IRAK1 Drives Intestinal Inflammation by
Promoting the Generation of Effector Th
Cells with Optimal Gut-Homing Capacity
This information is current as
of June 16, 2017.
Alexander F. Heiseke, Benjamin H. Jeuk, Anamarija
Markota, Tobias Straub, Hans-Anton Lehr, Wolfgang Reindl
and Anne B. Krug
J Immunol published online 11 November 2015
http://www.jimmunol.org/content/early/2015/11/11/jimmun
ol.1501874
http://www.jimmunol.org/content/suppl/2015/11/11/jimmunol.150187
4.DCSupplemental
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Supplementary
Material
Published November 11, 2015, doi:10.4049/jimmunol.1501874
The Journal of Immunology
IRAK1 Drives Intestinal Inflammation by Promoting the
Generation of Effector Th Cells with Optimal Gut-Homing
Capacity
Alexander F. Heiseke,* Benjamin H. Jeuk,* Anamarija Markota,* Tobias Straub,†
Hans-Anton Lehr,‡ Wolfgang Reindl,x and Anne B. Krug*
E
ffector Th cells producing inflammatory cytokines play an
important role in inducing and maintaining inflammatory
responses. Under the influence of environmental factors and
the local cytokine milieu, naive CD4+ T cells differentiate into
proinflammatory IFN-g–producing Th1 cells and IL-17–producing
*Institute for Immunology, Biomedical Center Munich, Ludwig Maximilians University, D-82152 Planegg-Martinsried, Germany; †Bioinformatics Core Unit, Biomedical
Center Munich, Ludwig Maximilians University, D-82152 Planegg-Martinsried, Gerur Pathologie, Medizin Campus Bodensee, Friedrichshafen, D-88048
many; ‡Institut f€
Friedrichshafen, Germany; and xKlinikum Mannheim II, Medizinische Klinik, D-68167
Mannheim, 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 Maximilians
University 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, and
wrote the manuscript; B.H.J. designed and performed experiments and analyzed
and interpreted data; A.M. performed experiments and analyzed data; T.S. analyzed
microarray data; H.-A.L. performed histological scoring, analyzed and interpreted
data, and critically revised the manuscript; W.R. contributed to the study concept and
design and critically revised the manuscript; and A.B.K. obtained funding, devised
the study concept, designed experiments, interpreted data, and wrote the manuscript.
The microarray data presented in this article have been submitted to the National Center
for 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 for
Immunology, 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, intraepithelial leukocyte; IRAK, IL-1R–associated kinase; ko, knockout; LPL, lamina propria leukocyte; MLN, mesenteric lymph node; mTOR, mammalian target of
rapamycin; RA, retinoic acid; ROR, RA-receptor–related orphan receptor; Treg,
regulatory T cell; wt, wild-type.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501874
Th17 cells or into Foxp3+ regulatory T cells (Tregs), which counteract
effector Th1 cell responses. The predominance of proinflammatory
effector Th cells and their selective tissue recruitment are critical
factors in autoimmune and inflammatory diseases. In inflammatory
bowel diseases (IBD) defects in the intestinal barrier function lead to
increased exposure of immune cells to intestinal bacteria, which are
sensed by TLRs and other pattern recognition receptors (1). Although
TLRs can have protective functions in epithelial cells (2), sustained
activation of TLRs in intestinal immune cells leads to aberrant innate
and adaptive immune responses and intestinal inflammation (3). Activation of TLRs (especially TLR2 and TLR4) in CD4+ T cells directly modulates Th cell differentiation and effector function (4).
Similarly, CD4+ T cells directly respond to the early proinflammatory
cytokine 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 the
colon, IL-1R signaling in CD4+ T cells was shown to promote Th17
cell 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 members. The adaptor protein MyD88 is recruited to TLRs and IL-1R
upon activation, forms oligomers, and binds to IRAK4 via the death
domain. IRAK4 associates with IRAK1 and phosphorylates IRAK1.
Autophosphorylation of IRAK1 is a critical step, which allows binding
of TNFR-associated factor 6 and detachment from the receptor complex. Further signaling events lead to the activation of transcription
factors, 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 mount
sufficient immune responses (11, 12). Furthermore, Irak1 deficiency
has not been described in immunodeficient human patients, suggesting that IRAK1 would be a safer therapy target than IRAK4.
