SMAD3 signaling pathway in Th17 CD4+ T cells

Journal of Autoimmunity 37 (2011) 198e208

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Journal of Autoimmunity journal homepage: www.elsevier.com/locate/jautimm

Coronin 1A is an essential regulator of the TGFb receptor/SMAD3 signaling pathway in Th17 CD4þ T cells Sandra Kaminski a, Natascha Hermann-Kleiter a, Marlies Meisel a, Nikolaus Thuille a, Shane Cronin b, Hiromitsu Hara b, c, Friedrich Fresser a, Josef M. Penninger b, Gottfried Baier a, * a b c

Experimental Cell Genetics, Department for Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Austria Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Department of Biomolecular Sciences, Saga University, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 May 2011 Received in revised form 25 May 2011 Accepted 26 May 2011

Transforming growth factor b (TGFb) plays a central role in maintaining immune homeostasis by regulating the initiation and termination of immune responses and thus preventing the development of autoimmune diseases. In this study, we describe an essential mechanism by which the actin regulatory protein Coronin 1A (Coro1A) ensures the proper response of Th17 CD4þ T cells to TGFb. Coro1A has been established as a key player in T cell survival, migration, activation, and Ca2þ regulation in naive T cells. We show that mice lacking Coro1a developed less severe experimental autoimmune encephalomyelitis (EAE). Unexpectedly, upon the re-induction of EAE, Coro1a/ mice exhibited enhanced EAE signs that correlated with increased numbers of IL-17 producing CD4þ cells in the central nervous system (CNS) compared to wild-type mice. In vitro differentiated Coro1a/ Th17 CD4þ T cells consistently produced more IL-17 than wild-type cells and displayed a Th17/Th1-like phenotype in regard to the expression of the Th1 markers T-bet and IFNg. Mechanistically, the Coro1a/ Th17 cell phenotype correlated with a severe defect in TGFbR-mediated SMAD3 activation. Taken together, these data provide experimental evidence of a non-redundant role of Coro1A in the regulation of Th17 CD4þ cell effector functions and, subsequently, in the development of autoimmunity. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: EAE Coronin 1A TGFb signaling Th17

1. Introduction Coronins, actin regulators belonging to the WD-repeat superfamily, are found in all eukaryotes and have been highly conserved over evolution [1,2]. Coronin 1a (Coro1A), which is almost exclusively expressed in hematopoietic lineages, has been studied in the immunological field over the last few years and established to play an important role during T lymphocyte activation, migration, survival, and calcium signaling [3e6]. Coro1a-deficient mice have reduced peripheral T cells due to both an impaired survival of naive cells and migration defects [3e5]. Both CD4þ and CD8þ T cell subsets are decreased in the periphery of Coro1a/ mice while thymic cellularity and subpopulations are similar to those observed in wildtype mice [3,4,7]. A nonsense mutation in Coro1a has been shown to

Abbreviations: CNS, central nervous system; LN, lymph nodes; EAE, experimental autoimmune encephalomyelitis; PDBu, phorbol-12,13-dibutyrate; SC, spinal cord; TGFb, transforming growth factor b1; TGFbR, transforming growth factor b receptor. * Corresponding author. Tel.: þ43 512 9003 70514; fax: þ43 512 9003 73510. E-mail address: [email protected] (G. Baier). 0896-8411/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2011.05.018

protect against systemic lupus in a mouse model [7]. Recent work has shown that Coro1a is mutated in a thymic egress-deficient mouse strain, but also in a patient with severe combined immunodeficiency (SCID) [6]. However, little is known about the role of Coro1A in CD4þ effector/memory T cell differentiation and function. Naive CD4þ T cells are able to differentiate in distinct subsets characterized by the expression of specific transcription factors and the secretion of signature cytokines [8]. Th1 cells are characterized by the expression of T-bet and STAT4 and produce IFNg while Th17 cells express RORg-t and produce IL-17 [9]. Th17 cells are critically involved in tissue inflammation and autoimmune diseases [10,11]. The important cytokines for Th17 differentiation include TGFb, IL-6, and/or IL-21 [12e17]. TGFb has been shown to play an essential role during Th17 differentiation by inhibiting Th1 markers and maintaining the Th17 phenotype [18e21]. Finally, in the absence of proinflammatory cytokines, TGFb drives the differentiation of naive CD4þ T cells in inducible regulatory T cells (iTreg) characterized by the expression of FoxP3 [12]. An increasing interest in how TGFb functions on T cells are regulated has revealed some of the molecular mechanisms involved at the level of signal transduction, epigenetics, transcription factor binding, and gene activation. The

