Differential Regulation of by IL-17 in Colonic Epithelial Cells Jimmy W. Lee, Ping Wang, Michael G. Kattah, Sawsan Youssef, Lawrence Steinman, Kathryn DeFea and Daniel S. This information is current as Straus of September 24, 2021. J Immunol 2008; 181:6536-6545; ; doi: 10.4049/jimmunol.181.9.6536 http://www.jimmunol.org/content/181/9/6536 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Regulation of Chemokines by IL-17 in Colonic Epithelial Cells1

Jimmy W. Lee,* Ping Wang,* Michael G. Kattah,† Sawsan Youssef,‡ Lawrence Steinman,†‡ Kathryn DeFea,2* and Daniel S. Straus2*

The IL-23/IL-17 pathway plays an important role in chronic inflammatory diseases, including inflammatory bowel disease. In inflammatory bowel disease, intestinal epithelial cells are an important source of chemokines that recruit inflammatory cells. We examined the effect of IL-17 on expression of HT-29 colonic epithelial cells. IL-17 strongly repressed TNF-␣-stimulated expression of CXCL10, CXCL11, and CCL5, but synergized with TNF-␣ for induction of CXCL8, CXCL1, and CCL20 mRNAs. For CXCL10, IL-17 strongly inhibited promoter activity but had no effect on mRNA stability. In contrast, for CXCL8, IL-17 slightly decreased promoter activity but stabilized its normally unstable mRNA, leading to a net increase in steady-state mRNA abundance. IL-17 synergized with TNF-␣ in transactivating the epidermal receptor (EGFR) and in activating ERK Downloaded from and p38 MAPK. The p38 and ERK pathway inhibitors SB203580 and U0126 reversed the repressive effect of IL-17 on CXCL10 mRNA abundance and promoter activity and also reversed the inductive effect of IL-17 on CXCL8 mRNA, indicating that MAPK signaling mediates both the transcriptional repression of CXCL10 and the stabilization of CXCL8 mRNA by IL-17. The EGFR kinase inhibitor AG1478 partially reversed the effects of IL-17 on CXCL8 and CXCL10 mRNA, demonstrating a role for EGFR in downstream IL-17 signaling. The overall results indicate a positive effect of IL-17 on chemokines that recruit neutrophils (CXCL8 and CXCL1), and Th17 cells (CCL20). In contrast, IL-17 represses expression of CXCL10, CXCL11, and CCR5, three http://www.jimmunol.org/ chemokines that selectively recruit Th1 but not other effector T cells. The Journal of Immunology, 2008, 181: 6536–6545.

rohn’s disease is an inflammatory bowel disease with a inhibit induction of Th17 cells, in some contexts the two cell types chronic relapsing and remitting course (1). The immu- seem to cooperate in initiating and/or perpetuating chronic inflam- C nopathology in Crohn’s disease was initially character- mation. For example, in human inflammatory diseases including ized as Th1 polarized, with high levels of the Th1 IFN-␥ Crohn’s disease (4) and psoriasis (6) and in the IL-10 knockout and low levels of the Th2 IL-4, IL-5, and IL-13 present mouse model of inflammatory bowel disease (13), high levels of in inflamed tissue (2). More recently, a newly defined class of IFN-␥ and IL-17 may be found together in the same specimens of ϩ by guest on September 24, 2021 IL-17-producing CD4 T cells, termed Th17, has been found to inflamed tissue. Further complicating the understanding of Th1- play a key role in inflammatory and autoimmune diseases, includ- Th17 interaction, a newly identified subset of T cells termed Th1/ ing Crohn’s disease (3–5). Human Th17 cells are characterized by Th17 has been found to have features of both Th1 and Th17, in- expression of the transcription factor ROR␥t, the IL-23 receptor, cluding production of both IFN-␥ and IL17 (4, 7, 12, 14). Finally, and the CCR6 (4, 6–9). Conditions that favor in addition to being produced by Th17 cells, IL-17 is also produced Th1 differentiation inhibit differentiation of Th17 cells. In partic- by cells that participate in the innate immune response, including ␥ Ϫ ular, the Th1 cytokine IFN- antagonizes the differentiation of , granulocytes, NK1.1 invariant NKT cells, and ␥␦ T naive mouse CD4ϩ T cells into Th17 cells (10, 11) and the induc- ϩ ϩ cells (15–18). tion of Th17 cells in human CD4 CD45RO memory pop- Chemokines play a central role in recruiting immune cells to ␥ ulations (12). Moreover, IFN- inhibits production of the CCR6 the site of inflammation, and the role of IL-17 in regulating CCL20, a chemokine that selectively recruits Th17 cells (9). chemokine production is complex, with positive effects exerted ␥ Despite the negative effect of IFN- on Th17 differentiation and on the production of some chemokines and negative effects ex- recruitment, the exact relationship between Th1 and Th17 cells erted on others. IL-17 synergizes with TNF-␣ in up-regulating remains poorly understood. Although Th1-polarizing conditions the expression of a subset of chemokines and cytokines includ- ing CXCL1, CXCL8, CCL20, and IL-6 (19–22). Whereas TNF-␣ induces transcription of the for CXCL1 and *Biomedical Sciences Division, University of California, Riverside, CA 92521; and ␬ †Program in Immunology and ‡Department of Neurology and Neurological Sciences, CXCL8 by activating the NF- B signaling pathway, IL-17 ap- Stanford University, Stanford, CA 94305 pears to act mainly by stabilizing their mRNAs (19, 20). In- Received for publication January 10, 2008. Accepted for publication August 20, 2008. triguingly, however, IL-17 negatively regulates TNF-␣-elicited The costs of publication of this article were defrayed in part by the payment of page production of other chemokines including CCL5 (RANTES) charges. This article must therefore be hereby marked advertisement in accordance (23, 24), CX3CL1 (fractalkine) (25), and CCL27 (cutaneous T with 18 U.S.C. Section 1734 solely to indicate this fact. cell-attracting chemokine or CTACK) (26). Moreover, in a 1 This work was supported by a Senior Investigator Award from the Crohn’s and Colitis Foundation of America (to D.S.S.) and National Institutes of Health Grants mouse model of allergic asthma, IL-17 functions as a negative R01-GM066151 (to K.D.) and R01-NS55997 (to L.S.). regulator by repressing expression of the chemokine 2 Address correspondence and reprint requests to Dr. Daniel S. Straus or Dr. Kathryn CCL11 (eotaxin) and Th2 chemokine CCL17 (- and ac- DeFea, Biomedical Sciences Division, University of California, Riverside, CA tivation-regulated chemokine or TARC) (27). Very little is 92521-0121. E-mail addresses: [email protected] and [email protected] known at present regarding the mechanism(s) for the repressive Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 effects of IL-17 on these chemokines. www.jimmunol.org The Journal of Immunology 6537 Downloaded from

