Leptin Receptor Signaling in T Cells Is Required for Th17 Differentiation This information is current as of May 2, 2015.

Bernardo S. Reis, Kihyun Lee, Melania H. Fanok, Cristina Mascaraque, Manal Amoury, Lillian B. Cohn, Aneta Rogoz, Olof S. Dallner, Pedro M. Moraes-Vieira, Ana I. Domingos and Daniel Mucida

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol published online 27 April 2015 http://www.jimmunol.org/content/early/2015/04/25/jimmun ol.1402996

Published April 27, 2015, doi:10.4049/jimmunol.1402996 The Journal of Immunology

Leptin Receptor Signaling in T Cells Is Required for Th17 Differentiation Bernardo S. Reis,*,1 Kihyun Lee,*,1,2 Melania H. Fanok,*,1,3 Cristina Mascaraque,*,1,4 Manal Amoury,* Lillian B. Cohn,† Aneta Rogoz,* Olof S. Dallner,‡ Pedro M. Moraes-Vieira,x Ana I. Domingos,{ and Daniel Mucida*

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nvironmental cues influence both innate and adaptive immune cell differentiation. Differentiating CD4+ T cells are among the most plastic leukocytes, showing a high degree of adaptation to the surrounding milieu (1–3). One of the factors proposed to influence immune cell adaptation to tissuespecific environments is the hormone leptin, which is highly expressed by adipocytes and primarily involved in the regulation of feeding behavior and metabolism (4). A large number of studies have proposed a direct effect of leptin on a range of immune cells, including macrophages, dendritic cells, neutrophils, NK cells, T cells, and B cells (5, 6). Using leptin-deficient mice (ob/ob), several groups have reported deficient T cell development in the thymus, activation upon stimulation, and effector T cell responses

*Laboratory of Mucosal Immunology, The Rockefeller University, New York, NY 10065; †Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065; ‡Laboratory of Molecular Genetics, The Rockefeller University, New York, NY 10065; xDivision of Endocrinology, Diabetes and Metabolism, Beth Israel Medical Deaconess Center, Harvard Medical School, Boston, MA 02215; and { Obesity Laboratory, The Gulbenkian Science Institute, 2780-156 Oeiras, Portugal 1

B.S.R., K.L., M.H.F., and C.M. contributed equally to this work.

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Current address: Graduate Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, New York, NY. 3 Current address: Sackler Institute of Graduate Biomedical Studies, New York University School of Medicine, New York, NY. 4 Current address: IBD Center, Laboratory of Immunology in Gastroenterology, Humanitas Clinical and Research Center, Milan, Italy.

ORCID: 0000-0002-0000-0452 (D.M.). Received for publication December 2, 2014. Accepted for publication March 23, 2015. This work was supported by an Ellison Medical Foundation New Scholar Award in Aging (to D.M.), an Irma T. Hirschl Award (to D.M.), a Crohn’s & Colitis Foundation of America Senior Research Award (to D.M.), and National Institutes of Health Grant R01 DK093674-02 (to D.M.). Address correspondence and reprint requests to Dr. Daniel Mucida, Laboratory of Mucosal Immunology, The Rockefeller University, 1230 York Avenue, New York, NY 10065. E-mail address: [email protected] Abbreviations used in this article: EAE, experimental allergic encephalomyelitis; LepR, leptin receptor; LP, lamina propria; MOG, myelin oligodendrocyte glycoprotein; ROR, retinoic acid–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.1402996

(7). Because the leptin receptor (LepR) signals through STAT3, many of leptin’s effects on these cells have been shown to be downstream of STAT3 phosphorylation, including induction of proliferation, prevention of apoptosis, and induction of proinflammatory responses (7, 8). Consistent with a STAT3-dependent effect, studies suggest that CD4+ T cells from ob/ob mice show a reduced in vitro IL-17–producing helper cell (Th17) differentiation and, conversely, that leptin in culture enhances de novo Th17 differentiation (9). A common caveat of in vivo studies describing pleiotropic effects of leptin in the immune system is the use of either ob/ob or LepR mutant mice (db/db) mice, which develop obesity very early after weaning and severe metabolic disorders during adult life (4), conditions that affect the immune system on their own. To avoid indirect metabolic defects and to address the effects of leptin signaling in T cells in a cell-intrinsic manner, we generated T cell–specific LepR conditional knockout mice, targeting all known isoforms of LepR (10). We found that Cd4(Dlepr) mice lack IL-17–producing CD4+ T cells in the intestinal lamina propria (LP) at steady-state. LepR was required for naive CD4+ T cells to differentiate into Th17 cells in vitro and in vivo. As a consequence, Cd4(Dlepr) mice displayed increased susceptibility to extracellular bacterial infection, but resistance to Th17-related inflammatory disorders. Importantly, the impact in Th17 responses observed in mice with T cell–specific conditional deletion of all LepR isoforms was broader and more severe than previously reported in studies using obese mice with total leptin or LepR deficiency. This study establishes cell-intrinsic roles for leptin signaling in the immune system and has major implications for the understanding of direct regulation of immune effector function under leptin-modulating conditions.