S. Kaminski et al. / Journal of Autoimmunity 37 (2011) 198e208

SMAD pathway is the canonical signaling pathway mediating TGFb activation [22]. Upon stimulation, TGFb dimers bind to the constitutively active type II receptor that, in turn, phosphorylates the type I receptor. The activated receptor complex phosphorylates receptor-regulated SMAD proteins (R-SMAD) SMAD2 and SMAD3. The R-SMADs oligomerize with the common SMAD4, and this complex enters the nucleus where it modulates target geneexpression [22e26]. The TGFb signaling pathway is closely linked to the cytoskeleton. TGFb modulates cell morphology and the actin cytoskeleton in a broad range of cell types [27e31], but TGFb signaling also seems to be dependent on the cytoskeleton, during processes such as receptor trafficking and nucleocytoplasmic shuttling of SMAD proteins [29,32]. Interestingly, other proteins involved in actin cytoskeleton regulation, such as Wiskott-Aldrich syndrome protein (WASP), have been established as regulators of adaptive immune responses. Mice lacking WASP exhibit immune deficiency as well as signs of autoimmune processes because WASP is required for the proliferation and suppressive function of, and TGFb production by, natural Treg cells (nTreg) [33,34]. Given the importance of TGFb in the regulation of the immune system, its link to the cytoskeleton, and the lack of knowledge concerning the function of Coro1A in CD4þ effector/memory T cells, we examined the role of Coro1A in the Th17 subset using in vivo and in vitro experimental approaches in Coro1a/ mice. Using an antigen-induced autoimmune disease model, we found that Coro1A is essential for the induction of experimental autoimmune encephalomyelitis (EAE). When re-challenged, Coro1a/ mice unexpectedly displayed increased clinical signs that correlated with higher IL-17 production compared to wild-type mice. Consistent with this result, differentiated Coro1a/ Th17 CD4þ T cells produced more IL-17 than their wild-type counterparts in vitro. RORg-t expression was not significantly different between the two genotypes. Interestingly, a population of in vitro differentiated Coro1a/ Th17 cells also expressed IFNg and T-bet, suggesting that Coro1A might be involved in the down-regulation of the Th1 subset. Finally, we showed that the heterogeneous Coro1a/ Th17 CD4þ T cell phenotype correlates with a defect in SMAD3-mediated TGFb receptor signaling. These results establish Coro1A as a positive regulator of TGFbR signaling and, subsequently, Th17 CD4þ T cell effector functions.

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USA). Additionally, 200 ng pertussis toxin (Sigma Aldrich) was injected intravenously on day 0 and day 2. EAE was scored according to a 0e4 scale as follows: 0, no clinical signs; 0.5, tail weakness; 1, completely limp tail; 1.5, limp tail and hindlimb weakness; 2, unilateral hindlimb paralysis; 2.5, bilateral partial hindlimb paralysis; 3, bilateral hindlimb paralysis; 3.5, complete hindlimb and unilateral forelimb paralysis; 4, death or moribund. For the reinduction of EAE, the same protocol was used for the second injection 35e50 days after the initial induction. 2.3. Preparation of mononuclear infiltrating cells Mononuclear infiltrating cells were isolated from the CNS and SC as described by Korn et al. (2007) [35]. Briefly, mice were perfused through the left cardiac ventricle, and the brain and SC were removed and flushed with PBS. Tissues were then digested with collagenase D (2.5 mg/ml; Roche Diagnostics) and DNaseI (1 mg/ml; Sigma) at 37  C for 45 min. Homogenate was passed through a 70 mm cell strainer and mononuclear cells isolated by percoll density centrifugation (70%e30%). Cells were removed from the interphase, washed, and resuspended in IMDM complete medium (10% FCS, 2 mM L-Glutamine and 50 U.ml1 penicillin/streptomycin). 2.4. In vitro Th17 differentiation Naive CD4þ T cells were isolated using the CD4þ CD62Lþ T cell isolation kit II (Miltenyi Biotech). Polarization of CD4þ T cells into Th17 cells was performed in complete IMDM medium supplemented with TGFb (5 ng/ml), IL-6 (20 ng/ml), IL-23 (10 ng/ml), antiIFNg, and anti-IL-4 (2 mg/ml) [36,37]. 2.5. Flow cytometry Cells were either not re-stimulated or stimulated with PDBu/ ionomycin (50 ng/ml; 500 ng/ml) or plate-bound anti-CD3 (2 mg/ ml) for 5 h in the presence of Golgi stop (BD Bioscience). The expression of CD4, IL-17, IFNg (BD Pharmingen), RORg-t, and T-bet (ebioscience) were analyzed by intracellular staining (Cytofix/ Cytoperm kit plus, BD Biosciences or ebioscience buffer) followed by FACS analysis (FACSCalibur, BD Biosciences). 2.6. Gene-expression analysis

2. Materials and methods 2.1. Mice

Total RNA was isolated with the Qiagen RNAeasy kit. First-strand cDNA synthesis was performed with oligo(dT) primers (Promega) with the Qiagen Omniscript RT kit according to the instructions of the supplier. Expression analysis for Il17a and Ifng was performed by real-time PCR on an ABI PRIM 7000 Sequence Detection System (Applied Biosystems) with TaqMan gene-expression assays and normalized to gapdh (Applied Biosystems).

