IRAK1 Drives Intestinal Inflammation by Promoting the Generation of ...

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Cells with Optimal Gut-Homing CapacityPromoting the Generation of Effector Th IRAK1 Drives Intestinal Inflammation by

and Anne B. KrugReindlMarkota, Tobias Straub, Hans-Anton Lehr, Wolfgang

Alexander F. Heiseke, Benjamin H. Jeuk, Anamarija

ol.1501874http://www.jimmunol.org/content/early/2015/11/11/jimmun

published online 11 November 2015J Immunol 

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The Journal of Immunology

IRAK1 Drives Intestinal Inflammation by Promoting theGeneration of Effector Th Cells with Optimal Gut-HomingCapacity

Alexander F. Heiseke,* Benjamin H. Jeuk,* Anamarija Markota,* Tobias Straub,†

Hans-Anton Lehr,‡ Wolfgang Reindl,x and Anne B. Krug*

IL-1R–associated kinase (IRAK) 1 is an important component of the IL-1R and TLR signaling pathways, which influence Th cell

differentiation. In this study, we show that IRAK1 promotes Th17 development by mediating IL-1b–induced upregulation of IL-23R

and subsequent STAT3 phosphorylation, thus enabling sustained IL-17 production. Moreover, we show that IRAK1 signaling fosters

Th1 differentiation by mediating T-bet induction and counteracts regulatory T cell generation. Cotransfer experiments revealed that

Irak1-deficient CD4+ T cells have a cell-intrinsic defect in generating Th1 and Th17 cells under inflammatory conditions in spleen,

mesenteric lymph nodes, and colon tissue. Furthermore, IRAK1 expression in T cells was shown to be essential for T cell accumu-

lation in the inflamed intestine and mesenteric lymph nodes. Transcriptome analysis ex vivo revealed that IRAK1 promotes T cell

activation and induction of gut-homing molecules in a cell-intrinsic manner. Accordingly, Irak1-deficient T cells failed to upregulate

surface expression of a4b7 integrin after transfer into Rag12/2 mice, and their ability to induce colitis was greatly impaired. Lack of

IRAK1 in recipient mice provided additional protection from colitis. Therefore, IRAK1 plays an important role in intestinal

inflammation by mediating T cell activation, differentiation, and accumulation in the gut. Thus, IRAK1 is a promising novel target

for therapy of inflammatory bowel diseases. The Journal of Immunology, 2015, 195: 000–000.

Effector Th cells producing inflammatory cytokines play animportant role in inducing and maintaining inflammatoryresponses. Under the influence of environmental factors and

the local cytokine milieu, naive CD4+ T cells differentiate intoproinflammatory IFN-g–producing Th1 cells and IL-17–producing

Th17 cells or into Foxp3+ regulatory T cells (Tregs), which counteracteffector Th1 cell responses. The predominance of proinflammatoryeffector Th cells and their selective tissue recruitment are criticalfactors in autoimmune and inflammatory diseases. In inflammatorybowel diseases (IBD) defects in the intestinal barrier function lead toincreased exposure of immune cells to intestinal bacteria, which aresensed by TLRs and other pattern recognition receptors (1). AlthoughTLRs can have protective functions in epithelial cells (2), sustainedactivation of TLRs in intestinal immune cells leads to aberrant innateand adaptive immune responses and intestinal inflammation (3). Ac-tivation of TLRs (especially TLR2 and TLR4) in CD4+ T cells di-rectly modulates Th cell differentiation and effector function (4).Similarly, CD4+ T cells directly respond to the early proinflammatorycytokine IL-1b, which is important for Th17 cell differentiation (5)and maintenance (6). In the murine T cell transfer model of colitis,which is characterized by accumulation of Th1 and Th17 cells in thecolon, IL-1R signaling in CD4+ T cells was shown to promote Th17cell accumulation in the colon and colitis development (7).The IL-1R–associated kinases (IRAK) regulate the expression of

inflammatory genes in response to TLR ligands or IL-1 family mem-bers. The adaptor protein MyD88 is recruited to TLRs and IL-1Rupon activation, forms oligomers, and binds to IRAK4 via the deathdomain. IRAK4 associates with IRAK1 and phosphorylates IRAK1.Autophosphorylation of IRAK1 is a critical step, which allows bindingof TNFR-associated factor 6 and detachment from the receptor com-plex. Further signaling events lead to the activation of transcriptionfactors, including NF-kB and IFN regulatory factors (8, 9).Whereas Irak4-deficient mice and humans are highly susceptible

to specific bacterial infections (10), Irak1-deficient mice mountsufficient immune responses (11, 12). Furthermore, Irak1 deficiencyhas not been described in immunodeficient human patients, sug-gesting that IRAK1 would be a safer therapy target than IRAK4.Irak1-deficient mice are less susceptible to systemic autoimmunityin a congenic lupus model (13) and are resistant against experi-mental autoimmune encephalomyelitis (14, 15), suggesting that

*Institute for Immunology, Biomedical Center Munich, Ludwig Maximilians Univer-sity, D-82152 Planegg-Martinsried, Germany; †Bioinformatics Core Unit, BiomedicalCenter Munich, Ludwig Maximilians University, D-82152 Planegg-Martinsried, Ger-many; ‡Institut f€ur Pathologie, Medizin Campus Bodensee, Friedrichshafen, D-88048Friedrichshafen, Germany; and xKlinikum Mannheim II, Medizinische Klinik, D-68167Mannheim, Germany

ORCIDs: 0000-0002-9071-7464 (B.H.J.); 0000-0002-6982-8506 (W.R.).