Materials and Methods Mice C57BL/6 CD45.1 and CD45.2, Ox40-Cre, Rag12/2, and stat3fl/fl mice were purchased from the Jackson Laboratories; Cd4-Cre mice were purchased from Taconic and maintained in our facilities. The leprfl/fl mice and LeptinLuciferase transgenic mice were generously provided by J. Friedman (The Rockefeller University). Several of these lines were interbred in our facilities to obtain the final strains described in the text. Mice were maintained at The Rockefeller University animal facilities under specific

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The hormone leptin plays a key role in energy homeostasis, and the absence of either leptin or its receptor (LepR) leads to severe obesity and metabolic disorders. To avoid indirect effects and to address the cell-intrinsic role of leptin signaling in the immune system, we conditionally targeted LepR in T cells. In contrast with pleiotropic immune disorders reported in obese mice with leptin or LepR deficiency, we found that LepR deficiency in CD4+ T cells resulted in a selective defect in both autoimmune and protective Th17 responses. Reduced capacity for differentiation toward a Th17 phenotype by lepr-deficient T cells was attributed to reduced activation of the STAT3 and its downstream targets. This study establishes cell-intrinsic roles for LepR signaling in the immune system and suggests that leptin signaling during T cell differentiation plays a crucial role in T cell peripheral effector function. The Journal of Immunology, 2015, 194: 000–000.

2 pathogen-free conditions, and sentinel mice from the Rag12/2 mouse colony were tested to be negative for Helicobacter spp. and Citrobacter rodentium. Mice were used at 7–12 wk of age for most experiments. Animal care and experimentation were consistent with the National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee at The Rockefeller University.

Abs and flow-cytometry analysis

In vitro T cell culture Naive (defined as CD4+CD252CD62hiCD44lo) T cells were sorted using FACSAria cell-sorter flow cytometer (Becton Dickinson) and cultured for 4.5 d in 96-well plates precoated with 2 mg/ml anti-CD3ε (17A2) and 1 mg/ml soluble anti-CD28 (37.51). Cells were then stimulated with indicated cytokines (10 ng/ml IL-1b, 20 ng/ml IL-6, 10 ng/ml IL-12, 10 ng/ml IL-23, 10 nM retinoic acid, 2 ng/ml TGF-b [regulatory T cell (Treg)], 0.2 ng/ml TGF-b [Th17]) in RPMI 1640 (Invitrogen) containing 10% FCS (Sigma), 1% L-glutamine (Life Technologies), 25 mM HEPES (Life Technologies), 1% essential amino acid mixture (Life Technologies), 5 mM 2-ME, and 1% pen-strep antibiotics (Life Technologies). Where indicated, cells were stimulated in serum-free media X-VIVO 20 (Lonza) supplemented with the after mentioned components. For in vitro block of leptin signaling, cells were incubated with 250 ng/ml mouse LepR fusioned to Fc portion of Ig (LepR:Fc chimera; R&D Systems). For restimulation experiments, cells were cultured for 4.5 d as described earlier and resuspended in new media containing the indicated cytokines for another 72 h.

Quantitative PCR Quantitative PCR was performed as previously described (11). rpl32 housekeeping gene was used to normalize samples. Primers used included: tbx21: forward 59-ATCCTGTAATGGCTTGTGGG-39, reverse 59-TCAACCAGCACCAGACAGAG-39; rpl32: forward 59-GAAACTGGCGGAAACCCA-39, reverse 59-GGATCTGGCCCTTGAACCTT-39; foxp3: forward 59-CCCATCCCCAGGAGTCTTG-39, reverse 59-ACCATGACTAGGGGCACTGTA-39; il17a: forward 59-TGAGAGCTGCCCCTTCACTT-39, reverse 59-ACGCAGGTGCAGCCCA-39; rorc: forward 59-CCGCTGAGAGGGCTTCAC-39, reverse 59-TGCAGGAGTAGGCCACATTACA-39; hif1a: forward 59-AAACTTCAGACTCTTTGCTTCG-39, reverse 59-CGGCGAGAACGAGAAGAA-39.

Experimental colitis model Colitis was induced after transfer of 5 3 105 sorted naive T cells into Rag12/2 mice, as previously described (11). For cotransfer experiments, 2.5 3 105 sorted naive T cells from Cd4(Dlepr) CD45.2 mice were injected together with 2.5 3 105 sorted naive T cells from C57BL/6 CD45.1 mice into Rag12/2 mice. Recipient mice were monitored regularly for signs of disease including weight loss, hunched posture, piloerection of the coat, and diarrhea, and analyzed at various times after the initial transfer or when they reached 80% of their initial weight.

C. rodentium infection Mice were infected with 2 3 108 C. rodentium per animal, as previously described (12). Bacteria were inoculated by gavage in recipient mice in a total volume of 200 ml sterile PBS. Postinfection, mice were followed daily for weight loss and CFUs in feces and liver. Mice were sacrificed and analyzed 18 d postinfection.