Irak1-deficient mice are less susceptible to systemic autoimmunity
in a congenic lupus model (13) and are resistant against experimental autoimmune encephalomyelitis (14, 15), suggesting that
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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 accumulation 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.
2
Foxp3, IL-13, IL-17A, a4b7 integrin, retinoic acid [RA]-receptor–related
orphan 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]) and
GolgiStop (0.14% [v/v]) (BD Biosciences) and stained intracellularly as
described (17). For proliferation analysis, T cells were labeled with 5 mM
CFSE 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 by
2 h resting and restimulation for 30 min in Th17 polarizing conditions with
or without IL-1b. STAT3 and Akt phosphorylation were measured using BD
Biosciences Phosflow reagents. A Gallios (Beckman Coulter, Krefeld, Germany) 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 and
CD45.12CD45.2+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
Full medium consisted of RPMI 1640 (PromoCell) supplemented with 1%
GlutaMAX-I (Invitrogen, Karlsruhe, Germany), 1% nonessential amino
acids, 1% penicillin/streptomycin, 1 mM sodium pyruvate solution (all from
PAA Laboratories), and 50 mM 2-ME (Sigma-Aldrich, Seelze, Germany).
Cell restimulation for FACS analysis was carried out in full medium
containing 20 ng/ml PMA (Sigma-Aldrich), 1 mg/ml ionomycin (SigmaAldrich), 0.2% (v/v) GolgiPlug, and 0.14% (v/v) GolgiStop (both from BD
Biosciences). Digestion medium consisted of RPMI 1640 (Invitrogen) containing 0.5 mg/ml collagenase D (II) and 0.1 mg/ml DNase I grade II (both
from Roche).
RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s
protocol. Total RNA was reverse transcribed to cDNA using SuperScript III
(Invitrogen) according to the manufacturer’s protocol. Quantitative real-time
PCR was performed using the StepOnePlus instrument and TaqMan primer
and probe sets (Life Technologies). Hypoxanthine phosphoribosyltransferase
1 was used for normalization. Quantitative analysis was performed using the
22DDCT method.
For microarray analysis, an Affymetrix GeneChip (mouse gene 2.0 ST
arrays) was used and performed by the Kompetenzzentrum Fluoreszente
Bioanalytik (Universität Regensburg, Regensburg, Germany). Microarray
data were processed using R/bioconductor (http://www.bioconductor.org).
We extracted gene expression levels applying the robust multi-array average procedure including between-array quantile normalization as provided by the oligo package. Control probesets were omitted from further
analyses. We also excluded probesets with 0 variance across all experiments 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 probeset
with the highest variation across all experiments. We subsequently performed differential expression analysis using the limma package based on
a linear model comprising the genotype and the experimental batch. Differentially regulated genes were called by computing the local false discovery
rate (locfdr package) on the moderated t statistic with a threshold of 0.2.
Microarray probeset mappings were based on GenBank data with a source date
stamp of March 13, 2014. Microarray data were deposited in the National
Center for Biotechnology Information Gene Expression Omnibus (http://www.
ncbi.nlm.nih.gov/geo/) data repository (accession no. GSE73875).
T cell transfer colitis and cotransfer experiments
Histology
Materials and Methods
Mice
Irak12/2 mice on the C57BL/6 background (at least 14 generations) were
provided by James Thomas (University of Texas Southwestern Medical
School) (11). Irak12/2, C57BL/6, CD45.1, Rag12/2, and Irak12/2/Rag12/2
mice (C57BL/6 background) were bred in our facility under specific
pathogen-free conditions. Experiments were approved by the local government authorities.
Media and reagents
Colitis was induced by transfer of 3 3 105 CD4+CD62L+ T cells into Rag12/2
mice as previously described (17). Body weight and consistency of stool
were monitored throughout the experiments to detect colitis induction. In
cotransfer 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 mesenteric
lymph 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 isolated
from splenocytes using the CD4+CD62L+ T cell isolation kit II (Miltenyi
Biotec, Bergisch Gladbach, Germany). For Th0, Th1, Th2, Th17, and Treg
differentiation, naive CD4+CD62L+ T cells were cultured with plate-bound
anti-CD3ε (5 mg/ml) and anti-CD28 (5 mg/ml) for up to 120 h with the
following cytokine cocktails: Th0, no additional cytokines; Th1, IL-12p70
(10 ng/ml; PeproTech); Th2, IL-4 (50 ng/ml; PeproTech); Th17, IL-1b (10
ng/ml; PeproTech), IL-6 (50 ng/ml; PeproTech), IL-23 (20 ng/ml; R&D
Systems), TGF-b (5 ng/ml; PeproTech); Tregs, IL-2 (200 U/ml; (PromoKine), TGF-b (5 ng/ml). For induction of gut-homing molecules, 10 nM
all-trans retinoic acid (Sigma-Aldrich) was added. Mesenteric lymph node
(MLN) cells and intraepithelial and lamina propria leukocytes (IELs and
LPLs) from intestine were isolated as previously described (17).