Received for publication August 20, 2015. Accepted for publication October 13,2015.

This work was supported by German Research Foundation Grants KR2199/3-2,KR2199/6-1, KR2199/9-1, SFB1054/TPA06, and GRK1482 (to A.B.K., A.F.H.,B.H.J., and A.M.) and by the Adele Hartmann Program of the Ludwig MaximiliansUniversity excellence initiative (to A.B.K., A.F.H., B.H.J., and A.M.).

A.F.H. designed and performed experiments, analyzed and interpreted data, andwrote the manuscript; B.H.J. designed and performed experiments and analyzedand interpreted data; A.M. performed experiments and analyzed data; T.S. analyzedmicroarray data; H.-A.L. performed histological scoring, analyzed and interpreteddata, and critically revised the manuscript; W.R. contributed to the study concept anddesign and critically revised the manuscript; and A.B.K. obtained funding, devisedthe study concept, designed experiments, interpreted data, and wrote the manuscript.

The microarray data presented in this article have been submitted to the National Centerfor Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE73875.

Address correspondence and reprint requests to Prof. Anne B. Krug, Institute forImmunology, Biomedical Center Munich, Ludwig Maximilians University,Großhaderner Strasse 9, D-82152 Planegg-Martinsried, Germany. E-mail address:[email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: IBD, inflammatory bowel disease; IEL, intraepi-thelial leukocyte; IRAK, IL-1R–associated kinase; ko, knockout; LPL, lamina pro-pria leukocyte; MLN, mesenteric lymph node; mTOR, mammalian target ofrapamycin; RA, retinoic acid; ROR, RA-receptor–related orphan receptor; Treg,regulatory T cell; wt, wild-type.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501874

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IRAK1 plays a nonredundant role on autoimmune and inflammatorydiseases. In vitro studies showed that Th17 differentiation is impairedwhereas Treg generation is enhanced in Irak1-deficient CD4+ T cells(16). Interference with IRAK1 could therefore restore the balance ofTregs and effector Th cells, which would be beneficial in autoimmuneand inflammatory diseases such as multiple sclerosis and IBD.In this study, we sought to define the role of IRAK1 signaling in

T cells for Th cell differentiation and effector function in the intestineduring development of colitis. We show that IL-1b promotes Th17cell development by inducing IL-23R expression in an IRAK1-dependent manner. Furthermore, our results indicate that IRAK1signaling in T cells promotes the generation of proinflammatory IFN-g– and IL-17–producing Th cells in vivo and is required for theiraccumulation in the colon during colitis development induced byT cell transfer. Irak1-deficient T cells showed a lower expression ofgenes involved in T cell activation, differentiation, and gut homing,which correlated with a reduced capacity to induce experimentalcolitis in mice. Thus, IRAK1-mediated signaling in CD4+ T cellsdrives their differentiation into proinflammatory effector cells and iscritical for their accumulation in the inflamed intestine.

Materials and MethodsMice

Irak12/2 mice on the C57BL/6 background (at least 14 generations) wereprovided by James Thomas (University of Texas Southwestern MedicalSchool) (11). Irak12/2, C57BL/6, CD45.1, Rag12/2, and Irak12/2/Rag12/2

mice (C57BL/6 background) were bred in our facility under specificpathogen-free conditions. Experiments were approved by the local govern-ment authorities.

Media and reagents

Full medium consisted of RPMI 1640 (PromoCell) supplemented with 1%GlutaMAX-I (Invitrogen, Karlsruhe, Germany), 1% nonessential aminoacids, 1% penicillin/streptomycin, 1 mM sodium pyruvate solution (all fromPAA Laboratories), and 50 mM 2-ME (Sigma-Aldrich, Seelze, Germany).Cell restimulation for FACS analysis was carried out in full mediumcontaining 20 ng/ml PMA (Sigma-Aldrich), 1 mg/ml ionomycin (Sigma-Aldrich), 0.2% (v/v) GolgiPlug, and 0.14% (v/v) GolgiStop (both from BDBiosciences). Digestion medium consisted of RPMI 1640 (Invitrogen) con-taining 0.5 mg/ml collagenase D (II) and 0.1 mg/ml DNase I grade II (bothfrom Roche).

T cell transfer colitis and cotransfer experiments

Colitis was induced by transfer of 33 105 CD4+CD62L+ T cells into Rag12/2

mice as previously described (17). Body weight and consistency of stoolwere monitored throughout the experiments to detect colitis induction. Incotransfer experiments, equal numbers (3 3 105) of wild-type (wt, CD45.1)and Irak12/2 (CD45.2) CD4+CD62L+ T cells were injected i.p. into Rag12/2

mice. For microarray analysis, T cells were FACS sorted from mesentericlymph nodes using a BD FACSAria (BD Biosciences, Heidelberg, Germany).Two recipient mice were pooled in each microarray experiment.