Leptin activity by in vivo imaging In vivo imaging of transgenic animals was performed using the Xenogen IVIS Lumina imaging system (Caliper). Anesthetized animals were injected

i.p. with luciferin (200 ml of stock 15 mg/ml in PBS). After 15–20 min, the animals were imaged in an imaging chamber and the photon image was analyzed by Living Image 3.0 software (Xenogen).

Phosphorylated and total STAT3 Western blot analysis Naive (defined as CD4+CD252CD62hiCD44lo) T cells were sorted using FACSAria cell sorter flow cytometer (Becton Dickinson) and rested for 30 min at 37˚C in serum-free medium. Cells were then stimulated with 20 ng IL-6 for 30 min and protein was extracted at 4˚C for 15 min using radioimmunoprecipitation assay buffer buffer plus Phospho Stop (04-906837-001; Roche) and proteinase inhibitor (539-134; Calbiochem). Cell protein extract was subjected to electrophoresis separation and transfer to polyvinylidene difluoride membrane. The membrane was blocked for 1 h with TBST 5% milk, incubated overnight with anti–phospho-STAT3 Ab (Y705; Cell Signaling Technology), and developed using secondary Ab conjugated to HRP. Anti–total STAT3 Ab (79D7; Cell Signaling Technology) was used as a control.

Induction of experimental allergic encephalomyelitis Female animals were immunized with 100 mg myelin oligodendrocyte glycoprotein (MOG) peptide emulsified in CFA 1:1 mixture intradermic in the flank. Animals were inoculated 4 h before and 2 d after immunization with 200 ng pertussis toxin (Sigma). Animals were monitored daily for weight loss and experimental allergic encephalomyelitis (EAE) symptoms. Animals were scored according to an established scoring system: level 1, limp tail; level 2, hind-leg weakness or partial paralysis; level 3, total hindleg paralysis; level 4, hind-leg paralysis and front-leg weakness or partial paralysis; level 5, moribund.

Preparation of intraepithelial and LP lymphocytes Intraepithelial and LP lymphocytes were isolated as previously described (11).

Statistics Statistical analyses were performed in GraphPad Prism software. Data were analyzed by applying one-way ANOVA or unpaired Student t test whenever necessary. For analysis of histological scores, nonparametric Mann– Whitney tests were used. A p value , 0.05 was considered significant.

Results LepR signaling is required for Th17 differentiation To study the cell-intrinsic role of leptin signaling on T cells, we generated CD4-driven lepr-conditional knockout mice Cd4(Dlepr). The Cd4-Cre construct was generated using T cell– specific minimal Cd4 enhancer/promoter, which avoids targeting of other CD4+ cell populations such as innate lymphoid cells (13). PCR analysis for “floxed” exon 1 of lepr confirmed deletion in CD4+ T, but not in B, cells isolated from Cd4(Dlepr) mice (Fig. 1A–C). Importantly, similarly to what was reported in the original study describing the generation of leprfl/fl mice (10), Cre-mediated excision of loxp-flanked lepr was variable and in some Cd4(Dlepr) mice (20–30%) we found no evidence for recombination. Due to this relatively high inefficiency of lepr excision (irrespective of the Cre line used), in most of the experiments described earlier, we FACS-sorted peripheral blood CD4+ T cells and B cells to confirm “floxed” lepr alleles prior to the analysis. As expected, in Cd4(Dlepr) mice with no evident excision of lepr, no phenotype was observed (data now shown). However, even after preanalyzing Cd4(Dlepr) mice for excised floxed lepr alleles, some experimental variation was noted, which may have been a consequence of incomplete excision of lepr in T cells. As expected, in Cd4(Dlepr) mice we found no signs of obesity (Fig. 1D) or the other gross metabolic defects described in ob/ob or db/db mice (data not shown). Overall, T cell populations were also relatively similar to those in Cre2 littermate controls in lymphoid and nonlymphoid tissues analyzed, although we found a small but significant decrease in the CD4/CD8 ratio in the mesenteric lymph nodes (Fig. 1E, 1F).

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Fluorescent-dye–conjugated Abs were purchased from BD Pharmingen (anti-CD4, 550954; anti-CD25, 553866; anti-CD103, 557495; anti–IL-17a, 559502; anti–T-bet, 561312) or eBioscience (anti-CD8a, 56-0081; antiCD44, 56-0441; anti-CD45.1, 25-0453; anti-CD45.2, 47-0454; antiCD62L, 48-0621; anti-TCRb, 47-5961; anti–IFN-g, 25-7311; anti–IL-22, 24-7221; anti–retinoic acid–related orphan receptor (ROR)gt, 12-6981; anti-Foxp3, 17-5773). Flow-cytometry data were acquired on an LSR-II flow cytometer (Becton Dickinson) and analyzed using FlowJo software package (Tree Star). Intracellular staining of Foxp3 was conducted using a Foxp3 Mouse Regulatory T cell Staining Kit (eBioscience). For flow-cytometric analysis of cytokine-secreting cells, cells were incubated in the presence of 100 ng/ml PMA (Sigma), 500 ng/ml Ionomycin (Sigma) for 4.5 h, and 10 mg/ml brefeldin A (Sigma) for the last 2.5 h before staining. Cell populations were first stained with Abs against the indicated cell-surface markers, followed by permeabilization in Fix/Perm buffer, and intracellular staining in Perm/Wash buffer (BD Pharmingen).