Coro1a/ mice were generated in the laboratory of J.M Penninger (IMBA, Vienna). Disruption of the mouse Coro1a gene was performed using a Cre/LoxP-mediated deletion. The deleted region included all coding sequences after exon 1. Efficient deletion in homozygous animals was verified by the complete loss of Coro1A expression (Fig. 1). Coro1a/ mice were born following the expected Mendelian frequency with no differences in growth, weight, viability and fertility. All experiments shown used mice that were backcrossed to C57BL/6 and wild-type littermates as control mice and maintained under specific pathogen-free (SPF) conditions. Experiments were performed using 6e12-week-old mice from a C57BL/6 background and complied with the current laws of Austria.

Naive and Th17 differentiated CD4þ T cells were stimulated with 5 ng/ml TGFb for the indicated time. Cells were lysed and nuclear extracts prepared and used in EMSA [36]. The following oligonucleotide was used as a probe: SBE 50 -G TCT AGA C CA-30 (Santa Cruz Biotechnology, Inc.).

2.2. MOG35e55 induced EAE

2.8. Western blot

Female mice were injected subcutaneously in the hind flank (100 ml) with 200 mg MOG35e55 peptide (PolyPeptide group, France) in complete Freund’s adjuvant (CFA) containing 5 mg/ml H37RA (Mycobacterium tuberculosis, Difco Laboratories, Detroit, Michigan,

Cells were stimulated with TGFb (5 ng/ml) for the indicated time, washed, and lysed in sample buffer. Whole cell extracts or nuclear extracts were electrophoresed on a NuPAGE gel (Invitrogen) and transferred to a PVDF membrane. Protein lysates were

2.7. Gel mobility-shift assay

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Fig. 1. Reduced severity of EAE and fewer CD4þ infiltrating cells in Coro1a-deficient mice. Induction of EAE in wild-type and Coro1a/ mice on a C57BL/6 background. Mice were immunized with MOG35e55 to induce EAE and disease severity scored. (A) The mean clinical score of five independent experiments (wild-type n ¼ 38, Coro1a/ n ¼ 26) over time. Only mice with clinical scores 0.5 are represented (wild-type n ¼ 35, Coro1a/ n ¼ 5). (B) Coro1A expression in whole cell lysates from wild-type and Coro1a/ T cells (spleen and LN) by western blot. (CeD) CD4þ cells were isolated from the brain, SC, and draining LNs and stimulated for 4 h with PDBu plus ionomycin in the presence of Golgi stop. Represented data are from two independent experiments with n ¼ 3 mice per group per experiment. (C) Mean absolute CD4þ cell numbers  SEM present in the brain, spinal cord (SC), and draining lymph nodes (LN) of immunized animals during the onset of disease (10e14 days). (D) Mean percent  SEM of gated CD4þ cells expressing IL-17 and IFNg. *p < 0.05; **p < 0.01; ***p < 0.001.

subjected to immunoblotting with antibodies from Cell Signaling against phospho-SMAD2, phospho-SMAD3 (C25A9), SMAD2 (L16D3), and SMAD3 (C67H9) and from Santa Cruz for T-bet, TGFbRI (H-100), Coro1A (14.1) and b-actin (C-11). 2.9. Statistical analysis The differences between wild-type and Coro1a/ were analyzed by an unpaired Student t test. Significant differences are indicated as: *p < 0.05; **p < 0.01; ***p < 0.001. 3. Results 3.1. Decreased severity and incidence of EAE in Coro1a/ mice To investigate the role of Coro1A in effector CD4þ T cells in vivo, particularly in Th17 CD4þ T cells, we used the well-established autoimmune model EAE [38]. Although EAE has been first described as a Th1 driven disease, it is now clear that Th17 cells are

the major effector cells in the development of active EAE induced by injection of MOG35e55 peptide [39e41]. The EAE developement is characterized by the expansion of MOG-specific CD4þ T cells in draining lymph nodes (LN) and by the infiltration of these cells into the CNS and the spinal cord leading to inflammation [35]. C57BL/6 wild-type and C57BL/6 Coro1a/ mice were immunized subcutaneously with MOG35e55 peptide in complete Freund’s adjuvant (CFA) on day 0 and intravenously injected with pertussis toxin (PT) on days 0 and 2. Coro1a/ mice displayed reduced disease severity, which was reflected by the mild scores observed in the knockout mice compared to the wild-type mice (Fig. 1A, B and Table 1). The incidence of disease was also reduced by 73% in Coro1a/ mice (19% vs. 92%). Compared to the wild-type mice, signs of paralysis were slightly delayed in the knockout mice (15  0.71 days vs. 12.97  0.36 days), indicating that early events, such as inflammatory cell recruitment or migration, might be decreased in Coro1a/ mice. To understand why Coro1a/ mice were resistant to EAE, the spinal cord (SC), central nervous system (CNS), and draining LN were examined. At disease onset, CNS and SC-infiltrating CD4þ cells