Cell isolation and culture conditions

For T cell transfer experiments and in vitro assays, T cells were isolatedfrom splenocytes using the CD4+CD62L+ T cell isolation kit II (MiltenyiBiotec, Bergisch Gladbach, Germany). For Th0, Th1, Th2, Th17, and Tregdifferentiation, naive CD4+CD62L+ T cells were cultured with plate-boundanti-CD3ε (5 mg/ml) and anti-CD28 (5 mg/ml) for up to 120 h with thefollowing cytokine cocktails: Th0, no additional cytokines; Th1, IL-12p70(10 ng/ml; PeproTech); Th2, IL-4 (50 ng/ml; PeproTech); Th17, IL-1b (10ng/ml; PeproTech), IL-6 (50 ng/ml; PeproTech), IL-23 (20 ng/ml; R&DSystems), TGF-b (5 ng/ml; PeproTech); Tregs, IL-2 (200 U/ml; (Promo-Kine), TGF-b (5 ng/ml). For induction of gut-homing molecules, 10 nMall-trans retinoic acid (Sigma-Aldrich) was added. Mesenteric lymph node(MLN) cells and intraepithelial and lamina propria leukocytes (IELs andLPLs) from intestine were isolated as previously described (17).

Flow cytometry

Cells were stained with fluorescently Abs as described previously (17). Abswere obtained from eBioscience (CCR9, CD3ε, CD4, CD8a, CD49d,

Foxp3, IL-13, IL-17A, a4b7 integrin, retinoic acid [RA]-receptor–relatedorphan receptor [ROR] gt, T-bet), BD Biosciences (CD45.2, CD62L,CD103, IL-4, IFN-g, Ki67, STAT3pY705, AktpS473), and BioLegend(CD45.1). T cells were stimulated with PMA (20 ng/ml)/ionomycin (1 mg/ml)(Sigma-Aldrich) for 6 h in the presence of GolgiPlug (0.2% [v/v]) andGolgiStop (0.14% [v/v]) (BD Biosciences) and stained intracellularly asdescribed (17). For proliferation analysis, T cells were labeled with 5 mMCFSE for 10 min at 37˚C. For detection of phosphorylated proteins by FACS,T cells were cultured for 3 d under Th17 polarizing conditions, followed by2 h resting and restimulation for 30 min in Th17 polarizing conditions withor without IL-1b. STAT3 and Akt phosphorylation were measured using BDBiosciences Phosflow reagents. A Gallios (Beckman Coulter, Krefeld, Ger-many) or FACSCanto flow cytometer (BD Biosciences) and FlowJo software(Tree Star, Stanford, CA) were used for analysis.

Cell sorting

Wt and Irak12/2 T cells were sorted from MLNs as CD45.1+CD45.22 andCD45.12CD45.2+CD3+CD4+ T cells 14 d after cotransfer into Rag12/2

mice using the FACSAria III (BD Biosciences). Purity was .99.5%.

RNA isolation, real-time PCR, and microarray

RNAwas isolated using TRIzol (Invitrogen) according to the manufacturer’sprotocol. Total RNA was reverse transcribed to cDNA using SuperScript III(Invitrogen) according to the manufacturer’s protocol. Quantitative real-timePCR was performed using the StepOnePlus instrument and TaqMan primerand probe sets (Life Technologies). Hypoxanthine phosphoribosyltransferase1 was used for normalization. Quantitative analysis was performed using the22DDCT method.

For microarray analysis, an Affymetrix GeneChip (mouse gene 2.0 STarrays) was used and performed by the Kompetenzzentrum FluoreszenteBioanalytik (Universitat Regensburg, Regensburg, Germany). Microarraydata were processed using R/bioconductor (http://www.bioconductor.org).We extracted gene expression levels applying the robust multi-array av-erage procedure including between-array quantile normalization as pro-vided by the oligo package. Control probesets were omitted from furtheranalyses. We also excluded probesets with 0 variance across all experi-ments and those with a median expression level of,4.5 in both conditions.In case of many probesets interrogating one gene, we only kept the probesetwith the highest variation across all experiments. We subsequently per-formed differential expression analysis using the limma package based ona linear model comprising the genotype and the experimental batch. Differ-entially regulated genes were called by computing the local false discoveryrate (locfdr package) on the moderated t statistic with a threshold of 0.2.Microarray probeset mappings were based on GenBank data with a source datestamp of March 13, 2014. Microarray data were deposited in the NationalCenter for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) data repository (accession no. GSE73875).

Histology

Colons were fixed as “Swiss rolls” in 4% paraformaldehyde and embeddedin paraffin. For histological scoring, sections of 3 mm were stained withH&E. Histological scoring was performed in a blinded fashion by thepathologist as described (17).

ELISA

IL-17A ELISA (eBioscience) was performed according to the manufac-turer’s protocol.

Statistical analysis

Data are shown as means 6 SEM. Student t test or a one-way ANOVAfollowed by appropriate post hoc testing were performed using SigmaStat(Systat Software, Erkrath, Germany). A p value ,0.05 was considered toindicate statistically significant differences.