FIGURE 1. Effect of IL-17 on chemokine expression. HT-29 cells were treated with IL-17 (50 ng/ml) for 2 h. TNF-␣ (25 ng/ml) was then added, and the cells were incubated for an additional 4 h. Cells were harvested, RNA was prepared, and the steady-state level of mRNA expression was determined by real-time PCR for the following genes: CXCL10 (A), CXCL11 (B), CCL5 (C), CXCL8 (D), CXCL1 (E), CCL20 (F), and I␬B␣ (G). The abundance of each mRNA was normalized to the abundance of ␤-actin mRNA, and the mean for untreated control cultures was set at 1.0. Each bar represents the mean Ϯ Ͻ of three cultures SE. Means with different letters were significantly different, p 0.05. http://www.jimmunol.org/

The chemokines CXCL9, 10, and 11 act via the CXCR3 recep- Cell culture tor, which is expressed by Th1 cells, and these three chemokines HT-29 CL19A human colorectal cancer cells were cultured in McCoy’s 5A play a role in recruiting Th1 cells to a site of inflammation (28, 29). medium supplemented with 10% FBS, penicillin (100 U/ml), and strepto- In inflammatory bowel disease, the epithelial cells lining the lumen mycin (100 ␮g/ml) (13). CD4ϩ human T cells enriched for IL-17-produc- of the gastrointestinal tract are an important source of chemokines ing cells were prepared as described previously (37) with minor modifi- cations. Briefly, CD4ϩ T cells were isolated from PBMCs from healthy (13, 30, 31). We show here that IL-17 represses TNF-␣-elicited ϩ by guest on September 24, 2021 volunteers using a RosetteSep human CD4 T cell isolation kit (StemCell CXCL10 and 11 in HT-29 colonic epithelial cells. Technologies) followed by a whole CD4ϩ T cell isolation kit (Miltenyi This repression is mainly transcriptional and requires activation of Biotec). Cells obtained in this manner consisted of 98% pure CD4ϩ T cells, the ERK and p38 MAPK signaling pathways by IL-17 in combi- of which approximately half were CD45RAϩ and half were CD45ROϩ. nation with TNF-␣. These results suggest that the accumulation of These cells were cultured with anti-CD3/anti-CD28-coated beads (Invitro- gen) in X-VIVO 15 medium (Lonza) supplemented with 10% FBS, IL-1␤, IL-17-producing cells in the inflamed mucosa would lead to de- IL-6, IL-23 (10 ng/ml each), anti-IL-4 and anti-IFN-␥ (5 ␮g/ml) (37). creased expression of Th1 chemotactic chemokines and attenuated Culture of the CD4ϩ T cells with this combination of cytokines in serum- recruitment of Th1 cells. In contrast, IL-17 synergizes with TNF-␣ containing medium results in enrichment for IL-17 producing cells (37), ϩ to increase expression of CCL20, a chemokine that would promote mainly arising from the CD45RO memory population (38, 39). recruitment of Th17 cells. assays Materials and Methods For collection of conditioned medium from HT-29 cells, densely confluent Materials monolayer cultures of HT-29 cells in McCoy’s 5A medium plus 0.5% FBS ␣ ␣ ϩ kinase inhibitors SB203580, U0126, and tyrphostin AG1478 were were treated with nothing (control), TNF- , IL-17, or TNF- IL-17 for 24 h. The medium was collected, centrifuged for 2 min at 13,200 rpm to obtained from Sigma-Aldrich. Stock solutions of the inhibitors were pre- Ϫ o pared in DMSO. Human recombinant TNF-␣ (210-TA) and IL-17A (317- remove cell debris, and stored at 80 C. Chemotaxis assays were performed in Transwells (5-␮ IL) were obtained fromR&DSystems. The RNA polymerase II inhibitor m pore size, 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB)3 was from Sigma- 6.5-mm diameter, 12-well plates; Corning). Cultured human T cells ␬ (300,000 cells in 100 ␮l of medium) were placed in the top well and 600 Aldrich. The NF- B-luciferase reporter had luciferase transcription under ␮ control of a promoter with three consensus NF-␬B binding sites cloned l of HT-29 conditioned medium was placed in the bottom well. The upstream from a TATA box (32). The CMV-␤gal plasmid had a bacterial Transwells were incubated at 37° for 3.5 h, after which the cells that mi- ␤-galactosidase gene under control of the CMV immediate-early enhancer grated to the lower chamber were collected by centrifugation and stimu- plus promoter (32). In other reporter constructs, luciferase transcription lated for 5 h with PMA (30 ng/ml; Sigma-Aldrich), ionomycin (750 nM; was under control of a fragment extending from Ϫ525 to ϩ 97 of the Sigma-Aldrich), and GolgiStop (as described in the manufacturer’s proto- CXCL10 gene promoter (CXCL10/luciferase reporter; obtained from R. col; BD Biosciences). Cells were then stained with allophycocyanin-Cy7- Ransohoff, Cleveland Clinic, Cleveland, OH) (33), a 1521-bp fragment of conjugated anti-human CD4 Ab (BD Biosciences; catalog no. 557871). the CXCL8 gene promoter (CXCL8/luciferase reporter, obtained from A. Next, cells were fixed with 4% paraformaldehyde and permeabilized using Keates, Beth Israel Deaconess Hospital, Boston, MA) (34), and a 400-bp a BD Cytofix/Cytoperm kit (BD Biosciences; catalog no. 554715). After region of the I␬B␣ promoter (obtained from K. Yamamoto, University of permeabilization, cells were stained with PE-conjugated anti-human IL-17 California, San Francisco, CA) (35, 36). (eBioscience; catalog no. 12-7179-73) and allophycocyanin-conjugated anti- human IFN-␥ (BD Biosciences; catalog no. 554702) Ab. Samples were subjected to FACS analysis using a BD LSR flow cytometer and FlowJo 3 Abbreviations used in this paper: DRB, 5,6-dichloro-1-␤-D-ribofuranosylbenzimi- software. The migration index was determined by setting the mean for the dazole; DAPI, 4,6-diamidino-2-phenylindole; EGFR, epidermal growth factor recep- percentage of cells migrating into medium from untreated HT-29 cells tor; ISRE, IFN stimulatory response element. at 1.0. 6538 REGULATION OF CHEMOKINES BY IL-17 Downloaded from

FIGURE 3. Effect of IL-17 on CXCL10, CXCL8, and I␬B␣ promoter activity. HT-29 cells were transfected with luciferase reporter constructs in which luciferase transcription was directed by the CXCL10 promoter (A), the CXCL8 promoter (B), the I␬B␣ promoter (C), or three consensus NF-␬B binding sites cloned upstream from a TATA box (D). The CMV- http://www.jimmunol.org/ ␤gal construct was cotransfected with each plasmid, and luciferase activity was normalized to ␤-galactosidase activity. Cells were treated with TNF-␣, IL-17, PMA, or combinations as indicated. Each bar represents the mean Ϯ SE of results obtained with at least four replicate cultures. Means with different letters were significantly different, p Ͻ 0.05.