Flow cytometry
Cells were stained with fluorescently Abs as described previously (17). Abs
were obtained from eBioscience (CCR9, CD3ε, CD4, CD8a, CD49d,
Colons were fixed as “Swiss rolls” in 4% paraformaldehyde and embedded
in paraffin. For histological scoring, sections of 3 mm were stained with
H&E. Histological scoring was performed in a blinded fashion by the
pathologist as described (17).
ELISA
IL-17A ELISA (eBioscience) was performed according to the manufacturer’s protocol.
Statistical analysis
Data are shown as means 6 SEM. Student t test or a one-way ANOVA
followed by appropriate post hoc testing were performed using SigmaStat
(Systat Software, Erkrath, Germany). A p value ,0.05 was considered to
indicate statistically significant differences.
Results
IRAK1 regulates Th17 differentiation by mediating IL-23R
expression in response to IL-1b
IRAK1 modulates signaling downstream of TLRs and IL-1R and
directly influences Th cell differentiation. In accordance with previous
data (6, 16), we found that lower frequencies of IL-17–producing cells
were generated from Irak1-deficient (knockout [ko]) CD4+ T cells
compared with wt T cells cultured under Th17 conditions, including
IL-1b and IL-23 (12.9 6 0.8 [wt] versus 5.7 6 0.5 [ko]; Fig. 1A),
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IRAK1 plays a nonredundant role on autoimmune and inflammatory
diseases. In vitro studies showed that Th17 differentiation is impaired
whereas Treg generation is enhanced in Irak1-deficient CD4+ T cells
(16). Interference with IRAK1 could therefore restore the balance of
Tregs and effector Th cells, which would be beneficial in autoimmune
and 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 intestine
during development of colitis. We show that IL-1b promotes Th17
cell development by inducing IL-23R expression in an IRAK1dependent manner. Furthermore, our results indicate that IRAK1
signaling in T cells promotes the generation of proinflammatory IFNg– and IL-17–producing Th cells in vivo and is required for their
accumulation in the colon during colitis development induced by
T cell transfer. Irak1-deficient T cells showed a lower expression of
genes involved in T cell activation, differentiation, and gut homing,
which correlated with a reduced capacity to induce experimental
colitis in mice. Thus, IRAK1-mediated signaling in CD4+ T cells
drives their differentiation into proinflammatory effector cells and is
critical for their accumulation in the inflamed intestine.
ROLE OF IRAK1 IN T CELL–DEPENDENT COLITIS
The Journal of Immunology
IRAK1 supports Th1 and inhibits Treg generation but is
dispensable for Th2 development
Besides Th17 cells, Th1, Th2, and Tregs are crucial mediators of
inflammation and immune homeostasis. When testing a possible
influence of IRAK1 on Th cell differentiation, we observed that
CD4+ T cells lacking IRAK1 also had a lower capacity to generate
IFN-g–producing Th1 cells in vitro (Fig. 1G), which was accompanied by reduced induction of T-bet (Fig. 1H), explaining the reduced IFN-g production. Confirming a previous report (16), we
also detected an important role of IRAK1 for Treg generation.
Lack of IRAK1 signaling in T cells reduced the frequency of
in vitro–generated Tregs (Fig. 1I). Finally, we tested whether
IRAK1 is also important for Th2 development. In contrast to its
role for Th1/Th17 and Treg development, we did not detect an
impact of IRAK1 on Th2 differentiation (Fig. 1J, Supplemental
Fig. 1E). These results indicate that Irak1 deficiency does not
have a general impact on Th cell development but rather has specific functions for individual subsets.