ResultsIRAK1 regulates Th17 differentiation by mediating IL-23Rexpression in response to IL-1b

IRAK1 modulates signaling downstream of TLRs and IL-1R anddirectly influences Th cell differentiation. In accordance with previousdata (6, 16), we found that lower frequencies of IL-17–producing cellswere generated from Irak1-deficient (knockout [ko]) CD4+ T cellscompared with wt T cells cultured under Th17 conditions, includingIL-1b and IL-23 (12.9 6 0.8 [wt] versus 5.7 6 0.5 [ko]; Fig. 1A),

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correlating with a lower IL-17A concentration in the supernatant(Supplemental Fig. 1A). Whereas proliferation and survival were notaffected under these conditions (Supplemental Fig. 1B, 1C), lower Akt(S473) phosphorylation was detected in Irak12/2 T cells (Fig. 1B),indicating lower activity of the Akt/mammalian target of rapamycin(mTOR) pathway, which was shown to promote Th17 induction in thepresence of IL-1b (6). In the absence of IL-1b, the frequency of wtIL-17+ T cells was reduced to similar levels as in Irak12/2 T cellscultured with or without IL-1b (Fig. 1C). Thus, the reduced IL-17production by Irak12/2 T cells is mostly due to their inability to re-spond to IL-1b. As a result of the abrogated IL-1b signaling, Irak12/2

T cells had il-23r mRNA expression levels comparable to those of wtT cells cultured without IL-1b (Fig. 1D) whereas RORgt expression inCD4+ T cells was induced by IL-1b independently of IRAK1 (Fig.1E, Supplemental Fig. 1D). STAT3 phosphorylation correlated withil-23r expression and was dependent on IL-1b and IRAK1 signaling(Fig. 1F). Taken together, these data demonstrate that IRAK1 mediatesupregulation of IL-23R in response to IL-1b. Thus, developing Th17cells are more responsive to IL-23, leading to enhanced STAT3phosphorylation, which in the presence of RORgt promotes sustainedIL-17 production.

IRAK1 supports Th1 and inhibits Treg generation but isdispensable for Th2 development

Besides Th17 cells, Th1, Th2, and Tregs are crucial mediators ofinflammation and immune homeostasis. When testing a possible

influence of IRAK1 on Th cell differentiation, we observed thatCD4+ T cells lacking IRAK1 also had a lower capacity to generateIFN-g–producing Th1 cells in vitro (Fig. 1G), which was accom-panied by reduced induction of T-bet (Fig. 1H), explaining the re-duced IFN-g production. Confirming a previous report (16), wealso detected an important role of IRAK1 for Treg generation.Lack of IRAK1 signaling in T cells reduced the frequency ofin vitro–generated Tregs (Fig. 1I). Finally, we tested whetherIRAK1 is also important for Th2 development. In contrast to itsrole for Th1/Th17 and Treg development, we did not detect animpact of IRAK1 on Th2 differentiation (Fig. 1J, SupplementalFig. 1E). These results indicate that Irak1 deficiency does nothave a general impact on Th cell development but rather has spe-cific functions for individual subsets.

Steady-state in vivo T cell development is not affected byIRAK1 deficiency

To test whether the effects of IRAK1 seen in in vitro differentiationassays have an impact on in vivo T cell development in the steady-state, we analyzed the frequencies of T cells, Tregs, and Th1/Th17cells in Irak12/2 mice and littermate controls (Fig. 2A). Neitherdifferences in overall CD3ε+/CD4+ T cell frequencies in spleen,MLNs, thymus, Peyer’s patches, colonic LPLs, and small intes-tinal LPLs (Fig. 2B) nor major differences in Treg frequencies(Fig. 2C) were detected in these tissues. With respect to Th1/Th17development, a trend toward lower frequencies of all three subsets

FIGURE 1. IRAK1 signaling in T cells promotes Th1/Th17 development. (A) Wt and Irak12/2 CD4+CD62L+ T cells were cultured under Th17

conditions. IL-17+CD4+ T cells were detected by intracellular IL-17A staining. Exemplary dot plots (72 h time point) and the time course of Th17 induction

(n = 3 independent experiments; mean 6 SEM; *p , 0.05, t test) are displayed. Numbers in the FACS plots indicate percentage of gated cells. (B) Akt

(S473) phosphorylation after restimulation in wt or Irak12/2 T cells cultured under Th17 conditions for 72 h (one representative of three independent

experiments). (C) Percentage of IL-17+ T cells cultured under Th17 condition with or without IL-1b for 72 h (n = 3 independent experiments; mean 6SEM; *p , 0.05, one-way ANOVA followed by a Bonferroni test). (D) Expression levels of il-23r mRNA on T cells cultured under Th17 condition with or

without IL-1b for 72 h (n = 3 independent experiments; mean6 SEM; *p, 0.05, one-way ANOVA followed by a Bonferroni test). (E) Induction of RORgt

in T cells cultured under Th17 condition with or without IL-1b for 72 h (one representative of three independent experiments). (F) STAT3 (pY705)

phosphorylation after restimulation in wt or Irak12/2 T cells cultured under Th17 conditions for 72 h (one representative of three independent experiments).

