMs isolated from WTand TTPDM mice. Data from at least threeexperiments were combined and values fromindividual mice (n � 8) were normalized to thecorresponding GAPDH followed by the mean WTwithin each experiment. The mean per group isshown as a black line; error bars, SEM. B, qRT-PCRanalysis of primary transcripts of inflammatorymRNAs in WT and TTPDM TAMs. Values fromindividual mice (n � 7) were normalized to GAPDHand mean WT within each experiment and arecombined from two experiments. Identical samplesfrom each tumor were processed in the absence ofreverse transcriptase to serve as controls forgenomic DNA contamination. Themean per group isshown as a black line; error bars, SEM. C, qRT-PCRanalysis of TTP target mRNAs in EG7 TAMpopulation A and C isolated from WT and TTPDM

mice. Data, expression values of individual mice (n�7) combined from two to four experiments. Valueswere normalized to the corresponding GAPDH andsubsequently to the mean ofWT TAM-C within eachexperiment. Mean is shown as a black line; errorbars, SEM. D, mRNA stability measured by qRT-PCRby comparing cells before and 90 minutes aftertranscriptional blockade by actinomycin D in EG7TAMs from WT and TTPDM mice. Values werenormalized to GAPDH and represent the percentageof cytokine mRNA remaining after actinomycin Dtreatment as mean and SEM. Expression values arefrom individual mice combined from two to threeexperiments (n � 8). Mean is indicated by a blackline; error bars, SEM.

Kratochvill et al.





Tracing tools argue that population "A" gives rise to the Ly6Cþ,MHCIIþpopulation "B,"which thendevelops into themature "C"fraction, consisting of cells that downregulate Ly6C and increaseexpression of MHCII (22, 23). The "C" macrophages increase inrelative proportion over time (Fig. 1A–C). The remainingMHCII-and Ly6C-negative myeloid infiltrates consist of eosinophil-likecells maintained independently of monocytes (population D;ref. 22). The developmental progression of tumor macrophagesandmacrophages from chronic peritonitis frommonocytes to theC fraction is similar to the "waterfall"model of in situmacrophagedevelopment observed in models where monocytes seed inflam-matory sites, arguing for a general mode of macrophage devel-opment under inflammatory conditions (24). For all subsequentexperiments we isolated TAMs from tumors at day 12, as at thisstage all TAM populations were most abundant and amenable todetailed ex vivo analysis.

A hallmark of chronic inflammation is constitutive cytokine andchemokine production promoted by the presence of the incitingentity. A crucial question in understanding the relationshipsbetween cytokine and chemokine-producing cells in chronic inflam-mation is whether negative feedback loops counteracting disease-causing inflammation are inactive, and how they become dysregu-lated. We therefore asked whether TAMs expressed inflammatorycytokines andwhether their expression patternwas linked to the "A,B, and C" development pathway. Although we found highestexpression of TNF and IL1a in the "B" fraction, all macrophagesubsets expressed readily detectable amounts of both cytokines (Fig.1D). For example, 36% of the "B" fraction were TNFþ (Fig. 1D).

AU-rich element-binding proteins are highly expressed in TAMsTTP is essential for mediating cytokine and chemokine

mRNA decay in inflammation. Indeed, global absence of TTP

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Figure 5.Reduced TTP-dependent mRNA decay coincides with chronic p38a activation in TAMs and PAMs. A, TTP expression analyzed by immunoblotting ofwhole cell lysates of EG7 TAMs and resting or LPS treated BMDMs. Blots were reprobed using anti-GRB2 antibody to confirm equal loading and represent 1 out of 3experiments. B, TNF mRNA stability determined by qRT-PCR by comparing cells before and 90 minutes after transcriptional blockade by actinomycin D inBMDMs and TAMs isolated from EG7 thymomas (T-TAM), neuroblastomas (N-TAM), or osteosarcomas (O-TAM). Values were normalized to GAPDH and representthe percentage of TNF mRNA remaining after actinomycin D treatment as mean and SEM of at least two experiments (n � 2). C, immunoblot analysis oftotal cell lysates of nonstimulated (ns) or 1 hour LPS-stimulated (LPS) BMDMs as well as TAMs isolated from different tumor models as in B, probed with anti–phospho-p38, anti-p38, or anti-GRB2 as loading control. The blot represents one of two experiments. D, p38 phosphorylation analyzed by immunoblotting of wholeEG7 TAM lysates from WT TAMs directly after isolation (�) or after resting them on tissue culture dishes (TC) for 3 hours. E and F, immunoblot analysis ofwhole EG7 TAM lysates (E) or BMDMs left untreated or stimulated with LPS for 1 and 3 hours (F) isolated from WT or p38aDM animals. Blots represent one of twoexperiments. G, whole EG7 TAM lysates from WT or p38aDH animals as in E analyzed for p38 and p-p38 protein expression by immunoblotting (n ¼ 2).H, flow-cytometry analysis of p-p38 in CD11bþ EG7 TAMs isolated from WT or p38aDH animals (n � 4). Representative plots are shown with unstained control asgray line.