ng/ml). Incubation was continued for 6 h following TNF-␣ addition, after which cells were harvested and assayed for luciferase and ␤-galactosidase FIGURE 2. Assay of chemokine activity in medium conditioned by un- activity as described previously (13). The luciferase activity was then by guest on September 24, 2021 treated HT-29 cells and cells treated with TNF-␣ and/or IL-17. Chemotaxis ␤ ϩ normalized to the -galactosidase activity of the same extract. In some assays were performed with human CD4 T cells using Transwells. The experiments cells were treated with PMA rather than TNF-␣ to activate ϩ Ϫ ϩ ϩ percentage of migrated cells that were IL-17 IFN-␥ (A), IL-17 IFN-␥ reporter expression. (B), and IL-17ϪIFN-␥ϩ (C) was determined by FACS analysis, and mi- gration index was calculated as described in Materials and Methods. The Phospho-ERK and tyrosine phosphorylation analyses actual mean percentage of cells migrating into medium from untreated Ϫ ϩ ϩ Ϫ Following treatment with cytokines, cells were harvested in 1% Triton HT-29 cells was 28.8% IL-17 IFN-␥ , 1.3% IL-17 IFN-␥ , and 1.3% X-100 lysis buffer in TBS (15 mM Tris, 150 mM NaCl (pH7.6)) with ϩ ϩ IL-17 IFN-␥ . Each bar represents the mean of results obtained with me- protease and phosphatase inhibitors and cleared by centrifugation. For dium from two independent HT-29 cultures, and error bars represent the phospho-ERK analysis, samples were stored in Laemmli sample buffer at range for the two determinations. Ϫ80°C. For epidermal growth factor receptor (EGFR) phosphorylation, samples were incubated with 4 ␮g/ml anti-EGFR (Upstate Biotech, catalog no. 06-847) for2hat4°C, after which immune complexes were isolated Gene expression experiments with 40 ␮l of protein A-agarose, washed, and eluted in Laemmli sample buffer. Protein samples were run on a 10% SDS-polyacrylamide gel and HT-29 cells were plated in 6-well plates at density of 3–5 million cells per transferred to Immobilon-FL (Millipore) membranes. For detection of well in McCoy’s 5A medium with 10% FBS plus antibiotics. Before treat- ERK, the membranes were probed with both anti-phospho-ERK (Cell Sig- ment with cytokines, cells were incubated in medium with 0.5% FBS over- naling Technology, catalog no. 9106S) and anti-total ERK-1 (Santa Cruz night. The next morning, cells were treated with TNF-␣ and/or IL-17. Biotechnology, catalog no. sc-93) Abs followed by IRDye 800CW- and Following treatment with cytokines, cells were harvested, and RNA was IRDye 680-conjugated secondary Abs (LI-COR Biosciences). For p38, the prepared using the RNeasy Mini kit (Qiagen). RNA samples (2 ␮g) were membranes were probed with both anti-phospho-p38 ( Tech- reverse-transcribed with Moloney murine leukemia virus reverse transcrip- nology, catalog no. 9211S) and anti-total p38 (Santa Cruz Biotechnology, tase (M0253S; New England Biolabs). Real-time PCR was performed us- catalog no. sc-535G) primary Abs followed by IRDye 800- (Rockland) and ing SYBR Green (Applied Biosystems) for detection and the standard Alexa Fluor 680-conjugated (Molecular Probes) secondary Abs. For EGFR curve method for quantification, as described previously (40). phosphorylation, membranes were probed with anti-PY99 (Santa Cruz Bio- tech, catalog no. sc-7020) and anti-EGFR. The membranes were then Promoter activity assay scanned using the Odyssey infrared imaging system (LI-COR Bio- sciences). Fold phosphorylation was determined from the integrated inten- For transfection