(G) Wt and Irak12/2 CD4+CD62L+ T cells were cultured under Th1 conditions. IFN-g+CD4+ T cells were detected by intracellular IFN-g staining.

Exemplary dot plots (96 h time point) and frequency (bar graph) of IFN-g+ T cells (n = 3 independent experiments; mean 6 SEM; *p , 0.05, t test) are

displayed. (H) Induction of T-bet in T cells treated as in (G). Left, Overlay histograms with mean fluorescence indicated. Right, Percentage of T-bet+CD4+

T cells (n = 3 independent experiments; mean 6 SEM; *p , 0.05, t test). (I) Wt and Irak12/2 CD4+CD62L+ T cells were cultured under Treg conditions.

Exemplary dot plots with the percentage of Tregs indicated (120 h time point) and the mean frequency of Foxp3+ T cells are displayed (n = 3 independent

experiments; mean6 SEM; *p, 0.05, t test). (J) Wt and Irak12/2 CD4+CD62L+ T cells were cultured under Th2 conditions. Exemplary dot plots with the

percentage of IL-4+ and/or IL-13+ cells are indicated (96 h time point).

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(IL-17 single-positive, IFN-g single-positive, IFN-g/IL-17 double-positive CD4+ T cells) was observed in Irak1-deficient mice insmall intestinal LPL fractions, yet this effect did not prove to bestatistically significant and no differences in the other organscould be detected.

Decreased Th1/Th17 development in Irak1-deficient T cells isdue to intrinsic effects of IRAK1 that also induce accumulationin the colon

To investigate the influence of IRAK1 signaling on Th1/Th17cell differentiation in an inflammatory setting, we cotransferredequal numbers of naive CD4+/CD62L+ T cells isolated from wt(CD45.1) and Irak12 /2 (CD45.2) mice into Rag12 /2 recipientmice (Fig. 3A). By cotransferring the T cells of both genotypesinto the same recipient, we circumvent possible effects of differentmicrobiota or different inflammatory microenvironments. Analy-sis of cytokine production upon restimulation 14 d after transferdemonstrated that IRAK1 promotes differentiation towardIFN-g– and/or IL-17–producing T cells in vivo in a cell-intrinsicfashion (Fig. 3A, 3B).Induction of gut-homing capacity is crucial for developing effector

Th cells to trigger intestinal inflammation. To compare the ability ofwt and Irak12/2 T cells to accumulate in the colon in vivo, weassessed the percentages of cotransferred wt and ko T cells in colon,MLNs, and spleen at different time points after transfer. The 1:1ratio of wt and ko T cells was maintained in spleen and MLNs ondays 3 and 7 after transfer, when T cells were slowly expanding andhad not yet entered the colon in significant numbers (SupplementalFig. 2A). After 14 d when mice had lost up to 20% of their bodyweight due to colitis, a significantly higher percentage of wt thanIrak12/2 T cells was found in colon LPL (mean 6 SEM, 72 64 [wt] versus 24 6 4% [ko], p = 5.2 3 1026) and IEL (75 6 4 [wt]versus 236 4% [ko], p = 3.93 1026) fractions as well as in MLNs(706 3 [wt] versus 276 3% [ko], p = 8.63 1026). This difference

was less apparent in the spleen (61 6 5 [wt] versus 35 6 5% [ko],p = 0.02) (Fig. 3C). Thus, IRAK1 plays a cell-intrinsic role forCD4+ T cell accumulation in the inflamed colon and MLNs.To further investigate the consequence of IRAK1 deficiency on

CD4+ T cell function in vivo, we compared the gene expressionpattern of cotransferred wt and Irak12/2 T cells sorted fromMLNs 14 d after transfer. Microarray analysis of wt and Irak12/2

T cells isolated from the MLNs of cotransferred Rag12/2 recip-ient mice (Fig. 3D) showed that mRNA expression levels of genesinvolved in T cell gut homing or retention (Itga1, Cd69), Th1/Th17 development (Klrd1, Acss2, Klrk1, Tnf, Stard5), and T cellactivation (Nr4a1, Fasl, Ier3, Ptpn4) are reduced in Irak12/2

T cells compared with wt T cells (Fig. 3E, Supplemental Table 1).Differential expression of genes involved in proliferation andapoptosis was not observed (unpublished data), suggesting that thereduced accumulation of Irak12/2 T cells in the inflamed colonand MLNs is rather due to impaired gut homing and retention.

IRAK1 signaling is required for efficient induction ofgut-homing integrin a4b7 and CD69 on CD4+ T cells

T cell homing to the intestine requires expression of a4b7 integrin,which binds to mucosal vascular adressin MAdCAM1 on highendothelial venules of MLNs, Peyer’s patches, and postcapillaryvenules of the intestine (18). Blockade or genetic deficiency ofa4b7 integrin prevents recruitment of T cells to the colon andcolitis development after transfer into Rag12/2 mice (19, 20). Wetherefore analyzed expression of a4b7 integrin and other mole-cules involved in gut homing and retention in cotransferred wt andIrak12/2 T cells 14 d after transfer (Fig. 4A, Supplemental Fig.2B). Whereas a clear induction of a4b7 integrin was seen in wtT cells isolated from MLNs and spleen, Irak12/2 T cells fromthese organs expressed much lower levels of a4b7 integrin. In thecolon, a4b7 integrin expression was reduced compared withMLNs and spleen as expected, but it was still found to be higher inwt than in Irak12/2 T cells. CD103 (integrin aE), CD49d (integrina4), and CCR9, which is critical for T cell homing to the smallintestine but not the colon, were similarly expressed in wt andIrak12/2 T cells. Both Irak12/2 and wt T cells stained positivelyfor Ki67 at a similarly high percentage, demonstrating that lack ofIRAK1 does not have a major impact on the proliferation of CD4+