S. Kaminski et al. / Journal of Autoimmunity 37 (2011) 198e208 Table 1 Clinical features of MOG35e55 induced EAE.

WT Coro1ae/e

No. of mice

Incidence

Mean EAE onset

Mean Max. score

38 26

35/38 (92%) 5/26 (19%)

12.97  0.36 15  0.71*

1.78  0.15 0.8  0.2**

Results are represented as mean  SEM and show the total number of individual mice in five independent experiments. *p < 0.05; **p < 0.01.

were isolated and analyzed by flow cytometry. Of note, as expected, no significant numbers of CD4þ cells were present in the brain and in the SC of healthy mice from both genotypes (data not shown). As shown Fig. 1C, counts of CD4þ cells were decreased in the brain, in which cells were almost absent, and in the SC of Coro1a/ mice compared to wild-type mice (w4-fold and w2,5-fold fewer cells, respectively). These results demonstrate that the lack of severity is due to fewer pathogenic cells infiltrating the brain in Coro1a/ mice. As described by others, Coro1a/ mice display a strong lymphopenia in secondary lymphoid organs [3e5,7]. Interestingly, while the number of CD4þ T cells was also decreased in the draining LN of EAE-induced Coro1a/ mice, their numbers were w10-fold higher than in naive Coro1a/ mice (Fig. S1). In contrast, the number of CD4þ T cells in draining LN of wild-type mice presented a w2-fold increase compared to healthy mice (Fig. S1). These results suggest that there is no defect in MOG35e55-induced CD4þ cell expansion or recruitment in Coro1a/ mice. Because IL-17 and IFNg are the two major cytokines involved in the development of EAE, we studied their expression in the CNS and SC-infiltrating CD4þ cells at the onset of disease [40,42]. Interestingly, the percentage of IL-17þ and IFNgþ cells were not significantly different in the brain between the two genotypes while their expression was strongly decreased in the SC of Coro1a/ mice compared to wild-type mice (Fig. 1D left and middle panels). Although in widely lower number, CD4þ cells from Coro1a/ LN showed an increased expression of both cytokines (Fig. 1D, right panel). Taken together, these data demonstrate that Coro1A is required for the initiation of EAE. 3.2. Coro1a/ mice have higher clinical scores after re-induction of EAE Different protocols are used to induce active EAE. One of them consists of a second injection after the mice fully recover. This protocol, the so-called re-induction of EAE, enables us to study the role of Coro1A in the development of EAE under strong inflammatory conditions. Therefore, Coro1a/ and wild-type mice were immunized a second time after full remission, following the same protocol as the first induction. Although, wild-type mice develop comparable scores after the first and second immunization of MOG35e55, Coro1a/ mice responded differently (Table 2). The disease incidence was significantly enhanced from 19% with one injection to 75% with re-induction, but it remained below the efficiency levels observed in the wild-type mice. The onset of disease was, again, slightly delayed (9.13  0.58 vs. 7.21  0.26 days in wt). More importantly, Coro1a/ mice were able to develop EAE with higher Table 2 Clinical features of MOG35e55 re-induced EAE.

WT Coro1ae/e

No. of mice

Incidence

Mean EAE onset

Mean Max. score

19 20

19/19 (100%) 15/20 (75%)

7.21  0.26 9  0.58**

1.72  0.24 2.55  0.21*

Results are represented as mean  SEM and show the total number of individual mice in four independent experiments. *p < 0.05; **p < 0.01.