experiments, HT-29 cells were plated at a density of 4 ϫ sity of individual bands using the LI-COR Odyssey software; phospho- 105 cells per 3.5-cm diameter well and transfected using Lipofectamine protein levels were normalized to total protein levels in each case. 2000 (Invitrogen). In all experiments, the plasmid pCMV-␤gal, in which the ␤-galactosidase gene is under control of the CMV enhancer plus pro- ERK nuclear localization moter, was cotransfected along with the luciferase reporter as an internal control for transfection efficiency and for the specificity of the effects ob- HT-29 cells were cultured on glass coverslips. Cells were transfected with served (13). To determine the effect of TNF-␣ and IL-17 on promoter an expression construct for rat ERK2 fused with GFP on the C terminus in activity, cells were transferred to medium with 0.5% serum for 14 h before the pEGFPN1 vector (41) using Lipofectamine 2000 (Invitrogen). Trans- cytokine addition. IL-17 (final concentration of 50 ng/ml) was added to fected cells were preincubated in IL-17 30 min before the addition of each culture 30 min before the addition of TNF-␣ (final concentration of 25 TNF-␣. Four hours after adding TNF-␣, coverslips were removed, the cells The Journal of Immunology 6539

were fixed, and the coverslips were mounted onto slides using Vectashield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). The cells were then imaged using an Eclipse TE2000U in- verted microscope (Nikon). Statistics For comparisons of more than two means, data were subjected to one-way ANOVA followed by the Student-Newman-Keuls multiple comparison test. For comparison of two means, data were analyzed by the t test. Results IL-17 negatively regulates some chemokine genes by repressing transcription and positively regulates others by enhancing mRNA stability The effect of IL-17, TNF-␣, and IL-17 plus TNF-␣ on mRNA abundance for six chemokines is shown in Fig. 1. Two distinct patterns of regulation by IL-17 were observed. For CXCL10, CXCL11, and CCL5, IL-17 alone had no significant effect on gene expression, but IL-17 strongly repressed the induction of these three genes by TNF-␣ (Fig. 1, A–C). For CXCL8, CXCL1, and Downloaded from CCL20, IL-17 alone had no significant effect on gene expression, FIGURE 4. Effect of IL-17 on CXCL10 and CXCL8 mRNA stability. but it strongly synergized with TNF-␣ in inducing expression of HT-29 cells were treated with IL-17 (50 ng/ml) (E) or not treated with IL-17 these three genes (Fig. 1, D–F). All six chemokine genes have at (F) for 30 min followed by addition of TNF-␣ (25 ng/ml) to all cultures. Cells least one binding site for NF-␬B in their promoter elements (33– were incubated for an additional 6 h. DRB (40 ␮M) was then added to each 36, 42–44), and in each case NF-␬B is thought to play an impor- culture, and cells were harvested at 0, 15, 30, 60, 120, and 360 min after the tant role in the induction of the genes by proinflammatory cyto- http://www.jimmunol.org/ addition of DRB. Abundance of CXCL8 (A) and CXCL10 (B) mRNA was kines, including TNF-␣. The effect of IL-17, TNF-␣, and IL-17 Ϯ determined by real-time PCR. Each point represents the mean SE of at least plus TNF-␣ on another well-studied NF-␬B target gene, that en- Mean differs significantly from mean of control ,ء .three replicate cultures coding I␬B␣, was also examined. IL-17 alone had no significant cultures not treated with IL-17, p Ͻ 0.05. by guest on September 24, 2021