T cells (Fig. 4A). Thus, the reduced capacity of Irak1-deficientT cells to accumulate in the colon during colitis is most likely dueto impaired a4b7 integrin–dependent homing to the inflamed co-lon. Confirming results of our microarray analysis CD69 wasexpressed at higher levels on the surface of wt than Irak12/2

T cells in the colon (Fig. 4A). Given the role of CD69 for retentionof memory T cells in the intestine in chronic infection (21), higherCD69 expression may contribute to preferential accumulation ofwt T cells in the colon.T cell intestinal homing capacity is imprinted by RA produced

by MLN dendritic cells. To investigate the influence of IRAK1signaling on the induction of gut-homing molecules, wt andIrak12/2 CD4+CD62L+ T cells were cultured under Th1, Th17,Treg, or neutral conditions (Th0) with or without IL-1b in thepresence or absence of RA (Fig. 4B). Induction of a4b7 integrinexpression by RAwas impaired in Irak12/2 T cells cultured underTh17 conditions irrespective of IL-1b addition to the culture.Irak12/2 T cells expressed lower levels of a4b7 integrin whencultured under Treg conditions, but they upregulated a4b7 integrinto a similar extent as did wt T cells when RA was included in theTreg culture. Expression of CCR9 and CD103 was not affected byIrak1 deficiency. We conclude that efficient induction of a4b7

integrin in developing Th17 cells by RA requires intact IRAK1signaling.

FIGURE 2. Normal T cell development in resting Irak1-deficient mice. (A)

Gating strategy to determine T cell frequencies and Foxp3 and Th1/Th17

development. (B) Frequencies of CD3ε+/CD4+ T cells in respective tissues.

(C) Frequencies of Fopx3+ T cells in respective tissues. (D) Frequencies of

IFN-g single-positive, IL-17 single–positive, and IFN-g/IL-17 double-positive

T cells in respective tissues. For (B)–(D), n = 4 mice. cLPL, colonic LPL;

siLPL, small intestinal LPL.

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Irak1-deficient T cells fail to induce severe colitis

To investigate whether IRAK1 signaling in T cells is critical forcolitis induction, wt and Irak12/2 CD4+CD62L+ T cells weretransferred separately into Rag12/2 mice and colitis activity wasassessed clinically and histologically. We also investigated thecontribution of IRAK1 signaling outside of the T cell compart-ment for colitis development by transferring wt or Irak12/2

T cells into Irak12/2/Rag12/2 recipients. Whereas Rag12/2

mice that received wt T cells had to be sacrificed early due tosevere colitis and weight loss, mice of the three other groups hada milder course of colitis and did not progress to severe colitisduring this time (Fig. 5A). Mice lacking IRAK1 and receivingIrak1-deficient T cells showed the mildest course of colitis in-duction (Fig. 5B). Histopathological signs of colitis, such asimmune cell infiltration and tissue destruction, were also lesspronounced in Rag12/2 recipients receiving Irak12/2 T cellsand in Irak12/2/Rag12/2 mice receiving either wt or Irak12/2

T cells, demonstrating a relevant role of IRAK1 for intestinalinflammation (Fig. 5C, 5D). FACS analysis of the transferredT cells showed that the reduced colitis activity was associatedwith reduced induction of Th1/Th17 cells in MLNs and spleen(Fig. 5E, 5F), consolidating the results found in cotransfer ex-periments. In contrast, the percentage of Foxp3+ Tregs in MLNsduring colitis was increased in the absence IRAK1 signaling(Supplemental Fig. 3). In summary, these results show thatblocking IRAK1 signaling has the potential to inhibit colitisinduction by interfering with effector Th cell differentiation andaccumulation in the intestine.

DiscussionThe generation of effector Th1 and Th17 cells depends on signalsderived from bacteria or proinflammatory cytokines such as IL-1b,

which directly influence CD4+ T cells via TLRs or cytokinereceptors. These signals promote Th1/Th17 cell differentiationwhile overriding the Treg induction program, which is dominant

in barrier organs such as the intestine in the steady-state, butsuppressed during intestinal inflammation. In this study, we in-vestigated the function of IRAK1, which is one of the critical

components of the TLR and IL-1R signaling pathway, and definedits T cell–intrinsic role for Th cell differentiation and its functionduring T cell–dependent intestinal inflammation.In this study, we found that Irak1-deficient CD4+ T cells had a

reduced ability to generate Th17 cells with sustained IL-17 pro-duction, which correlated with their inability to upregulate IL-23R

expression in response to IL-1b stimulation. Similar results wereobtained with Irak4-deficient T cells in an earlier report (15). Ithas been reported that IRAK1 directly phosphorylates STAT3 in