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scores (2.55  0.21) than their wild-type counterparts (1.72  0.24; Fig. 2A and Table 2). These results correlated with the presence of the same numbers of CD4þ cells in the brain of wild-type and Coro1a/ mice (Fig. 2B). Indeed, the number of brain-infiltrating CD4þ cells was w10-fold higher in Coro1a/ mice than after the first induction. As observed after the first immunization, the draining LN of Coro1a/ mice contained less CD4þ cells than in wild-type mice but their number increased in the same range as in wild-type mice (w3-fold and w2-fold, respectively) (Fig. 2B and Fig. S1). We next investigated expression of IL-17 and IFNg in the CNS and SC-infiltrating CD4þ as well as in the draining LN of re-immunized animals. We found that the expression of IFNg in the CNS and both IL-17 and IFNg in the SC are comparable between the two genotypes (Fig. 2C and D). However, CNS-infiltrating CD4þ cells from Coro1a/ produced significantly more IL-17 than the wild-type cells (39.25%  3.52 vs. 25.1%  6.39 in wt). Moreover, the median values, which are more robust against outliers, were also calculated (Table 3). As shown in Table 3, single IL-17þ CD4þ cells as well as double IL-17þ/ IFNgþ CD4þ cells were increased in the brain and, to a lesser extent, in the SC from Coro1a/ mice when compared to wild-type mice. These results may account for the higher scores observed in Coro1a/ mice. Interestingly, as shown after the first induction, CD4þ cells present in the draining LN of Coro1a/ mice, were able to produce significantly more IL-17 and IFNg (Fig. 2C and D, lower panels and Table 3). FoxP3 expression was found to be comparable in the draining LNs of both genotypes, excluding a major defect in Treg differentiation (data not shown). Collectively, these results indicate that Coro1A may play a role during the re-induction of EAE, modulating the pathological phenotype of Th17 cells. Furthermore, these data suggest a functional segregation of Coro1A with distinct roles in naive vs effector/memory CD4þ T cell subsets. 3.3. Coro1a/ CD4þ T cells differentiate under Th17 conditions but exhibit increased levels of IL-17 and IFNg in vitro In order to understand the effects of Coro1a deficiency on cytokines production during Th17 differentiation, we polarized naive Coro1a/ and wild-type CD4þ T cells under Th17 conditions in vitro. We then investigated the expression of IL-17 and IFNg in the differentiated cells using FACS analysis. Because Coro1a/ T cells have a defect in calcium signaling that can be overcome by stimulation with PDBu/ionomycin [4,5], cells were re-stimulated either with PDBu/ionomycin or anti-CD3. Th17 CD4þ T cells from Coro1a/ mice produced significantly more IL-17 than the wildtype cells under basal conditions (without re-stimulation) or when stimulated with anti-CD3, but not when stimulated with PDBu/ionomycin (Fig. 3A and B and Fig. S2). Coro1a/ Th17 CD4þ T cells displayed an approximately 4-fold and 2-fold increase in the percentage of total IL-17þ cells compared to wild-type cells, both under basal conditions and upon re-stimulation with anti-CD3, respectively (Fig. S2). Interestingly, under all conditions and proportional to the strength of stimulation, Coro1a/ Th17 CD4þ T cells produced more IFNg than their wild-type counterparts. Similar observations were made for mRNA expression of Il17a and Ifng after 3 days of differentiation (Fig. 3C). We also observed the appearance of a population of Coro1a/ Th17 CD4þ T cells expressing both IL-17 and IFNg cytokines (Fig. 3A and B). Taken together, these results show that the resistance of Coro1a/ mice to the induction of EAE is not caused by abrogated Th17 cell differentiation or reduced IL-17 production. These data confirm our in vivo observations showing increased levels of IL-17 in CNS-infiltrating CD4þ cells and both IL-17 and IFNg in CD4þ cells from the draining LN of Coro1a/ mice. Furthermore, CD4þ T cells lacking Coro1a appear unable to properly regulate IFNg and IL-17 expression during Th17 differentiation in vitro.

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Fig. 2. Coro1a/ mice develop EAE after re-induction. Mice were immunized a first time as in Fig. 1 and re-immunized 35e40 days later using the same protocol. (A) The graph represents the mean clinical score over time for four independent experiments (wild-type n ¼ 19, Coro1a/ n ¼ 20). Only mice with clinical scores 0.5 are represented (wild-type n ¼ 19, Coro1a/ n ¼ 15). (BeD) Data are from two independent experiments with n ¼ 3 mice per group per experiment. (B) Mean absolute CD4þ cell number in the brain, spinal cord (SC), and draining lymph nodes (LN). (C) Representative FACS plots of IL-17 and IFNg expression in gated CD4þ cells isolated from the brain, SC, and draining LNs of EAE mice during the onset of disease (7e10 days after the second injection). Cells were isolated and stimulated for 4 h with PDBu plus ionomycin in the presence of Golgi stop. (D) Mean percent  SEM of gated CD4þ cells expressing IL-17 and IFNg or both cytokines in the brain, SC or draining LNs. *p < 0.05; **p < 0.01; ***p < 0.001.

3.4. Differentiated Coro1a/ Th17 CD4þ T cells show normal RORg-t expression and increased levels of the Th1 marker T-bet in vitro To determine whether Coro1a/ Th17 CD4þ T cells were properly differentiated, we investigated the expression of the Th17specific transcription factor RORg-t. In order to correlate the expression of IFNg with the Th17 subset, we studied its expression

relative to RORg-t. After differentiation under Th17 polarizing conditions, cells were re-stimulated, or not, and the intracellular expression of RORg-t and IFNg measured by flow cytometry. The expression of RORg-t was not significantly different between both genotypes (Fig. 4A and B), indicating that the increased expression of IL-17 in Coro1a/ Th17 differentiated cells is not caused by an abnormal level of RORg-t expression. Moreover, the majority of IFNgþ CD4þ T cells also expressed RORg-t, confirming the potential

S. Kaminski et al. / Journal of Autoimmunity 37 (2011) 198e208 Table 3 Median percent of cytokine-producing CD4þ cells after MOG35e55 re-induced EAE.