FIGURE 5. Effect of IL-17 on ERK and p38 MAPK activity. HT-29 cells were treated for the indicated times with TNF-␣ (E), IL-17 (), or TNF-␣ plus IL-17 (F). Western blots were prepared and analyzed for phospho-ERK and total ERK (A) or phospho-p38 and total p38 (B). Results for phospho-ERK were normalized to total ERK (C), and those for phospho-p38 were normalized to total p38 (D). In C and D, each point represents the mean of two experiments; error bars show the range for the two experiments. 6540 REGULATION OF CHEMOKINES BY IL-17

FIGURE 6. Effect of IL-17 on ERK nuclear localization. HT-29 cells were transfected with a GFP-ERK ex- pression construct and left untreated or treated with TNF-␣, IL-17, or TNF-␣ plus IL-17 for 4 h. Fixed cells were stained and photographed. Cells from five representative fields of vision are shown for each treatment. GFP-ERK labeling is in green, DAPI-stained nu- clei are in red, and merged GFP-ERK and DAPI nuclear stain are colocalized in yellow. NTC, No treatment control. Downloaded from http://www.jimmunol.org/ effect on expression of the I␬B␣ gene, but it augmented TNF-␣- 4A). In contrast, CXCL10 mRNA was very stable, and its stability induced expression (Fig. 1G). was unaffected by IL-17 (Fig. 4B). Taken together with the results We next used a functional assay to examine the effect of TNF-␣ presented in Figs. 1 and 3, these results indicate that in the HT-29 and IL-17 on chemokine production by HT-29 cells. The HT-29 cells IL-17 represses the expression of some chemokine genes by cells were treated with TNF-␣ and/or IL-17 for 24 h. The condi- a transcriptional mechanism and increases the expression of others tioned medium was harvested and used as a source of chemokines by increasing mRNA stability. in chemotaxis assays of cultured human T cells. (Fig. 2). Consis- tent with the effect of TNF-␣ and IL-17 on CCL20 mRNA ex- pression, TNF-␣ stimulated production of chemokine(s) that were by guest on September 24, 2021 chemoattractive to Th17 cells, and chemokine production was en- hanced by the coaddition of IL-17 (Fig. 2A). Interestingly, the same response was observed with IL-17ϩ IFN-␥ϩ cells, which also express CCR6, the receptor for CCL20 (4) (Fig. 2B). In the same unconcentrated conditioned medium samples, there was not a sufficient amount of chemokines that attract Th1 cells for us to detect the effect of TNF-␣ or IL-17 on Th1-attractive chemokine production (Fig. 2C). To determine whether the effects illustrated in Fig. 1 involved transcriptional or posttranscriptional regulation of gene expres- sion, the effect of IL-17 and TNF-␣ was determined in cells transfected with luciferase reporter constructs in which lucif- erase transcription was directed by the CXCL10, CXCL8, or I␬B␣ promoter. IL-17 alone had no significant effect on the CXCL10 reporter, but it strongly repressed TNF-␣-induced ex- pression (Fig. 3A), consistent with a transcriptional mechanism for the repressive effect of IL-17 on CXCL10 mRNA. IL-17 also modestly repressed TNF-␣-induced expression of the CXCL8 and I␬B␣ reporters (Fig. 3, B and C), suggesting that the augmentation of TNF-␣-induced expression of CXCL8 and I␬B␣ mRNA was posttranscriptional. The effect of IL-17 on the expression of a reporter in which luciferase expression is di- rected by three consensus NF-␬B binding sites cloned upstream FIGURE 7. ERK and p38 MAPK inhibitors reverse the effects of IL-17 ␮ D on CXCL10 and CXCL8 gene expression. SB203580 (10 M), U0126 (1 from a TATA box was also determined (Fig. 3 ). In this func- ␮ ␬ M), or DMSO vehicle was added to cultures 30 min before cytokine tional assay, IL-17 alone had no significant effect on NF- B ␣ ␣ addition. IL-17 was added, followed immediately by TNF- , and incuba- activity, and it had a small negative effect on TNF- or PMA- tion was continued for 6 h. Cells were harvested, and abundance of ␬ elicited activation of NF- B. CXCL10 (A) or CXCL8 (B) mRNA was quantified by real-time PCR. The The effect of IL-17 on CXCL10 and CXCL8 mRNA stability is mean of the untreated control cultures was set at 1.0. Each bar represents shown in Fig. 4. CXCL8 mRNA was very unstable in HT-29 cells, the mean Ϯ SE of results obtained with three replicate cultures. Means with and it was significantly stabilized in cells treated with IL-17 (Fig. different letters were significantly different, p Ͻ 0.05. The Journal of Immunology 6541 Downloaded from