LepR IS REQUIRED FOR Th17 CELL DIFFERENTIATION

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To address whether LepR is involved in the physiological regulation of CD4+ Th cell differentiation in the gut, we analyzed Foxp3-expressing Tregs and IL-17A–producing Th17 cells in the intestinal LP of Cd4(Dlepr) mice. Although we did not find differences in the frequency of Foxp3+ Treg cells in the thymus or peripheral tissues between the groups (Fig. 1G), the frequency of Foxp3+ Tregs was slightly increased in the LP of Cd4(Dlepr) mice, and these mice also exhibited a trend toward higher frequency of IFN-g–producing CD4+ T cells in the LP (Fig. 2A). However, we found ∼50% reduction in the frequency of IL-17A– producing CD4+ T cells in the small intestine LP of Cd4(Dlepr) mice (Fig. 2A). The earlier data suggest a cell-intrinsic role for the LepR in effector CD4+ T cell differentiation. Recent studies propose that leptin enhances IL-6– and TGF-b– induced Th17 differentiation in vitro, while blocking leptin/LepR results in the opposite effect (9, 14, 15). Accordingly, in vivo administration of leptin exacerbates disease in a collageninduced arthritis model (15). Using different Th17-conditioning cytokines, we indeed observed reduced Th17 differentiation of naive CD4+ T cells isolated from db/db mice, both under “nonpathogenic” (IL-6 and TGF-b, 30% reduction) and “pathogenic” (IL-6 and IL-23, 60% reduction) (16) conditions (Fig. 2B). Reduced Th17 programming from db/db-derived cells was confirmed by lower levels of RORgt expression (Fig. 2C). To directly assess the cell-specific requirement for LepR signaling in this Th pathway, avoiding possible effects of db/db-related metabolic disorders in developing or mature T cells, we performed in vitro T cell differentiation studies using naive CD4+ T cells from Cd4(Dlepr) or Cre2 littermate control mice. Cd4(Dlepr)-derived T cells differentiated toward Th1 and Treg cells as efficiently as control Cre2 cells (Fig. 2D). However, differentiation toward either nonpathogenic or pathogenic Th17 cells was severely reduced (around 80%) in Cd4(Dlepr) cells (Fig. 2E, 2F). Consistently, rorc, hif1a, and il17a mRNA levels were also reduced in Th17differentiated Cd4(Dlepr)-derived T cells (Fig. 2G). These data were confirmed using serum-free media for Th17 differentiation,

suggesting that LepR signaling might influence CD4+ T cell polarization even in the absence or low levels of leptin (Fig. 2H). In addition, blocking leptin–LepR interaction using a recombinant LepR/Fc chimera did not affect Th17 polarization under serumfree conditions, indicating that T cell–derived leptin does not play a significant role in this process (Fig. 2H). To address whether LepR expression could also directly influence activated CD4+ T cells in the process of differentiation toward Th17, we crossed leprfl/fl with Ox40-Cre mice, restricting Cre expression to activated/memory CD4+ T cells (11). We found that Ox40(Dlepr)-derived cells were also impaired in Th17 differentiation (Fig. 2I). Exogenous leptin enhanced RORgt+ expression in T cells from Cre2 control cells but had no effect in cells from Ox40(Dlepr) mice (Fig. 2I). As expected (2), no Th17 differentiation was observed in STAT3 conditional knockout mice [Cd4(Dstat3)], and exogenous leptin had no effect on Cd4(Dstat3) CD4+ T cells (Fig. 2I). ELISAs for secreted IL-17A and IL-22 corroborated the RORgt data (data not shown). The earlier data establish a crucial role for LepR expression in the differentiation of Th17 cells in vitro and in vivo, and suggest a role for STAT3 in this process. Impaired IL-6–mediated STAT3 phosphorylation in the absence of LepR Complete Th17 differentiation is a stepwise process that involves multiple cytokine signaling pathways, such as TGF-b–, IL-1b–, IL-6–, IL-21–, and IL-23–mediated signaling, and several transcription factors, such as RORa, RORgt, and STAT3 (1, 2). LepR signaling is also associated with STAT3 phosphorylation in different cell types, including immune cells (6). To address possible mechanisms involved in the impaired Th17 differentiation observed in T cells from Cd4(Dlepr) mice, we analyzed both upstream and downstream molecules involved in Th17 differentiation. At 48 h after initial activation, both IL-6Ra and IL-23R expression levels were slightly but reproducibly reduced in Cd4(Dlepr) cells under Th17 differentiating conditions