Median (%)

Brain SC LN

WT Coro1ae/e WT Coro1ae/e WT Coro1ae/e

IL-17þ

IFNgþ

IL-17þ/IFNgþ

IL-17þ total

IFNgþ total

12.9 30.2 8.4 12.7 1.2 5.1

23.9 21.7 31.1 30.2 5.5 11.4

6.5 11.8 6.6 9.3 0.2 1.1

19.4 37.4 18 24.5 1.5 6.1

35.9 37.6 43.3 44.9 5.7 12.4

Results are from two independent experiments with n ¼ 3 mice per group per experiment.

of Coro1a/ Th17 differentiated cells to produce aberrantly IFNg (Fig. 4A and B). Because IFNg expression is regulated by the Th1-specific transcription factor T-bet, we analyzed its expression in differentiated Coro1a/ Th17 cells. Consistent with IFNg production, T-bet expression was enhanced in Coro1a/ CD4þ/RORg-tþ Th17 cells (Fig. 4C and D). These results emphasize the importance of Coro1A for the down-regulation of Th1 markers and its role in controlling IL-17 production during Th17 cell differentiation.

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3.5. Coro1a-deficient Th17 cells exhibit a defective SMAD3-mediated TGFbR signaling These observations prompted us to study the mechanistic basis underlying the de-regulation of IL-17 and IFNg in Coro1a/ Th17 CD4þ T cells. Given the central role of TGFb in Th17 differentiation, we investigated the potential effects of Coro1a-deficiency on the TGFbR signaling pathway. Thus, we studied the effects of TGFb stimulation on SMAD2/3 DNA binding activity in naive CD4þ T cells using electrophoretic mobility shift assay (EMSA). No differences were found between Coro1a/ and control CD4þ T cells stimulated with TGFb (Fig. 5A, left panel). The same experiment was then performed with Th17 CD4þ T cells. Nuclear extracts from differentiated Th17 CD4þ T cells, either restimulated with TGFb or not, were analyzed. Importantly, Coro1a/ Th17 CD4þ T cells showed a strong SMAD2/3 DNA binding activity defect compared to control cells (Fig. 5A, right panel). Western blot analysis of whole cell extracts from Th17 CD4þ T cells showed no difference in the expression of SMAD2 and SMAD3 (Fig. 5B). Similarly, the amount of TGFbRI and TGFbRII were not affected in Coro1a/ Th17 cells (Fig. 5C, left panel). We also analyzed the cell surface expression of TGFbRII during Th17

Fig. 3. Coro1a-deficient CD4þ T cells differentiate into Th17 cells but exhibit a Th17/Th1-like cytokine phenotype in vitro. Naive CD4þ T cells isolated from Coro1a/ and control mouse spleens and LNs were differentiated under Th17 polarization conditions for 5 days. On day 5, the cells were re-stimulated for 5 h with plate-bound CD3 or PDBu/ionomycin (or not re-stimulated) in the presence of Golgi stop. Production of IL-17 and IFNg was determined by intracellular staining followed by flow cytometric analysis. (A) Representative FACS plots gated on CD4þ cells out of three independent experiments with similar results. Numbers in the quadrant represent the percentage of cytokine-producing CD4þ T cells for each condition. (B) Mean percent  SEM of gated CD4þ cells expressing IL-17 and IFNg or both cytokines from at least three independent experiments. (C) After 3 days of differentiation, Th17 CD4þ T cells were processed for mRNA quantification of the indicated genes by real-time PCR under basal conditions. Wild-type values were set as 1. Data from three independent experiments were normalized and are expressed as fold differences relative to wild-type cells. *p < 0.05; **p < 0.01; ***p < 0.001.

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Fig. 4. In vitro Th17 polarized CD4þ T cells from Coro1a-deficient mice display the same amount of RORg-t and increased expression of Th1 marker T-bet. Naive CD4þ T cells isolated from Coro1a/ and control mouse spleens and lymph nodes (LNs) were differentiated under Th17 polarization conditions for 5 days. On day 5, cells were re-stimulated for 5 h with plate-bound anti-CD3 or PDBu/ionomycin (or not re-stimulated) in the presence of Golgi stop. The expression of RORg-t, IFNg, and T-bet was assessed by intracellular staining followed by FACS analysis. (A) Representative FACS plots gated on CD4þ cells from at least three independent experiments. (B) Mean percent  SEM of gated CD4þ cells expressing RORg-t and IFNg or both from at least three independent experiments. (C) Representative FACS histograms showing the expression of T-bet in gated CD4þ/RORg-tþ cells for each stimulation condition. (D) Mean percent  SEM of Th17 polarized CD4þ/RORg-tþ cells expressing T-bet from two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

differentiation using FACS. TGFbRII surface expression levels in Coro1a/ Th17 CD4þ T cells were comparable to that of wild-type cells (Fig. 5C, right panel). Because none of the critical components of the TGFbR signaling pathway appeared to be downregulated, we next investigated the activation and translocation of SMAD2 and SMAD3 in the nucleus. Th17 CD4þ T cells were

stimulated as already described and the levels of phosphoSMAD2, phospho-SMAD3, SMAD2, and SMAD3 were assessed by Western blot. Though SMAD2 phosphorylation seemed to be normal in both genotypes, we observed a strong SMAD3 activation defect in Coro1a/ Th17 CD4þ T cells (Fig. 5D and E). Consistent with these results and in contrast to Coro1a/ naive