FIGURE 8. ERK and p38 MAPK inhibitors partially reverse the effect http://www.jimmunol.org/ of IL-17 on CXCL10 promoter activity. Cells were transfected with the CXCL10/luciferase and CMV-␤gal. SB203580 (10 ␮M), U0126 (1 ␮M), or DMSO vehicle was added to cultures 30 min before cytokine addition. IL-17 was added, followed immediately by TNF-␣, and incubation was continued for 6 h. Cells were harvested and extracts were assayed for luciferase and ␤-galactosidase activity. Each bar represents the mean Ϯ SE of results obtained with four or five cultures. Means with different letters were significantly different, p Ͻ 0.05. by guest on September 24, 2021 Transcriptional repression and mRNA stabilization by IL-17 both depend on ERK and p38 MAPK activation Although the signaling pathways for IL-17 action are not completely understood, evidence indicates that some effects are mediated by activation of ERK and/or p38 MAPK (19). The effect of TNF-␣, IL-17, and TNF-␣ plus IL-17 on ERK (Fig. 5, A and B) and p38 MAPK (Fig. 5, C and D) was determined in HT-29 cells. FIGURE 9. The EGFR inhibitor tyrphostin AG1478 partially reverses In these cells, TNF-␣ transiently activated both ERK and p38 the effects of IL-17 on CXCL10 and CXCL8 mRNA abundance and MAPK, whereas IL-17 alone had very little effect. The combina- CXCL10 promoter activity. A and B, AG1478 (5 ␮M) or DMSO vehicle tion of IL-17 and TNF-␣ gave synergistic transient activation and was added to cultures 30 min before cytokine addition. IL-17 was added, ␣ also prolonged activation of both ERK and p38 (Fig. 5). followed immediately by TNF- , and incubation was continued for 6 h. Cells were harvested, and abundance of CXCL10 (A) or CXCL8 (B) The effect of IL-17 on nuclear localization of ERK was exam- mRNA was quantified by real-time PCR. The mean of the untreated control ined by transfecting HT-29 cells with a GFP-ERK expression con- cultures was set at 1.0. Each bar represents the mean Ϯ SE of results struct. The results indicated that the combination of IL-17 and obtained with three cultures. C, Cells were transfected with the CXCL10/ TNF-␣ gave persistent (4 h) nuclear localization of ERK, whereas luciferase and CMV-␤gal. AG1478 (5 ␮M) or DMSO vehicle was added neither cytokine alone had this effect (Fig. 6). Taken together, the to cultures 30 min before cytokine addition. IL-17 was added, followed results presented in Figs. 5 and 6 indicate that IL-17 synergizes immediately by TNF-␣, and incubation was continued for 6 h. Cells were with TNF-␣ in activating ERK and p38 and also in promoting harvested, and extracts were assayed for luciferase and ␤-galactosidase persistent nuclear localization of ERK. activity. Each bar represents the mean Ϯ SE of results obtained with four We next used the selective protein kinase inhibitors SB203580, or five cultures. which inhibits p38, and U0126, which inhibits the ERK signaling pathway, to determine whether either or both kinases mediate the effects of IL-17 on CXCL10 and CXCL8 gene expression. The construct. The results (Fig. 8) indicated that both SB203580 and results (Fig. 7) indicated that SB203580 reversed both the repres- U0126 partially reversed repression by IL-17 of the CXCL10 pro- sive effect of IL-17 on CXCL10 and the inductive effect on moter. Finally, U0126 slightly increased the activity of the CXCL8. U0126 reversed the repressive effect of IL-17 on CXCL10 promoter above that seen with TNF-␣ alone (Fig. 8B), CXCL10 and partially reversed the inductive effect on CXCL8. To and the combination of TNF-␣ plus IL-17 plus U0126 gave stron- determine whether the inhibitors reversed the repressive effect of ger induction of CXCL10 mRNA than TNF-␣ alone (Fig. 7A). IL-17 on CXCL10 promoter activity, experiments were performed This result suggested that the relatively high baseline of ERK sig- with HT-29 cells transfected with the CXCL10/luciferase reporter naling in the HT-29 cells, attributable to the presence of a BRAF 6542 REGULATION OF CHEMOKINES BY IL-17

FIGURE 10. IL-17 cooperates with TNF-␣ in transactivating EGFR. A, HT-29 monolayers were left untreated or treated with cytokines for 5 min and4hasfollows: TNF-␣ alone, IL-17 alone, or TNF-␣ plus IL-17. Epidermal growth factor (EGF) (5 ng/ml for 5 min) was included as a positive control. Cell lysates were immunoprecipi- tated with IgG (far left lane, as a negative control) or anti-EGFR, followed by SDS- PAGE and Western analysis with anti-PY99 (upper panel) or anti-EGFR (lower panel).