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FIGURE 1. leprfl/fl genotyping, excision validation, and general analysis of Cd4(Dlepr) mice. (A) PCR for plox-flanked lepr allele in the ear tissue from WT (+/+), heterozygous (+/fl), and homozygous (fl/fl) mice. (B) PCR for plox-flanked lepr allele from peripheral blood–sorted CD4+ T cells and B cells from WT homozygous [Cd4(Dlepr)] mice. (C) PCR for lepr-excised allele in sorted CD4+ T cells and B cells from the peripheral blood of Cd4(Dlepr). (D) Body weight of Cd4(Dlepr) mice and littermate control at 7 (left panel) and 12 (right panel) wk of age. (E) Total leukocyte, CD4+, and CD8+ T cells, (F) CD4+/CD8+ T cell ratio, and (G) Foxp3-expressing cells (among CD4+ T cells) in the indicated tissues from leprfl/fl (Ctrl) and Cd4(Dlepr) mice. Pooled data are from at least three independent experiments (n = 4–5/group/experiment, error bars = SEM). *p , 0.05. Primers for genotyping: LepR: forward 59-TCTAGCCCTCCAGCACTGGAC-39, reverse1 59-GTCACCTAGGTTAATGTATTC-39. Primer for excision validation: LepR: reverse2 59-GCAATTCATATCAAAACGCC-39.

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LepR IS REQUIRED FOR Th17 CELL DIFFERENTIATION

(Fig. 3A). In addition, p-STAT3, analyzed by flow cytometry, was significantly impaired in Cd4(Dlepr)-derived cells (Fig. 3B). Western blot analysis confirmed an impairment in p-STAT3 in Cd4(Dlepr)-derived cells after 4 d under Th17 differentiating conditions (Fig. 3C). Similarly, sorted naive CD4+ T, but not B, cells from Cd4(Dlepr) mice showed reduced p-STAT3 30 min after exposure to IL-6 in serum-free media, further suggesting a role for LepR signaling in Th17 polarization even in the absence of evident leptin–LepR interaction (Fig. 3D). The earlier data suggest that the LepR conditions or tunes IL-23R– and IL6Ra–mediated STAT3 expression and phosphorylation. Overall, these results indicate that the observed defects in Th17 differentiation in Cd4(Dlepr) mice are associated with impaired IL-6– mediated STAT3 function. STAT3 counteracts both STAT5- (17) and Foxp3-mediated (18, 19) transcription, whereas CD4+ T cells deficient for STAT3 show enhanced expression of Th1-related genes and a Treg-related program, depending on the cytokine milieu (20). Indeed, we observed that naive CD4+ T cells from Cd4(Dstat3) mice readily expressed Foxp3 when cultured with TGF-b even in the presence of IL-6 (Fig. 3E). We therefore reasoned that because of their reduced p-STAT3 upregulation, Cd4(Dlepr)-derived T cells could be skewed toward Th1 and Treg programs when stimulated under Th17 conditions. Although Th1 differentiation was unaltered in Cd4(Dlepr), we observed that Th1-related effector molecules such as IFN-g and T-bet were upregulated in naive CD4+ T from Cd4(Dlepr) mice differentiated under Th17 conditions (Fig. 3F). Furthermore, Foxp3 expression was also increased in Cd4(Dlepr)

T cells differentiated under TGF-b–containing Th17 conditions from naive cells (Fig. 3G). These results indicate that Cd4(Dlepr) CD4+ T cells fail to produce the appropriate p-STAT3 levels to undergo complete Th17 differentiation, resulting in alternative helper differentiation toward Treg and Th1 programs. Cd4(Dlepr) mice show impaired extracellular bacteria clearance and intestinal Th17 differentiation Next, we asked whether local intestinal responses to infection were affected in Cd4(Dlepr) mice. We used the murine analog of enteropathogenic E. coli infection, C. rodentium, which requires IL17/IL-22–producing T cells for clearance (12, 21). Cd4(Dlepr) mice showed severely impaired clearance of C. rodentium in feces, particularly after day 10 of infection, when clearance is T cell dependent (22) (Fig. 4A). These results closely resembled the reduced clearance found in Cd4(Dstat3) mice (Fig. 4B), further supporting the conclusion that the Cd4(Dlepr) phenotype is a consequence of impaired p-STAT3 under Th17 conditions. In addition, Cd4(Dlepr) mice showed roughly 100-fold increase in C. rodentium CFUs recovered from the liver when compared with Cre2 littermate controls (Fig. 4C). Analysis of cytokine production by intestinal CD4+ T cells at days 10 and 18 postinfection showed a drastic reduction in the levels of IL-17 and IL-22, but not IFN-g, in Cd4(Dlepr) mice (Fig. 4D, 4E). Similar to what was observed after in vitro Th17 differentiation, intestinal RORgt+ T cell pools were significantly reduced in Cd4(Dlepr) mice, although we did not observe differential T-bet expression between the groups (Fig. 4F). In contrast, conditional deletion of LepR in