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Fig. 5. Coro1a-deficient Th17 cells exhibit a defect in SMAD-mediated TGFbR signaling. (A) CD4þ T cells or Th17 polarized CD4þ T cells isolated from Coro1a/ and wild-type mice were stimulated with TGFb (5 ng/ml) for the indicated times. The SMAD DNA-binding activity was analyzed in nuclear extracts of naive CD4þ T cells (left panel) and Th17 CD4þ T cells (right panel). The same results were obtained in three independent experiments (B) Total amount of SMAD family proteins and TGFbRI and RII in Th17 CD4þ T cells detected by Western blot of whole cell lysate. A representative blot out of three independent experiments with similar results is shown (C) Expression of TGFbRI and RII in Th17 CD4þ T cells detected by Western blot of whole cell lysate (left panel). Cell surface expression of TGFbRII during Th17 differentiation detected by FACS analysis (right panel). Graph represents specific fluorescence intensity/cell  SEM from three independent experiments. (D) Nuclear extracts were used to determine the amount of phosphorylated and total SMAD2 and SMAD3 proteins using Western blot. A representative blot out of three independent experiments with similar results is shown. (E) Graphs represent the normalized nuclear amounts of respective proteins from Th17 CD4þ T cells, expressed as a percentage of the maximum induction following stimulation. Data are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

CD4þ T cells (data not shown) where SMAD DNA-binding activity was unchanged, a decreased phosphorylation of SMAD3 was also obtained with whole cell extracts from Coro1a/ Th17 CD4þ T cells (data not shown). Since SMAD3 translocation is dependent

on its phosphorylation state, these results suggest that Coro1A is essential for the activation-inducing translocation of SMAD3. These data collectively suggest that Coro1A acts as a positive regulator of TGFbR signaling in Th17 CD4þ T cells.

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4. Discussion Coro1A was first discovered as an actin binding protein and has been shown to be a positive regulator in naive T cells during activation, migration, and survival processes, but its role in the effector/memory subset remains unclear [3e5,7]. In this study, we demonstrate that Coro1A regulates Th17 CD4þ T cell function via the TGFbR/SMAD3 signaling pathway. According to a very recent report, we show that Coro1a/ mice fail to fully develop EAE when immunized with MOG35e55 [43]. However, we demonstrate for the first time that Coro1a/ mice are hypersusceptible to EAE development after re-immunization. We showed that the lack of severity observed after the first immunization is due to fewer pathogenic cells infiltrating the brain and SC of the immunized Coro1a/ mice. As demonstrated in vivo and in vitro, Coro1a/ Th17 cells are able to produce both IL-17 and IFNg, the key cytokines involved during EAE induction. These data suggest that the decreased clinical signs observed in Coro1a/ mice may most likely be due to a defect in the recruitment of pathological cells into the CNS and SC of immunized animals rather than a defect in differentiation. In agreement with our results, Föger et al. showed that Coro1a/ T cells have a migration defect to CCL19, which is essential for the homing of T cells in secondary lymphoid organs and more importantly for the migration of pathological mononuclear cells to the CNS site, during EAE [3,44,45]. The reduced CD4þ cells infiltration observed in Coro1a/ mice could also result from a reduced CD4þ cell production after immunization. As shown in the draining LN, active induction of EAE shows higher proportional increase of CD4þ cells in Coro1a/ compared to wild-type mice, which makes the last hypothesis very unlikely. However, the decreased severity of the disease observed in Coro1a/ mice could also be explained by the fewer overall numbers of CD4þ cells contained in draining LN of healthy Coro1a/ mice. Indeed, one could speculate that they are too few to produce the required number of MOG35e55-specific CD4þ T cells for proper EAE development and consequent inflammation. Consistent with this hypothesis, Siegmund et al. showed that cell transfer of wild-type MOG35e55-specific CD4þ T cells into Coro1a/ mice was able to restore susceptibility to EAE only when injected with high cell amounts [43]. Furthermore, we show that Coro1a/ mice display increased disease severity when subjected to the re-induction of EAE. Consistent with higher clinical scores, numbers of Coro1a/ CNS-infiltrating CD4þ cells were equal to those observed in wild-type mice. The increased numbers of Coro1a/ CD4þ cells migrating to the brain after re-induction of EAE correlated with a rise of CD4þ cells present in the draining LN, which then reached the levels of CD4þ T cells contained in draining LN of wild-type mice after the first immunization. The reduced disease incidence observed in Coro1a/ mice, after reinduction of EAE, could be explained by the fact that some mice were still not able to produce the required amount of pathogenic cells. These results confirm that the overall counts of CD4þ T cells are the restrictive elements for EAE progression in these mice. Importantly, we show that pathogenic Th17 CD4þ cells from Coro1a/ mice are potent and produce even more IL-17 than wildtype cells, whereas the levels of single IFNg-producing cells remain comparable in the brain between both genotypes. These data imply that Coro1A negatively regulates IL-17 production in vivo. In vitro Th17 differentiation experiments confirmed this hypothesis because Th17 CD4þ T cells from Coro1a/ mice exhibited increased IL-17 under basal and CD3 re-stimulated conditions. The increased IL-17 levels are not the consequence of an increased RORg-t expression in these cells, suggesting that the differentiation in Coro1a/ Th17 cells is normal but that Coro1A exerts a downregulatory effect on IL-17 production.