Steady-state in vivo T cell development is not affected by
IRAK1 deficiency
To test whether the effects of IRAK1 seen in in vitro differentiation
assays have an impact on in vivo T cell development in the steadystate, we analyzed the frequencies of T cells, Tregs, and Th1/Th17
cells in Irak12/2 mice and littermate controls (Fig. 2A). Neither
differences in overall CD3ε+/CD4+ T cell frequencies in spleen,
MLNs, thymus, Peyer’s patches, colonic LPLs, and small intestinal LPLs (Fig. 2B) nor major differences in Treg frequencies
(Fig. 2C) were detected in these tissues. With respect to Th1/Th17
development, 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 6
SEM; *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; mean 6 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; mean 6 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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correlating with a lower IL-17A concentration in the supernatant
(Supplemental Fig. 1A). Whereas proliferation and survival were not
affected 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 the
presence of IL-1b (6). In the absence of IL-1b, the frequency of wt
IL-17+ T cells was reduced to similar levels as in Irak12/2 T cells
cultured with or without IL-1b (Fig. 1C). Thus, the reduced IL-17
production by Irak12/2 T cells is mostly due to their inability to respond 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 wt
T cells cultured without IL-1b (Fig. 1D) whereas RORgt expression in
CD4+ T cells was induced by IL-1b independently of IRAK1 (Fig.
1E, Supplemental Fig. 1D). STAT3 phosphorylation correlated with
il-23r expression and was dependent on IL-1b and IRAK1 signaling
(Fig. 1F). Taken together, these data demonstrate that IRAK1 mediates
upregulation of IL-23R in response to IL-1b. Thus, developing Th17
cells are more responsive to IL-23, leading to enhanced STAT3
phosphorylation, which in the presence of RORgt promotes sustained
IL-17 production.
3
4
ROLE OF IRAK1 IN T CELL–DEPENDENT COLITIS
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 for
CD4+ 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 expression
pattern of cotransferred wt and Irak12/2 T cells sorted from
MLNs 14 d after transfer. Microarray analysis of wt and Irak12/2
T cells isolated from the MLNs of cotransferred Rag12/2 recipient mice (Fig. 3D) showed that mRNA expression levels of genes
involved in T cell gut homing or retention (Itga1, Cd69), Th1/
Th17 development (Klrd1, Acss2, Klrk1, Tnf, Stard5), and T cell
activation (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 and
apoptosis was not observed (unpublished data), suggesting that the
reduced accumulation of Irak12/2 T cells in the inflamed colon
and MLNs is rather due to impaired gut homing and retention.
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.
(IL-17 single-positive, IFN-g single-positive, IFN-g/IL-17 doublepositive CD4+ T cells) was observed in Irak1-deficient mice in
small intestinal LPL fractions, yet this effect did not prove to be
statistically significant and no differences in the other organs
could be detected.
Decreased Th1/Th17 development in Irak1-deficient T cells is
due to intrinsic effects of IRAK1 that also induce accumulation
in the colon
To investigate the influence of IRAK1 signaling on Th1/Th17
cell differentiation in an inflammatory setting, we cotransferred
equal numbers of naive CD4+/CD62L+ T cells isolated from wt
(CD45.1) and Irak12/2 (CD45.2) mice into Rag1 2 /2 recipient
mice (Fig. 3A). By cotransferring the T cells of both genotypes
into the same recipient, we circumvent possible effects of different
microbiota or different inflammatory microenvironments. Analysis of cytokine production upon restimulation 14 d after transfer
demonstrated that IRAK1 promotes differentiation toward
IFN-g– and/or IL-17–producing T cells in vivo in a cell-intrinsic
fashion (Fig. 3A, 3B).
Induction of gut-homing capacity is crucial for developing effector
Th cells to trigger intestinal inflammation. To compare the ability of
wt and Irak12/2 T cells to accumulate in the colon in vivo, we
assessed the percentages of cotransferred wt and ko T cells in colon,
MLNs, and spleen at different time points after transfer. The 1:1
ratio of wt and ko T cells was maintained in spleen and MLNs on
days 3 and 7 after transfer, when T cells were slowly expanding and
had not yet entered the colon in significant numbers (Supplemental
Fig. 2A). After 14 d when mice had lost up to 20% of their body
weight due to colitis, a significantly higher percentage of wt than
Irak12/2 T cells was found in colon LPL (mean 6 SEM, 72 6
4 [wt] versus 24 6 4% [ko], p = 5.2 3 1026) and IEL (75 6 4 [wt]
versus 23 6 4% [ko], p = 3.9 3 1026) fractions as well as in MLNs
(70 6 3 [wt] versus 27 6 3% [ko], p = 8.6 3 1026). This difference
T cell homing to the intestine requires expression of a4b7 integrin,
which binds to mucosal vascular adressin MAdCAM1 on high
endothelial venules of MLNs, Peyer’s patches, and postcapillary
venules of the intestine (18). Blockade or genetic deficiency of
a4b7 integrin prevents recruitment of T cells to the colon and
colitis development after transfer into Rag12/2 mice (19, 20). We
therefore analyzed expression of a4b7 integrin and other molecules involved in gut homing and retention in cotransferred wt and
Irak12/2 T cells 14 d after transfer (Fig. 4A, Supplemental Fig.