B, Phospho-tyrosine levels (normalized to Downloaded from total EGFR) were calculated and depicted in the bar graph as fold change over baseline (untreated) for each condition (mean Ϯ SEM p Ͻ 0.05 ,ء .(for three different experiments compared with unstimulated control. http://www.jimmunol.org/

mutation in these cells (45), might have a repressive effect on experiments indicate that the effects of IL-17 on CXCL8 and CXCL10 gene transcription that was relieved by U0126. In sum, CXCL10 gene expression are in part mediated by EGFR by guest on September 24, 2021 the results presented in Figs. 7 and 8 demonstrated that the effects transactivation. of IL-17 on both CXCL8 and 10 gene expression were mediated, at least in part, by the ERK and p38 MAPK signaling pathways. Discussion We show here that IL-17 strongly represses expression of the Biological effects of IL-17 are mediated in part by EGFR CXCR3 chemokines CXCL10 and 11 by HT-29 colonic epithelial signaling cells. In addition, consistent with results reported previously with The results described above indicated that the effects of IL-17 on other cells (23, 24), IL-17 also represses CCL5 expression by chemokine gene expression were partly mediated through the ERK HT-29 cells. CXCR3 is expressed on Th1 but not Th2 or Th17 signaling pathway. The activation of ERK by IL-17 in HT-29 cells cells; thus the CXCR3 ligands CXCL10 and 11 are selectively is consistent with several previous studies performed with other chemotactic for Th1 cells (28). CCL5 is also chemotactic for Th1 cells (46, 47); however, the mechanism by which IL-17 activates but not Th2 cells (49). Interestingly, negative regulation of ERK is not known. Recent studies have demonstrated that in hu- CX3CL1 by IL-17 in ocular epithelial cells has also been reported man cells IL-17A acts via IL-17RA and IL-17RC receptors (48). (25). Like CXCL10, CXCL11, and CCL5, CX3CL1 is selectively IL-17RA has a very large cytoplasmic domain, and the IL-17 re- chemotactic for Th1 cells and acts as an amplifier of polarized Th1 ceptor family is not homologous to other known cytokine recep- responses (50). It appears, therefore, that accumulation of IL-17- tors. We considered the possibility that in colonic epithelial cells producing cells at the site of inflammation would attenuate subse- IL-17 binding to its receptor might trans-activate the EGFR, re- quent recruitment of Th1 cells. There are two contexts in which sulting in activation of the ERK signaling pathway. To test the this could occur. First, in the intestinal innate immune response hypothesis that EGFR mediated the effects of IL-17, we tested the IL-23 produced by dendritic cells and stimulates pro- ability of the specific EGFR kinase inhibitor tyrphostin AG1478 to duction of a family of proinflammatory cytokines, including reverse the effects of IL-17 on CXCL8 and CXCL10 gene expres- TNF-␣ and IL-17, by ␥␦ T cells and other innate immune cells (5, sion. The results indicated that AG1478 partially reversed the ef- 15–18). Under these conditions it may be desirable to inhibit or fects of IL-17 on CXCL10 and CXCL8 mRNA abundance (Fig. 9, delay recruitment of Th1 cells. Second, IL-17 produced by Th17 A and B) and CXCL10 promoter activity (Fig. 9C). Direct assays cells as they accumulate at the site of inflammation may negatively of EGFR phosphorylation after 5 min and4hofcytokine treat- regulate the recruitment of Th1 cells. Consistent with the notion of ment showed that IL-17 and TNF-␣ synergized to promote phos- a feedback loop in which IL-23 and IL-17 negatively regulate the phorylation of EGFR at 4 h (Fig. 10). The activation of EGFR by IL-12/IFN-␥ network, the administration of an anti-IL-17 mAb IL-17 plus TNF-␣ at 4 h was consistent with the prolonged acti- worsens the course of dextran sodium sulfate-induced colitis, the vation of the downstream ERK signaling pathway. Thus, IL-17 in elimination of IL-23 worsens the development of T cell-mediated combination with TNF-␣ transactivates EGFR, and the inhibitor trinitrobenzene sulfonic acid-induced colitis, and donor Th17 cells The Journal of Immunology 6543

FIGURE 11. Sequences of CXCL10, CXCL11, and CCL5 gene promoters. Sequences are from the database (genome.ucsc.edu). NF-␬B binding sites, ISREs, and TATA-like elements are bold and underlined. The NF-␬B binding sites and ISREs of the CXCL10 and CCL5 promoter elements have been described previously (33, 42). Likely NF-␬B and ISRE elements in the CXCL11 promoter were located by searching for sequences resembling the consensus elements.

negatively regulate Th1 differentiation and ameliorate acute graft- contrast, relatively little is known regarding the mechanism for vs-host disease (51–53). Similarly, in a mouse model of allergic stabilization of mRNAs by the ERK signaling pathway. asthma, IL-17 functions as a negative regulator by repressing ex- As illustrated in Fig. 3, IL-17 modestly repressed TNF-␣-me- pression of the eosinophil chemokine CCL11 (eotaxin) and the diated activation of the CXCL8 and I␬B␣ promoters, two well Downloaded from Th2 chemokine CCL17 (thymus- and activation-regulated chemo- known NF-␬B targets (36), as well TNF-␣-mediated activation of kine or TARC) (27). Taken together, these results are consistent an NF-␬B reporter. In contrast, IL-17 more strongly repressed the with negative regulation by IL-17 of both Th1 and Th2 CXCL10 promoter. These results are consistent with a general recruitment. weak interference with TNF-␣ activation of NF-␬B targets plus We also confirm and extend the previous observation that IL-17 additional selective repression of the CXCL10 promoter. The mo-