Another major characteristic of the phenotype of Coro1a/ Th17 CD4þ T cells polarized in vitro is a higher proportion of IFNgpositive cells that correlated with higher T-bet expression compared to wild-type cells. These results are consistent with our in vivo observations in the draining LN and the presence of increased IL-17/IFNg double producing CD4þ cells in the CNS and in the SC of the majority of Coro1a/ mice during re-induction of EAE. Although Th17 effector cells were first described as a part of the Th1 subset, that they constitute an individualized major CD4þ T cell subset is now clear [46e48]. To explain the Th17/Th1-like phenotype displayed by Coro1a/ Th17 CD4þ T cells, we were able to demonstrate that TGFbR signaling was altered in these cells. According to our results, TGFbR signaling is known to inhibit Th1 differentiation through the down-regulation of STAT4 and T-bet signaling [19,49,50]. In addition, despite specific signals being responsible for Th1 or Th17 differentiation, recent work has shown that committed Th17 cells are able to acquire numerous characteristics of the Th1 cell subset in the absence of TGFb [21,51]. In agreement, we found that effector Coro1a/ Th17 CD4þ T cells display a strong defect in SMAD2/3 DNA binding activity compared to wild-type cells. This alteration correlated with impaired SMAD3 activation and could explain the emergence of aberrant IFNgþ cells among Coro1a/ Th17 CD4þ T cells. Accordingly, Takimoto et al. reported that the TGFb-mediated suppression of IFNg was partially impaired in both Smad2/ T cells and in Smad3/ T cells, and fully abrogated in the double knockout T cells [52,53]. Furthermore, it has been shown that Th17 cell population is heterogeneous. Indeed, Ghoreschi et al. demonstrated that Th17 cells differentiated with IL-1b, IL-6 and IL-23, in the absence of TGFb, were able to produce more IFNg and expressed higher levels of T-bet than Th17 cells differentiated in the presence of TGFb [54]. Interestingly and in agreement to our results, these cells were more pathogenic in an adoptive transfer model of EAE when compared to those differentiated with TGFb [54]. How Coro1A is specifically involved in the SMAD3-mediated TGFb signaling pathway remains unclear. Although SMAD2 and SMAD3 are highly homologous, they can be regulated by distinct pathways and modulate the transcription of different target genes [29,55e57]. Several WD-40 repeat proteins, other than Coro1A, have been shown to associate with TGFbR, such as the TGFbreceptor-interacting protein 1 (TRIP-1), the regulatory Ba subunit of protein phosphatase 2A (PP2A), and STRAP, which can also associate with SMAD2 and SMAD3 [58e60]. However, the specific roles of SMAD2 and SMAD3 proteins during Th17 differentiation are still unclear. Smad2/ mice are embryonic-lethal and Smad3/ mice display strong inflammatory diseases that correlate with constitutively activated TGFbresistant T cells [61,62]. Studies of conditional Smad2 knockout and Smad3 knockout mice differed in their conclusions relative to their roles in Th17 differentiation. Among those, an indirect regulation of Th17 cell development by SMAD2 and SMAD3 signaling was reported [53]. Furthermore, other reports showed that SMAD2 positively regulates Th17 generation and that, in agreement with our observations, the absence of SMAD3 enhances IL-17 production [63e65]. Martinez et al. also demonstrated that SMAD3 is able to bind directly to RORg-t and inhibit its DNA binding activity [64]. This mechanism could very likely explain the enhanced IL-17 production in Coro1a/ Th17 cells. Finally, and in agreement to our results, SMAD2 and SMAD3 seem to be dispensable for RORg-t induction [53]. 5. Conclusion Taken together, our results demonstrate that Coro1a/ mice develop exacerbated EAE under strong inflammatory conditions underlining a previously unrecognized role of Coro1A in

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downregulating both IL-17 and IFNg expression in Th17 cells. Mechanistically, our findings reveal an essential function of Coro1A in the TGFbR signaling pathway in Th17 effector cells. Dissecting the detailed signaling mechanisms of this non-redundant role of Coro1A in Th17 cells may contribute to an improved understanding of the molecular and cellular processes that lead to autoimmunity and inflammation.

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