2B). Whereas a clear induction of a4b7 integrin was seen in wt
T cells isolated from MLNs and spleen, Irak12/2 T cells from
these organs expressed much lower levels of a4b7 integrin. In the
colon, a4b7 integrin expression was reduced compared with
MLNs and spleen as expected, but it was still found to be higher in
wt than in Irak12/2 T cells. CD103 (integrin aE), CD49d (integrin
a4), and CCR9, which is critical for T cell homing to the small
intestine but not the colon, were similarly expressed in wt and
Irak12/2 T cells. Both Irak12/2 and wt T cells stained positively
for Ki67 at a similarly high percentage, demonstrating that lack of
IRAK1 does not have a major impact on the proliferation of CD4+
T cells (Fig. 4A). Thus, the reduced capacity of Irak1-deficient
T cells to accumulate in the colon during colitis is most likely due
to impaired a4b7 integrin–dependent homing to the inflamed colon. Confirming results of our microarray analysis CD69 was
expressed at higher levels on the surface of wt than Irak12/2
T cells in the colon (Fig. 4A). Given the role of CD69 for retention
of memory T cells in the intestine in chronic infection (21), higher
CD69 expression may contribute to preferential accumulation of
wt T cells in the colon.
T cell intestinal homing capacity is imprinted by RA produced
by MLN dendritic cells. To investigate the influence of IRAK1
signaling on the induction of gut-homing molecules, wt and
Irak12/2 CD4+CD62L+ T cells were cultured under Th1, Th17,
Treg, or neutral conditions (Th0) with or without IL-1b in the
presence or absence of RA (Fig. 4B). Induction of a4b7 integrin
expression by RA was impaired in Irak12/2 T cells cultured under
Th17 conditions irrespective of IL-1b addition to the culture.
Irak12/2 T cells expressed lower levels of a4b7 integrin when
cultured under Treg conditions, but they upregulated a4b7 integrin
to a similar extent as did wt T cells when RA was included in the
Treg culture. Expression of CCR9 and CD103 was not affected by
Irak1 deficiency. We conclude that efficient induction of a4b7
integrin in developing Th17 cells by RA requires intact IRAK1
signaling.
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IRAK1 signaling is required for efficient induction of
gut-homing integrin a4b7 and CD69 on CD4+ T cells
The Journal of Immunology
5
Irak1-deficient T cells fail to induce severe colitis
Discussion
To investigate whether IRAK1 signaling in T cells is critical for
colitis induction, wt and Irak12/2 CD4+CD62L+ T cells were
transferred separately into Rag12/2 mice and colitis activity was
assessed clinically and histologically. We also investigated the
contribution of IRAK1 signaling outside of the T cell compartment 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 to
severe colitis and weight loss, mice of the three other groups had
a milder course of colitis and did not progress to severe colitis
during this time (Fig. 5A). Mice lacking IRAK1 and receiving
Irak1-deficient T cells showed the mildest course of colitis induction (Fig. 5B). Histopathological signs of colitis, such as
immune cell infiltration and tissue destruction, were also less
pronounced in Rag12/2 recipients receiving Irak12/2 T cells
and in Irak12/2/Rag12/2 mice receiving either wt or Irak12/2
T cells, demonstrating a relevant role of IRAK1 for intestinal
inflammation (Fig. 5C, 5D). FACS analysis of the transferred
T cells showed that the reduced colitis activity was associated
with reduced induction of Th1/Th17 cells in MLNs and spleen
(Fig. 5E, 5F), consolidating the results found in cotransfer experiments. In contrast, the percentage of Foxp3+ Tregs in MLNs
during colitis was increased in the absence IRAK1 signaling
(Supplemental Fig. 3). In summary, these results show that
blocking IRAK1 signaling has the potential to inhibit colitis
induction by interfering with effector Th cell differentiation and
accumulation in the intestine.