cooperates with TNF-␣ to up-regulate expression of other chemo- lecular mechanisms for these effects and the role of p38 and ERK http://www.jimmunol.org/ kines, including CXCL8, CXCL1, and CCL20 (19–22). TNF-␣ signaling remain to be elucidated. Regarding the slight general plus IL-17 induced a remarkable 845-fold increase in CCL20 interference with TNF-␣ activation of NF-␬B, the synergistic in- mRNA abundance (Fig. 1). CCL20 and its receptor CCR6 play a duction of I␬B␣ by IL-17 in combination with TNF-␣ is of inter- role in lymphorganogenesis in the intestine and are also thought to est. The NF-␬B inhibitor I␬B␣ is rapidly degraded in response to participate in the inflammatory response (54). Recent studies have TNF-␣, leading to nuclear entry and transcriptional activation by demonstrated that CCR6 is expressed on human Th17 and Th1/ NF-␬B. As an NF-␬B target, I␬B␣ is subsequently resynthesized, Th17 cells (4, 7, 9). CCL20 stimulates calcium flux (4) and is leading to termination of the NF-␬B response. Because the coad- chemotactic (9) for Th17 but not Th1 cells. Thus, our results sug- dition of IL-17 with TNF-␣ potentiates the induction of I␬B␣,itis gest that accumulation of IL-17-producing cells at the site of in- possible that NF-␬B-mediated transcription is halted earlier or by guest on September 24, 2021 flammation would establish a positive feedback loop leading the more completely, thereby explaining the modest general inhibitory recruitment of additional IL-17-producing Th17 cells. Interest- effect of IL-17 on NF-␬B targets. ingly, the CCL20 receptor CCR6 is expressed on CD4ϩCD25high The molecular mechanism for selectively stronger repression by Foxp3ϩ Treg cells, and CCL20 is chemotactic for these cells as IL-17 of the CXCL10 promoter is also of considerable interest and well as for Th17 cells (55). The recruitment of Treg cells along remains to be explored further. It is possible that this selectivity is with Th17 cells may serve to restrain the inflammatory response, related to the specific coactivator complexes, including Bcl-3 and thereby preventing severe tissue damage. CARM1, required for the induction of CXCL10 gene expression Consistent with an earlier report (19), CXCL8 is encoded by an by TNF-␣ (57, 58). The CXCL11 gene is located on the long arm unstable mRNA. The induction of CXCL8 gene expression by of 4, in tandem with and ϳ10 kb telomeric to the IL-17 was posttranscriptional and involved CXCL8 mRNA stabi- CXCL10 gene (59) Although CXCL11 transcription has not been lization. TNF-␣ induction of the CXCL8 luciferase reporter was as thoroughly studied as CXCL10 transcription, it is of interest to actually slightly decreased in IL-17-treated cells, although we can- note that the CXCL11 promoter has an NF-␬B site with exactly the not conclusively rule out a possible positive effect of IL-17 on same sequence as the distal CXCL10 NF-␬B site and a sequence CXCL8 transcription mediated by a cis-acting element outside the resembling a likely IFN stimulatory response element (ISRE) lo- 1521-bp CXCL8 core promoter used for construction of the re- cated upstream of this site (Fig. 11). CCL5 gene transcription is porter. In contrast, our results indicate that CXCL10 is encoded by also repressed by IL-17 (23), and the CCL5 promoter has an a very stable mRNA, the decay of which is not affected by IL-17. NF-␬B site with two ISREs located upstream of this site. (Fig. 11). Rather, the negative effect of IL-17 on CXCL10 gene expression oc- Thus, it is possible that strong transcriptional repression of curs via transcriptional repression, in agreement with the previously CXCL10, CXCL11, and CCL5 by IL-17 is related to this shared reported repression by IL-17 of CCL5 gene transcription (23). promoter configuration. Intriguingly, although IL-17 induces CXCL8 by stabilizing its The effects of IL-17 on chemokine gene expression were par- mRNA and negatively regulates CXCL10 by repressing its tran- tially reversed by the specific EGFR tyrosine kinase inhibitor tyr- scription, both forms of regulation are dependent on the p38 and phostin AG1478, suggesting that IL-17 signaling involves activa- ERK MAPK signaling pathways. Like many other unstable tion of the EGFR kinase. Consistent with this hypothesis, IL-17 mRNAs, CXCL8 mRNA contains an AU-rich element in its 3Ј- cooperated with TNF-␣ in transactivating EGFR. The IL-17RA untranslated region. Stabilization of AU-rich element-containing receptor has a very long cytoplasmic domain, and signaling path- mRNAs by p38 MAPK is thought to result from phosphorylation ways activated by IL-17 receptors are not completely understood. of the mRNA-destabilizing protein tristetrapolin by the p38-acti- To our knowledge, this is the first observation implicating EGFR vated kinase MK-2 and a consequent change in the subcellular activation in IL-17 signaling, and this may account for the effect of location of tristetrapolin mediated by binding to 14-3-3 (56). In IL-17 on ERK activity. Many G protein-coupled receptors activate 6544 REGULATION OF CHEMOKINES BY IL-17

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