T cells cultured with TGF-b and IL-6 (16), yet we found that inthe presence of IL-23, IRAK1 signaling acts downstream of IL-1bto promote sustained IL-17 production. It was shown recently thatIL-1 additionally promotes STAT3 tyrosine phosphorylation in-

duced by IL-23 by repressing suppressor of cytokine signaling 3, anegative regulator of the STAT3 pathway (22). In line with ourfinding that IRAK1 signaling is not required for upregulation of

RORgt expression by IL-1b, it was shown that lack of MyD88 inCD4+ T cells impairs Th17 cell generation without affectingRORgt expression (23). Chang et al. (23) also demonstrated that

FIGURE 3. Irak1-deficient T cells show

reduced Th1/Th17 differentiation in vivo

and fail to accumulate in the intestine.

(A) Schematic representation of wt

(CD45.1) and Irak12/2 (CD45.2) CD4+

CD62L+ T cells cotransferred in equal

numbers into Rag12/2 recipient mice

with representative dot pots showing

Th1/Th17 analysis in the indicated organs.

(B) Frequencies of IFN-g single-positive,

IL-17 single-positive, and IFN-g/IL-17

double-positive T cells isolated from

mice cotransferred with wt (CD45.1)

and Irak12/2 (CD45.2) CD4+CD62L+

T cells 14 d after transfer (n = 12 mice

from three independent experiments with

four mice per group; mean 6 SEM; *p ,0.05, t test). (C) Frequencies of wt (CD45.1)

and Irak12/2 (CD45.2) T cells 14 d after

transfer in spleen, MLNs, and colon tissue

(n = 5 independent experiments with four

mice per group; mean 6 SEM; *p , 0.05,

t test). (D) Sorting of T cells from MLNs

14 d after T cell transfer for microarray

analysis. The percentages of gated cells are

indicated. (E) Heat map of genes with sig-

nificantly higher expression in wt than in

Irak12/2 T cells sorted from MLNs 14 d

after transfer (n = 4 individual experiments

with cells from two mice pooled in each).

Gene Expression Omnibus accession no.

GSE73875.

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the MyD88-mediated activation of the mTOR signaling pathwayby IL-1b promotes IL-23R induction and sustains IL-17 produc-tion. Indeed, we could show that full activation of the Akt/mTORpathway by IL-1b also requires IRAK1 signaling in CD4+ T cells.These results are in agreement with the findings of Gulen et al. (6)

who demonstrated that activation of the Akt/mTOR pathway byIL-1b promotes Th17 cell differentiation. We also observed areduction in the percentage of CD4+ T cells expressing T-bet andproducing IFN-g in Irak1-deficient T cells cultured under Th1polarizing conditions. These results suggest that IRAK1 may also

FIGURE 4. Decreased induction of gut-homing re-

ceptors on Irak12/2 T cells in MLNs and colon tissue.

(A) Wt (CD45.1) and Irak12/2 (CD45.2) CD4+/CD62L+

T cells were cotransferred in equal numbers into Rag12/2

recipient mice and analyzed 14 d after transfer. Overlay

histograms show the surface expression levels of the in-

dicated molecules and Ki67 staining in CD3ε+/CD4+ wt

and Irak12/2 T cells (gray line, wt; black line, ko; shaded,

unstained control). Results of one representative of two

experiments are shown. (B) CD4+/CD62L+ wt and

Irak12/2 T cells were cultured under Th0, Th1, Th17, or

Treg conditions with or without all-trans RA (ATRA) and

with or without IL-1b. The expression of gut-homing

receptors was analyzed by FACS. Bars represent the

percentages of cells positive for the depicted markers (n =

3 independent experiments; mean 6 SEM; *p , 0.05,

t test).

FIGURE 5. IRAK1 signaling promotes colitis devel-

opment in the T cell transfer model. (A) Body weight

development of Rag12/2 and Rag12/2/Irak12/2 recipient

mice upon transfer of wt or Irak12/2 CD4+/CD62L+

T cells (one of three independent experiments with four

to five mice in each group is displayed; mean 6 SEM;

*p , 0.05, one-way ANOVA followed by a Bonferroni

test). (B) Cumulative data of endpoint body weights of

mice from (A) (n = 3 independent experiments with

four to five mice per group; horizontal lines indicate

mean values; *p , 0.05, one-way ANOVA followed by

a Bonferroni test). (C) Representative H&E stainings of

colon tissue section of mice from (A). Original mag-

nification 310. (D) Cumulative histological colitis

score of mice from (A) (n = 3 independent experiments

with three to four mice per group; mean 6 SEM; *p .0.05, one-way ANOVA followed by a Bonferroni test).

(E) Representative dot plots of intracellular cytokine

stainings of MLN T cells of mice from (A) restimulated

with PMA/ionomycin. Percentages are indicated in the

quadrants. (F) Frequencies of IFN-g single-positive,

IL-17 single-positive, and IFN-g/IL-17 double-positive

CD4+ T cells isolated from MLN and spleen (n = 3

independent experiments with three to four mice in

each group; mean6 SEM; *p, 0.05, one-way ANOVA

followed by a Bonferroni test).