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45.1

CD45.2H

Figure 6.p38a regulates TTP expression and activity in TAMs. A, EG7 TAM RNA fromWT or p38aDH tumor-bearing mice was analyzed by qRT-PCR. Expression values fromindividual mice combined from three experiments (n � 13) were normalized to GAPDH and the mean WT within each experiment is depicted as meanindicated by black lines; error bars, SEM. B and G, mRNA stability measured by qRT-PCR as in Fig. 4D in EG7 TAMs fromWT(Mapk14flox/flox) and p38aDH animals (B)and in unstimulated (�) or SB203580 (SB)-treated WT EG7 TAMs (G). SB treatment was started concomitant to actinomycin D-imposed transcriptionalblockade. Values from individual mice were normalized to GAPDH from either three to five (n� 10, B) or one of two (n� 7; G) experiments, respectively. Black lines,mean per group; error bars, SEM. C, cytokine expression analyzed by flow cytometry of EG7 TAMs (gated on CD11bþ) isolated from WT or p38aDH mice.Values from individual mice are the percentage of CD11bþ cells (n¼ 6) representing one of two experiments with the mean depicted as a black line; error bars, SEM.D, cytokine production evaluated by intracellular flow cytometry in lethally irradiated mice reconstituted with a mixture of WT (CD45.1) and p38aDH (CD45.2)bone marrow (n ¼ 4). Depicted values represent percentage of positive cells of CD11bþ TAMs gated for CD45.1 or CD45.2 as shown (left); error bars, SEM. E, EG7tumor mass 12 days posttransplantation of EG7 cells into WT or p38aDH animals. (Continued on the following page.)

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in all cells causes systemic inflammatory pathology becausemultiple inflammatory cytokines, including TNF and IL23, areproduced in excess (13, 14). TTP's target mRNAs form ahierarchy dependent in part on the number and type of AUelements in the mRNA 30-untranslated region (25). As bothTNF and IL1a mRNAs are TTP targets (6, 15, 26), but highlyexpressed in tumor inflammation-associated macrophages, wesuspected sustained cytokine production by TAMs was causedby failure of TTP expression. Paradoxically, we found TTPmRNA and protein was highly expressed in TAMs, and expres-sion increased as cells developed to the "C" population (Fig. 2Aand B). Although TTP was expressed, cytokine production washigh (Fig. 1D). High expression of TTP was not an artifact of ourexperimental system because high amounts of the Zfp36 mRNA(encoding TTP) and TTP protein were detectable in macro-phages from different cancer types, and were expressed athigher amounts than LPS-stimulated BMDMs (Fig. 2C). Inter-estingly, mRNAs encoding two TTP-related zinc finger mRNA–destabilizing proteins, Zfp36L1 and Zfp36L2, were also highlyexpressed in tumor macrophages, being among the top 2%genes (Fig. 2D; F. Kratochvill and colleagues, manuscript sub-mitted; GEO accession number GSE59047). Given TTP,Zfp36L1, and Zfp36L2 were highly expressed in TAMs, butcytokine expression was correspondingly high, we concludedthat mRNA degradation processes are ineffective at eliminatinginflammatory cytokine mRNAs in tumor macrophages.

TTP limits the production of inflammatory cytokines inTAMs

Wenext askedwhether TTPwas required for regulating cytokineand chemokine production. To do this, we generated TAMs frommice where TTP is specifically and efficiently eliminated frommacrophages and neutrophils using the LysM-Cre deleter (calledTTPDMmice; Fig. 3A). This tactic enabled us to avoid the effects ofsystemic inflammation in the Zfp36�/�mice where the absence ofTTP increases numerousproinflammatory cytokines and results inearly death (13, 14). We examined tumors in the TTPDMmice andfound they were smaller and contained more dying cells ascomparedwith tumors fromcontrolmice (Fig. 3B–D). TAMs fromTTPDM mice had increased numbers of macrophages positive forTNF, Ccl3, IL1a, and IL1b (Fig. 3E and F). In addition, TAMsisolated from TTPDM animals secreted approximately 2.5-foldmore TNF when rested on tissue culture dishes for 6 hours thanWT TAMs (Fig. 3G). We further compared the TNF production inTAMs with resting or LPS stimulated BMDMs. TAMs producedmore TNF than resting BMDMs and approximately one fourth ofTNF secreted by an equivalent number of homogeneous BMDMsstimulated for 3 hours with LPS (Fig. 3G). Production of the anti-inflammatory cytokine and TTP target IL10 (27)was unaffected interms of number of IL10 producing macrophages or intensity ofstaining (Fig. 3E and F). Collectively, these data suggest TTP isrequired to temper the already high cytokine and chemokineproduction in the inflammatory milieu of chronic inflammation.