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FIGURE 2. LepR signaling is required for Th17 cell differentiation. (A) Frequency of Foxp3-, IFN-g–, and IL-17A–expressing cells among CD4+ T cells in the LP of leprfl/fl (control) or Cd4-Cre+leprfl/fl [Cd4(Dlepr)] mice. Plots are representative of two to three independent experiments (n = 4–8/group). (B–H) Sorted naive CD4+ T cells from db/db and WT (B and C) or from leprfl/fl (control) and Cd4(Dlepr) mice (D–H); mice were cultured with plate-bound antiCD3ε and soluble anti-CD28 in the presence of the indicated cytokines. (B) Expression of intracellular IL-17A. (C and F) Expression of intracellular RORgt. Numbers indicate mean fluorescence intensity (MFI) of RORgt+ cells for each group. (D) Expression of intracellular IFN-g and Foxp3 by cells cultured with Th1- or Treg-polarizing conditions, respectively. Bar graphs depict mean 6 SEM of biological replicates pooled from at least eight independent experiments. (E) Expression of intracellular IL-17A and GM-CSF by cells with Th17-polarizing conditions. Bar graphs depict mean 6 SEM (numbers depict % of suppression between groups) of biological replicates pooled from at least eight independent experiments (each dot represents an independent experiment). *p , 0.05. (G) Expression of mRNA for rorc, hif1a, and il17a after 0, 6, and 48 h of culture with Th17-polarizing conditions. Representative data (mean 6 SEM of technical replicates) from at least five independent experiments. (H) Expression of intracellular IL-17A, IFN-g, and IL-22 by CD4+ T cells cultured under TGF-b+IL-6 conditions (in the presence or not of recombinant mouse LepR Fc chimera) with serum-free media. (I) Sorted naive CD4+ T cells from leprfl/fl (control), Ox40(Dlepr), or Cd4(Dstat3) mice were cultured as in (H) in the presence or not of leptin. (H and I) Representative data (mean 6 SEM of technical replicates) from three independent experiments.

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CD11c-expressing cells [Cd11c(Dlepr)] did not affect C. rodentium clearance or cytokine production by intestinal T cells, ruling out the possibility that LepR-deficient CD4+ dendritic cells contributed significantly to the phenotype (Fig. 4G, 4H). The earlier results indicate that in vivo protective Th17/Th22 responses to pathogens require LepR expression by T cells. Previous studies have postulated that changes in leptin secretion could be correlated with the peak of pathogenic autoimmune CD4+ T cell responses (23). We then asked whether local or systemic changes in leptin production are associated with induction of Th17 differentiation in responses to intestinal infection. To assess leptin production in vivo, we used a transgenic mouse strain that expresses a luciferase reporter gene under the control of leptin regulatory sequences (24). Animals infected with C. rodentium did not show changes in leptin-reporter activity when compared with noninfected control animals in any of the time points analyzed (Fig. 4I, 4J). These data suggest that dynamic changes in leptin secretion are not associated with in vivo Th17 differentiation during C. rodentium infection. In addition to its effects on Th17 differentiation, STAT3 is also involved in regulation of cell survival (25). To address whether

CD4+ T cells from Cd4(Dlepr) mice have reduced capacity to expand and, at the same time, to evaluate helper differentiation in another model of intestinal inflammation, we used the T cell transfer model of colitis. To directly investigate their expansion under lymphopenic conditions, we cotransferred wild-type (WT) CD45.1+ and Cd4(Dlepr) CD45.2+ sorted naive CD4+ T cells into Rag12/2 host mice at a 1:1 ratio. Eight weeks posttransfer, Cd4(Dlepr) cells represented only 20% of donor cells in different tissues examined, indicating reduced proliferative and/or survival under these conditions (Fig. 5A), and reflecting the more subtle altered CD4+/CD8+ T cell ratio observed in the donor mouse in the absence of competition for the CD4 T cell niche. We attributed this reduced frequency of CD4+ T cells from Cd4(Dlepr) mice to their reduced survival, rather than to a defective proliferation, because BrdU incorporation was similar to WT CD45.1 cells (Fig. 5B). Consistent with these data, naive CD4+ T cells from Cd4(Dlepr) animals, contrary to WT controls, were unable to trigger colitis when adoptively transferred into Rag12/2 host mice (Fig. 5C, 5D). Finally, even with impaired survival of transferred cells, Cd4(Dlepr)-derived cells clearly lacked a Th17 phenotype while showing normal or enhanced IFN-g production (Fig. 5E), in

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FIGURE 3. Impaired IL-6–induced STAT3 phosphorylation in Cd4(Dlepr) T cells. (A–C, F, and G) Sorted naive CD4+ T cells from leprfl/fl (control) or Cd4(Dlepr) mice were cultured with plate-bound anti-CD3ε and soluble anti-CD28 in the presence of the indicated cytokines. (A) Expression of IL-6Ra and IL-23R was analyzed after 48 h. Numbers indicate mean fluorescence intensity (MFI) for each group. Flow cytometry (B) and Western blot (C) for total and p-STAT3. Numbers indicate MFI for each group. WT CD4+ T cells differentiated under Th1 skewing conditions (IL-12 + anti–IFN-g) were used as negative control for p-STAT3 staining (neg). Representative data from at least three independent experiments. (D) Western blot for total and p-STAT3 in sorted CD4+ T and B cells from leprfl/fl (control) or Cd4(Dlepr) mice after 30-min incubation with IL-6. (E) Frequency of Foxp3-expressing cells from stat3fl/fl (control) or Cd4(Dstat3) mice cultured as in (A) in the presence of TGF-b 6 IL-6. Representative data (mean 6 SEM of technical replicates) from two independent experiments. (F) Frequency of IFN-g– producing and T-bet–expressing cells. (G) Frequency of Foxp3-expressing cells (left) and expression of mRNA (right) for foxp3. Graphs depict mean 6 SEM of biological replicates pooled from five independent experiments. *p , 0.05.