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be involved in signaling downstream of the IL-12 receptor. Apossible link could be the direct phosphorylation of STAT3 byIRAK1 in the nucleus (24) as STAT3 cooperates with STAT4 inIL-12R signaling (25).IRAK1 expression in T cells was also required for full differ-

entiation of proinflammatory Th1/Th17 effector cells in vivo. Incompetitive cotransfer experiments with Irak12/2 and wt T cells, wecould demonstrate a cell-intrinsic effect of IRAK1 signaling oneffector Th cell differentiation. Ahern et al. (26) reported a similareffect of IL-23R signaling on the generation of pathogenic IFN-g/IL-17 double–producing T cells during colitis. This is in agreementwith the close link between IRAK1 signaling and IL-23R signalingestablished in our in vitro experiments.We observed that induction of Foxp3+ Tregs in vitro by TGF-b

was more efficient in Irak1-deficient CD4+ T cells. This could bedue to increased activation of the Foxp3 promoter by NFAT/Smad3complexes in the absence of IRAK1 signaling, which was reported(16). Furthermore, reduced activation of the Akt/mTOR pathway inIrak12/2 CD4+ T cells in the presence of IL-1 could explain theenhanced expression of Foxp3.Competitive transfer experiments demonstrated a cell-intrinsic

requirement of IRAK1 in T cells for accumulation in the colonduring colitis development. This could be a cell-intrinsic effect inthe true sense or reflect that Irak12/2 T cells interact with APCs ina different manner than wt T cells, leading to reduced differenti-ation and accumulation in the inflamed colon.We found no evidence for impaired survival or proliferation of

CD4+ T cells lacking IRAK1 in our experiments in vitro andin vivo, suggesting that survival effects of IL-1R signals in T cells(7) may depend on other IRAKs. Global gene expression analysisof cotransferred wt and ko T cells from MLNs during colitisrevealed IRAK1-dependent expression of genes involved in T cellactivation, but also in memory T cell retention in the intestine,such as CD69 and integrin a1 (21). Although a4 and b7 integrinexpression did not show differential expression on mRNA level inthe microarray, a marked reduction in the expression of thegut-homing integrin a4b7 on the surface of transferred Irak12/2

T cells was observed during colitis. Effector T cells rely on a4b7

integrin for homing to the inflamed mucosa of the small and largeintestine, and blockade of a4b7 integrin selectively inhibits ac-cumulation of pathogenic effector T cells in the intestine (19, 20,27, 28). The importance of preventing T cell homing to the in-testine in the treatment of IBD was underscored by recent clinicaltrials demonstrating efficacy of the a4b7 integrin blocking Abvedolizumab in the therapy of ulcerative colitis (29). Our resultssuggest that Irak1-deficient T cells fail to accumulate in the colonduring colitis due to, at least in part, impaired upregulation ofa4b7 integrin expression. Indeed, a reduced induction of a4b7

integrin expression by RA was detected in Irak12/2 CD4+ T cellscultured under Th17 conditions including IL-1b.We found that Irak1-deficient T cells also expressed markedly

lower levels of CD69 than did wt T cells in the colon LPL and IELfractions after cotransfer. Induction of CD69 prevents lymphocyteegress from lymphoid organs by inhibiting the surface expression ofsphingosine-1-phosphate receptor (30). A subset of CD8+ memoryT cells generated in response to infection migrates to the intestine,where they become resident memory cells. Upon arrival in the in-testine, these cells downregulate a4b7 integrin expression and up-regulate aE integrin (CD103) and CD69 expression, which togetherallow their retention in the intestine (21, 31). Similarly, it was foundthat CD69 and CD103 are also involved in recruitment and reten-tion of pathogen-specific CD8+ T cells in the mucosa of the lung(32). We observed that the failure of Irak12/2 T cells to accumulatein the inflamed colon correlates with reduced CD69 upregulation.

Therefore, CD69 may also be involved in CD4+ T cell accumulationin the inflamed colon in this setting.In the T cell transfer colitis model, we dissected the role of

IRAK1 signaling in transferred T cells and in the recipient mice,which lacked T and B lymphocytes for colitis development. Theseexperiments demonstrated that IRAK1 expression in T cells isrequired for induction of severe colitis and that IRAK1 expressionoutside of the T cell compartment, for example in APCs, innatelymphoid cells, intestinal epithelial cells, and stroma cells (11, 33),additionally contributes to development of T cell–dependent co-litis. This was also suggested by reduced susceptibility of Irak1-deficient mice to acute DSS-induced colitis (Ref. 34 and unpublisheddata). Our study shows that IRAK1 signaling drives intestinal in-flammation by promoting the generation of proinflammatory ef-fector T cells with optimal gut-homing and retention capacity.These findings provide a rationale for considering IRAK1 as atherapy target in inflammatory bowel diseases.

AcknowledgmentsWe thank James Thomas for providing Irak1-deficient mice. We thank

Silvia Ahlig and Regina Dorin for excellent technical assistance. This

work is part of the doctoral thesis of B.H.J.

DisclosuresThe authors have no financial conflicts of interest.

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