WhenTTP is genetically eliminated inmacrophages, cytokine, andchemokine output further increases relative to the already highamounts in controls, which is linked to increased cell death anddecreased tumor size.

TTP-dependent mRNA decay is limited in TAMsTTP increases the instability of AU-richmRNAs (6). Given high

expression of TTP inmacrophages associatedwith chronic inflam-mation, we asked whether TTP regulated AU-richmRNA stability.We measured steady-state mRNA amounts of AU-rich inflamma-tory mRNAs in the total CD11bþ myeloid fraction from tumorsand found no differences between controls and TTPDM cells (Fig.4A). TTP has also been implicated in regulating the transcriptionstatus of certain genes, possibly compensating for alterations atthemRNA stability level (28).We therefore assessed transcriptionby detecting the unspliced mRNA primary transcripts of inflam-matory mRNAs in WT and TTPDM TAMs. Similar to total mRNAamounts we detected no significant changes between control andTTPDM cells (Fig. 4B). Furthermore, focusing on the TAM subsetswhere TTP expression is highest, we also failed to detect significanteffects on total amounts of normally unstable TTP targets with theexception of the TNF mRNA, which is known to be affected byTTP-dependent mRNA decay even under conditions where TTPfunction is limited (Fig. 4C; ref. 15).Whenwe estimated the decayrates of TTP targets in control or TTPDMTAMs,we found significanteffects on stability only for TNF and KC, the AU-rich mRNAs thatshow the highest effects of TTP activity inmodel systems (Fig. 4D;ref. 15). However, the data in Fig. 4A–C indicate effects of TTP onmRNAstabilityweremarginal, as the total quantity of TNF andKCmRNAs were equivalent between control and TTPDM TAMs.Therefore, TTP controls chemokine and cytokine production ata level above transcription ormRNA stability. Thus, tumormacro-phages had high TTP protein, and TTP was required to inhibitinflammatory cytokine and chemokine production, but not theircognate mRNA amounts or stability.

p38a is constitutively activated in NRIIf TTP is highly expressed in chronic inflammation, why is AU-

rich mRNA degradation inefficient? To begin to answer thisquestion, we noted TTP activity toward its AU-rich mRNA sub-strates is negatively regulated by phosphorylation on specificserine and threonine residues (9). Phosphorylation of TTP could,therefore, account for why AU-rich mRNA quantity and stabilitywere unaffected in macrophages. To test this, we assessed pTTP inTAMs and observed a characteristic "smear" of TTP protein similarto that found in LPS-stimulated BMDMs (Fig. 5A; ref. 8). In thelatter, pTTP increases to stabilize inflammatory cytokine andchemokine mRNAs, enhancing their production, after whichactive (unphosphorylated) TTP rises to eliminate AU-richmRNAsto return macrophages to a "ground" state (7, 10, 15). In accor-dance with high amounts of pTTP associated with inactive TTP-dependent mRNA decay, the stability of the TNF mRNA wasincreased in TAMs isolated from different tumor models

(Continued.) Values from individual mice combined from three experiments are shown, with the black line representing the mean; error bars, SEM. F, qRT-PCRanalysis of TTP and TTP family members in EG7 TAMs from WT and p38aDH mice. Expression values from individual mice (n � 14) were normalized to GAPDHand the correspondingmeanWT from three independent experiments. Mean, black line; error bars, SEM. H, mRNA decaymeasured inWT and TTPDM EG7 TAMswithand without SB treatment as in G. Values of individual mice are depicted as the percentage of mRNA stability after SB treatment relative to thecorresponding stability in untreated WT or TTPDM TAMs. Values were normalized to GAPDH and the corresponding untreated TAM sample (n � 9) and werecombined from two experiments. Black line, mean; error bars, SEM.






compared with resting BMDMs where TTP activity is known to behigh (Fig. 5B; ref. 15).