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LepR IS REQUIRED FOR Th17 CELL DIFFERENTIATION

line with the in vitro findings. These results establish a cellintrinsic role for LepR signaling in the modulation of intestinal Th17 differentiation and protective function. Cd4(Dlepr) mice are resistant to EAE development To evaluate whether pathogenic Th17 responses were also impaired in Cd4(Dlepr) beyond the intestine, we used an MOG-induced model of experimental autoimmune encephalomyelitis (EAE), which depends on RORgt (26), IL-6Ra, and IL-23R signaling, as well as on STAT3 expression by CD4+ T cells (27). We found that Cd4(Dlepr) mice are highly resistant to EAE development, determined both by the absence of weight loss during the course of the experiment and by the absence of EAE clinical manifestations (Fig. 6A). Although the total cell number in systemic and local tissues was similar between the groups, CD4+ T cell infiltrate into the spinal cord and consequently IL-17–producing and IFN-g– producing CD4+ T cells were reduced (roughly a 5-fold reduction) in Cd4(Dlepr) mice (Fig. 6B, 6C). Consistently, the frequency of RORgt+ CD4+ T cells was 2- to 10-fold diminished in the draining lymph nodes and in the spinal cord of Cd4(Dlepr) mice, respectively (Fig. 6D). In this model, we also observed increased frequency of Foxp3+ Tregs in the cellular infiltrate of Cd4(Dlepr) mice (Fig. 6D).

These results define an intrinsic role for LepR in the generation of pathogenic Th17 cells.

Discussion In this study we demonstrated a T cell–intrinsic requirement for LepR signaling in several aspects of Th17 differentiation in the absence of any observable systemic metabolic disorder. In mice with T cell–specific ablation of LepR, we described an impaired STAT3 phosphorylation, RORgt expression, and IL-17/IL-22 secretion in vitro and in vivo. These effects did not appear to be restricted to a particular Th17 population, because they were observed in both “pathogenic” and “nonpathogenic” in vitro Th17 conditions, as well as in natural protective and pathogenic T cell populations in vivo. Along these lines, our results suggest that LepR plays a broad role in STAT3-dependent T cell differentiation. For instance, it might influence the downstream signaling of IL-23R, which is also STAT3 dependent and plays significant roles in the onset of EAE by inducing an inflammatory milieu, which includes IFN-g, TNF-a, and IL-17 (28, 29). Previous studies have postulated that changes in leptin secretion could be correlated with the peak of pathogenic autoimmune CD4+ T cell responses (23). Additional studies have demonstrated that activation of the leptin–mammalian target of rapamycin axis is

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FIGURE 4. LepR signaling is required for protective and inflammatory intestinal Th17 responses. (A–F) Mice were orally infected with C. rodentium and analyzed 10 and 18 d postinfection (p.i.). (A and C–F) Data from infected leprfl/fl (control) and Cd4(Dlepr) mice. (B) Data from infected stat3fl/fl (control) and Cd4(Dstat3) mice. (G and H) Data from infected leprfl/fl (control) and Cd11c(Dstat3) mice. CFU of C. rodentium from fecal pellets throughout the infection (A, B, and G) or liver (C) at day 18 p.i. Pooled data from five (A and C–F) or two (B) independent experiments, n = 3–5/group/experiment (error bars = SEM). (D) Expression of IL-17A, IL-22, and IFN-g or (E) RORgt and T-bet by CD4+ T cells in the large intestine LP (LPL) and epithelial (IEL) compartments 10 d p.i. (E) Numbers indicate MFI of RORgt+ or T-bet+ cells for each group. (F and H) Frequency of IL-17A– and IFN-g–producing cells among CD4+ T cells in the LPL 18 d p.i. (I and J) Data from noninfected (control) and infected leptin-luciferase transgenic animals. Leptin-luciferase transgenic animals were infected with 2 3 109 CFU C. rodentium intragastrically, and luciferase activity was measured using the Xenogen IVIS Lumina imaging system. (I) In vivo imaging of a representative leptin-luciferase transgenic animal showing adipose-specific luciferase activity on infected (C. rodentium) or not infected (Ctrl) animals. (J) Quantification of in vivo luciferase activity using Living Image 3.0 software (mean 6 SEM). *p , 0.05, **p , 0.01.