The generation of effector Th1 and Th17 cells depends on signals
derived from bacteria or proinflammatory cytokines such as IL-1b,
which directly influence CD4+ T cells via TLRs or cytokine
receptors. These signals promote Th1/Th17 cell differentiation
while overriding the Treg induction program, which is dominant
in barrier organs such as the intestine in the steady-state, but
suppressed during intestinal inflammation. In this study, we investigated the function of IRAK1, which is one of the critical
components of the TLR and IL-1R signaling pathway, and defined
its T cell–intrinsic role for Th cell differentiation and its function
during 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 production, which correlated with their inability to upregulate IL-23R
expression in response to IL-1b stimulation. Similar results were
obtained with Irak4-deficient T cells in an earlier report (15). It
has been reported that IRAK1 directly phosphorylates STAT3 in
T cells cultured with TGF-b and IL-6 (16), yet we found that in
the presence of IL-23, IRAK1 signaling acts downstream of IL-1b
to promote sustained IL-17 production. It was shown recently that
IL-1 additionally promotes STAT3 tyrosine phosphorylation induced by IL-23 by repressing suppressor of cytokine signaling 3, a
negative regulator of the STAT3 pathway (22). In line with our
finding that IRAK1 signaling is not required for upregulation of
RORgt expression by IL-1b, it was shown that lack of MyD88 in
CD4+ T cells impairs Th17 cell generation without affecting
RORgt expression (23). Chang et al. (23) also demonstrated that
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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 significantly 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.
6
ROLE OF IRAK1 IN T CELL–DEPENDENT COLITIS
the MyD88-mediated activation of the mTOR signaling pathway
by IL-1b promotes IL-23R induction and sustains IL-17 production. Indeed, we could show that full activation of the Akt/mTOR
pathway 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 by
IL-1b promotes Th17 cell differentiation. We also observed a
reduction in the percentage of CD4+ T cells expressing T-bet and
producing IFN-g in Irak1-deficient T cells cultured under Th1
polarizing conditions. These results suggest that IRAK1 may also
FIGURE 5. IRAK1 signaling promotes colitis development 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 magnification 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; mean 6 SEM; *p , 0.05, one-way ANOVA
followed by a Bonferroni test).
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FIGURE 4. Decreased induction of gut-homing receptors 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 indicated 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).
The Journal of Immunology
Therefore, CD69 may also be involved in CD4+ T cell accumulation
in 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. These
experiments demonstrated that IRAK1 expression in T cells is
required for induction of severe colitis and that IRAK1 expression
outside of the T cell compartment, for example in APCs, innate
lymphoid cells, intestinal epithelial cells, and stroma cells (11, 33),
additionally contributes to development of T cell–dependent colitis. This was also suggested by reduced susceptibility of Irak1deficient mice to acute DSS-induced colitis (Ref. 34 and unpublished
data). Our study shows that IRAK1 signaling drives intestinal inflammation by promoting the generation of proinflammatory effector T cells with optimal gut-homing and retention capacity.
These findings provide a rationale for considering IRAK1 as a
therapy target in inflammatory bowel diseases.
Acknowledgments
We 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.
Disclosures
The authors have no financial conflicts of interest.
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be involved in signaling downstream of the IL-12 receptor. A
possible link could be the direct phosphorylation of STAT3 by
IRAK1 in the nucleus (24) as STAT3 cooperates with STAT4 in
IL-12R signaling (25).
IRAK1 expression in T cells was also required for full differentiation of proinflammatory Th1/Th17 effector cells in vivo. In
competitive cotransfer experiments with Irak12/2 and wt T cells, we
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effector Th cell differentiation. Ahern et al. (26) reported a similar
effect of IL-23R signaling on the generation of pathogenic IFN-g/
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established in our in vitro experiments.
We observed that induction of Foxp3+ Tregs in vitro by TGF-b
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complexes in the absence of IRAK1 signaling, which was reported
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Competitive transfer experiments demonstrated a cell-intrinsic
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during colitis development. This could be a cell-intrinsic effect in
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a different manner than wt T cells, leading to reduced differentiation and accumulation in the inflamed colon.
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CD4+ T cells lacking IRAK1 in our experiments in vitro and
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(32). We observed that the failure of Irak12/2 T cells to accumulate
in the inflamed colon correlates with reduced CD69 upregulation.
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ROLE OF IRAK1 IN T CELL–DEPENDENT COLITIS