TTP inhibitionwas reported to bemediated byp38aMAPK, thepredominant MAPK in macrophages, and to be blocked byphosphatases, which act on p38a, and thus indirectly promoteTTP function (26, 29). We therefore investigated how the p38a–TTP cycle works in chronic inflammation. First, we observedoverall high p-p38a in tumor-derived macrophages despite vary-ing degrees of total p38a (Fig. 5C). p38a activation was consti-tutive and not a transient effect from the isolation procedure aswecould still detect strong p38a phosphorylation in TAMs rested ontissue culture dishes for 3 hours (Fig. 5D). Second, when wegenetically eliminated p38a in macrophages with the LysM-Credeleter(p38aDM) as previously reported (30), we observed neg-ligible depletion of p38a amounts, arguing conclusions drawnusing this deleter system are difficult to interpret (Fig. 5E and F).Therefore, for subsequent experiments, we adjusted our tacticsand used the pan-hematopoietic deleter Tie2-Cre (p38aDH),which produced a complete elimination of p38a (Fig. 5G andH).

p38a drives chronic inflammation posttranscriptionallyWhen we measured inflammatory cytokine and chemokine

mRNA amounts in p38a-deficient TAMs, we observed no dif-ferences between controls and knockouts for transcripts such asTNF, IL1a, and IL6 and only minor differences for other selectedtranscripts (Fig. 6A). In the same system, mRNA stability inp38a-deficient TAMs was reduced for TNF and IL1bmRNA only(Fig. 6B). However, despite unaltered mRNA amounts p38a-deficient cells had a substantial reduction in the amounts ofcertain inflammatory cytokines such as TNF and IL1a, suggest-ing p38a is required for translation of inflammatory mediators(Fig. 6C, Supplementary Fig. S1). To exclude the possibility thatthe detected alterations are due to p38a deletion in nonmyeloidcells caused by the use of Tie2-Cre, we performed mixed radi-ation chimera experiments with 1:1 mixtures of control andp38aDH bone marrow. In chimeric mice only p38a-deficientmacrophages had reduced cytokine production, confirmingp38a primarily acts in a macrophage-autonomous way in ourchronic inflammation models (Fig. 6D). Returning to thep38aDH mice, we found the reduction in inflammatory cyto-kines made by p38a-deficient TAMs correlated with an increasein overall tumor size; the opposite of the phenotype observed inthe TTPDM mice (Fig. 6E compared with Fig. 3B).

As mentioned above, the mRNA stability of several TTP targettranscripts was unaffected by p38a deletion (Fig. 6A and B).However, in accordance with in vitro studies (8), expression ofTTP as well as the TTP-related proteins ZFP36L1 and ZFP36L2was p38a dependent (Fig. 6F). Given a straightforward geneticmeans to address the role of p38a in regulating TTP activity wasnegated by the p38a dependence of TTP expression, we used apharmacologic approach to block p38a. When we blockedp38a activity using the kinase inhibitor SB203580, we foundAU-rich mRNAs were rapidly degraded (Fig. 6G). In TTP-defi-cient cells SB203580 treatment only partially increased mRNAdecay. For instance, in WT TAMs, SB203580 treatment reducedTNF mRNA stability to approximately 43% of untreated TAMscompared with 64% in TTPDM TAMs, suggesting that p38aregulates mRNA stability in a TTP-dependent and -independentmanner (Fig. 6H).Two conclusions can be drawn from theseexperiments. First, TTP-dependent mRNA decay in TAMs isblocked by p38a, consistent with previous data generated from

in vitro systems. However, TTP is still able to limit proteinproduction of target mRNAs, thereby counteracting inflamma-tion. Thus, in tumor inflammation, TTP's mRNA-degradingfunction is negated by p38a, but TTP is still required to temperinflammatory mediator output. Second, p38a is a driver ofchronic inflammation at the posttranscriptional level. Our datasuggest that p38a is constitutively activated in TAMs andresistant to the known negative feedback mechanismsdescribed for acute inflammatory responses (12, 31, 32).

DiscussionGenetic experiments have established the basic parameters

of TNF production in scenarios where the inflammatory signalis limited. First, p38a cooperates within the TLR signalingpathway to initiate Tnf expression and TNF production (33). Inthe subsequent resolving phase, a step-wise process cooperatesto extinguish TNF production once TLR signaling tapers: DUSPproteins dephosphorylate p38a; the decreased p38a activityreleases TTP inhibition (induced by TLR signaling) to degradethe TNF mRNA and IL10 (induced by TLR signaling) amplifiesTTP expression because Zfp36 is a STAT3-dependent target ofIL10 signaling (7, 12, 31, 32). Using conditional knockoutapproaches to minimize the effects of systemic inflammation,we found that in TAMs none of the step-by-step processes inTNF regulation occurs. Instead, the tumor microenvironmentenforces constitutive p38a activation, leading to blockade ofTTP activity and abatement of TNF mRNA removal.