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involved in the modulation of Treg proliferation in vivo (30). An interesting possibility is that physiological changes in leptin levels, for instance, during circadian phases, are associated with modulation of STAT3-dependent Th17 differentiation (31). However, using a leptin-reporter strain, we were unable to observe changes in leptin levels during the course of C. rodentium infection. Nevertheless, we cannot exclude the possibility that a dynamic regulation of local leptin concentration modulates the strength of Th17 differentiation, for instance, in the microenvironment surrounding T cells within the intestinal LP. A surprising aspect of experiments shown in this study is the suggestion of a broader role for LepR signaling in T cells when responding to IL-6 that could be independent of leptin binding. This is supported by the observation that Cd4(Dlepr)-derived T cells have impaired STAT3 phosphorylation and Th17 differentiation even in the absence of any exogenous source of leptin (serum-free experiments). This possibility requires further investigation to define, for instance, possible structure or signaling convergence between IL-6Ra/IL-23R and LepR pathways.

Previous studies performed in obese ob/ob or db/db mice, or studies that either blocked leptin/LepR interaction or administered exogenous leptin to mice all concur with a prominent role for LepR pathway in the regulation of Th17 differentiation (9, 14, 15). In addition, a recent study showed that in models of obesity, including high-fat-diet–fed ob/ob and db/db mice, IL-22 responses and C. rodentium clearance are impaired (32). The impact observed in Cd4(Dlepr) mice regarding Th17 differentiation was broader and more severe than previously reported in studies using obese mice with total leptin or LepR deficiency (9, 14, 15, 32). Given the genome location of the main Th17-related genes, such as il6ra (chr3), rorc (chr3), stat3 (chr11), stat5 (chr11), and gp130 (chr13), although hypothetically possible, it is unlikely that excision of lepr (chr4) directly interfered with these genes. Although there are six described isoforms of the LepR, the db/db mutation results in virtually no expression of leprb (33). Collectively, those observations suggest that LepRb is the main LepR isoform involved in the regulation of Th17 responses in models of obesity. Supporting this notion, we did not observe any Th17-related

FIGURE 6. LepR signaling in CD4+ T cells is required for EAE development. (A–D) leprfl/fl (control) and Cd4(Dlepr) mice were immunized with MOG peptide for EAE induction and analyzed 14 d postimmunization. (A) Body weight (left panel) and disease progression (right panel). (B) Total leukocyte cell number in the spleen, draining lymph nodes, and spinal cord. (C) Total CD4+ T cell number (left panel) and IL-17– or IFN-g–producing CD4+ T cell number (right panel) in the spinal cord. (D) Frequency of RORgt or Foxp3expressing cells among CD4+ T cells in the spinal cord and draining lymph nodes (dLN). Representative data from three independent experiments (n = 3–5/group; error bars = SEM). *p , 0.05, **p , 0.01, ***p , 0.001.

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FIGURE 5. LepR signaling in CD4+ T cells is required for transfer colitis development. (A–D) Sorted naive CD4+ T cells from WT CD45.1+ and Cd4(Dlepr) CD45.2+ mice were cotransferred at 1:1 ratio (A and B) or single-transferred (C–E) to Rag12/2 host mice and analyzed 40–60 d after transfer. (A) Frequency of CD45.1+ and CD45.2+ donor T cells and (B) BrdU+ cells recovered from spleen, mesenteric lymph node (mLN), and large intestine LP (LPL) of recipient mice. Animals were injected with BrdU 18 h before analysis. (C) Body weight of recipient animals after transfer of indicated cells. (D) H&E staining of the proximal colon of recipient mice. Original magnification 320. (E) Expression of IL-17 and IFN-g by CD4+ T cells from indicated tissues of the host animals. Representative data from two independent experiments with similar results (error bars = SEM). *p , 0.05.

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16.

Acknowledgments

20.

We are indebted to Klara Velinzon for sorting cells and members of the Nussenzweig Laboratory (The Rockefeller University) and employees at The Rockefeller University for continuous assistance. We thank J. Friedman for insightful discussions and for providing the leprfl/fl strain, and members of the Friedman Laboratory (The Rockefeller University), particularly Z. Li, for providing the Obra2/2 mice. We thank J. Idoyaga (Stanford University, Stanford, CA) for valuable assistance in setting up the EAE model. We thank members of the Laboratory of Mucosal Immunology (The Rockefeller University), particularly V. Pedicord and D. Esterhazy, for discussions, critical reading, and editing of the manuscript.

Disclosures

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15.

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The authors have no financial conflicts of interest. 25.

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phenotype in Obra2/2 mice, which lack LepRa, the other relatively long, and potentially functional, LepR isoform (34) (data not shown). Nonetheless, the fact that results performed in db/db mice (15) showed a less pronounced defect in Th17 differentiation than the one described in this article using Cd4(Dlepr) mice leaves open the possibility that other isoforms of LepR play a synergistic role. Alternatively, it is possible that the mutant LepRb isoform present in db/db mice retains previously uncharacterized function that is independent of leptin binding, as suggested earlier for IL6R signaling. Our findings indicate that LepR signaling regulates Th17 responses independently of obesity-induced metabolic defects, suggesting a novel physiological checkpoint for Th cell differentiation and possibly identifying an additional target for therapeutic intervention in Th17-related diseases, and further substantiating the notion that inflammatory responses are contingent to nutritional state.