TTP is generally considered to primarily control AU-richmRNAstability (34). Although TTP also contributes to blocking trans-lation of some mRNAs (35), our experiments suggest these twofunctions are differentially regulated within the tumor microen-vironment. We found mRNA stability and total amounts of TTPtarget mRNAs were marginally affected in the absence of TTP.These results contrast with in vitro studieswhere TTP influences thestability and amounts of hundreds of AU-rich mRNAs (15, 27).However, the absence of TTP caused inflammatory proteinamounts to increase, leading to increased death in the tumormicroenvironment and decreased tumor volume. Therefore, inTAMs, we suggest that TTP mainly regulates steps beyond mRNAdecay such as translation. A further implication of our studies isthe two functionsof TTP canbe separatedby the inhibitory actionsof chronic p38a activity: TTP expression is constitutively high inTAMs and its effects on mRNA stability are blocked by p38aphosphorylation. However, in contrast with in vitro studies ofacute inflammation where p38a blocks both TTP-dependentmRNA decay and translation (35), in TAMs TTP can retain itsinhibitory activity on cytokine production. Our results, therefore,increase the understanding of gene and protein regulation incomplex environments.

Our data indicate that p38a is constitutively active during NRIand drives the production of inflammatory cytokines at theposttranscriptional level. What signals lead to p38a activationin TAMs? Multiple NRI models have shown endogenous "alar-mins" drive TLR and other inflammatory signaling. Alarminsinclude S100a8/a9, IL1a and HMGB1. Release of any of thesecan provoke TLR/IL1R signaling, leading to Tnf expression (36).However, a consequence of ongoing TLR/IL1R is activation ofp38a phosphorylation and subsequent TTP deactivation, leadingto further TNF production and potential for additive TNFRsignaling to perpetuate the overall response.


Kratochvill et al.




Our data implicate p38a activity as the nexus of inflammatorysignaling in TAMs, regardless of what occurs downstream in thepathway. As long as p38a is active, all the subsequent stepsleading to signaling abatement cannot occur in a step-wise man-ner. When we targeted p38a activity with the kinase inhibitorSB203580, inflammatorymRNAswere greatly reduced. Therefore,p38a is a central checkpoint in chronic inflammation. Given NRIinflammation occurs in virtually all chronic diseases, our resultspoint to targeting two aspects of the disease: First, the incitingentity must be reduced, modified, or eliminated, coincident withtargeting upstream inflammatory signaling at the level of p38a.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: F. Kratochvill, P.J. MurrayDevelopment of methodology: F. KratochvillAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.E. Qualls, L.-A. Van De VeldeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Kratochvill, P.J. MurrayWriting, review, and/or revision of the manuscript: F. Kratochvill, N. Gratz,J.E. Qualls, P.J. Murray

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. Kratochvill, L.-A. Van De Velde, H. Chi,P. KovarikStudy supervision: P.J. MurrayOther (performed experiments): N. Gratz

AcknowledgmentsThe authors thank Perry J. Blackshear for generously providing the TTPKO and

Michael A. Dyer for the Th-MYCN transgenic and Rbflox/flox;p53flox/flox;Osx-Creþmouse stains. The authors thank Martine F. Roussel for providing theglioma tumor model as well as Lidija Barbaric, Parker Ingle, Greig Lennon, andRichard Cross for technical assistance and Derek Gilroy and Roland Lang fordiscussion and advice.

Grant SupportF. Kratochvill is supported by the Austrian Science fund (J3309-B19). This

work was supported by The Hartwell Foundation (P.J. Murray), Alex's Lemon-ade Stand Foundation (P.J. Murray), NIH grants CA189990 (P.J. Murray),CA138064 (J.E. Qualls), NCI grant P30 CA21765, and the American LebaneseSyrian Associated Charities.

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked advertise-ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 22, 2015; revised April 21, 2015; accepted May 8, 2015;published OnlineFirst July 16, 2015.

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2015;75:3054-3064. Published OnlineFirst July 16, 2015.Cancer Res Franz Kratochvill, Nina Gratz, Joseph E. Qualls, et al. Manner

Independent−Tumor-Associated Macrophages in an mRNA Decay Tristetraprolin Limits Inflammatory Cytokine Production in

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