Leptin Receptor Signaling in T Cells Is Required for Th17 Differentiation

Environmental 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 tissue-specific 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 (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(Δlepr) 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(Δlepr) 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.

C57BL/6 CD45.1 and CD45.2, Ox40-Cre, Rag1^−/−, and stat3^fl/fl mice were purchased from the Jackson Laboratories; Cd4-Cre mice were purchased from Taconic and maintained in our facilities. The lepr^fl/fl mice and Leptin-Luciferase 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 pathogen-free conditions, and sentinel mice from the Rag1^−/− 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.

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-CD8α, 56-0081; anti-CD44, 56-0441; anti-CD45.1, 25-0453; anti-CD45.2, 47-0454; anti-CD62L, 48-0621; anti-TCRβ, 47-5961; anti–IFN-γ, 25-7311; anti–IL-22, 24-7221; anti–retinoic acid–related orphan receptor (ROR)γt, 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 μg/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).

Naive (defined as CD4⁺CD25⁻CD62^hiCD44^lo) 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 μg/ml anti-CD3ε (17A2) and 1 μg/ml soluble anti-CD28 (37.51). Cells were then stimulated with indicated cytokines (10 ng/ml IL-1β, 20 ng/ml IL-6, 10 ng/ml IL-12, 10 ng/ml IL-23, 10 nM retinoic acid, 2 ng/ml TGF-β [regulatory T cell (Treg)], 0.2 ng/ml TGF-β [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 μM 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 was performed as previously described (11). rpl32 housekeeping gene was used to normalize samples. Primers used included: tbx21: forward 5′-ATCCTGTAATGGCTTGTGGG-3′, reverse 5′-TCAACCAGCACCAGACAGAG-3′; rpl32: forward 5′-GAAACTGGCGGAAACCCA-3′, reverse 5′-GGATCTGGCCCTTGAACCTT-3′; foxp3: forward 5′-CCCATCCCCAGGAGTCTTG-3′, reverse 5′-ACCATGACTAGGGGCACTGTA-3′; il17a: forward 5′-TGAGAGCTGCCCCTTCACTT-3′, reverse 5′-ACGCAGGTGCAGCCCA-3′; rorc: forward 5′-CCGCTGAGAGGGCTTCAC-3′, reverse 5′-TGCAGGAGTAGGCCACATTACA-3′; hif1a: forward 5′-AAACTTCAGACTCTTTGCTTCG-3′, reverse 5′-CGGCGAGAACGAGAAGAA-3′.

Colitis was induced after transfer of 5 × 10⁵ sorted naive T cells into Rag1^−/− mice, as previously described (11). For cotransfer experiments, 2.5 × 10⁵ sorted naive T cells from Cd4(Δlepr) CD45.2 mice were injected together with 2.5 × 10⁵ sorted naive T cells from C57BL/6 CD45.1 mice into Rag1^−/− 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.

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

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 μl 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).

Naive (defined as CD4⁺CD25⁻CD62^hiCD44^lo) 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 (04-906-837-001; 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.

Female animals were immunized with 100 μg 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 hind-leg paralysis; level 4, hind-leg paralysis and front-leg weakness or partial paralysis; level 5, moribund.

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

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.

To study the cell-intrinsic role of leptin signaling on T cells, we generated CD4-driven lepr-conditional knockout mice Cd4(Δlepr). 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(Δlepr) mice (Fig. 1A–C). Importantly, similarly to what was reported in the original study describing the generation of lepr^fl/fl mice (10), Cre-mediated excision of loxp-flanked lepr was variable and in some Cd4(Δlepr) 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(Δlepr) mice with no evident excision of lepr, no phenotype was observed (data now shown). However, even after preanalyzing Cd4(Δlepr) 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(Δlepr) 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 Cre⁻ 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).

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(Δlepr) 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(Δlepr) mice, and these mice also exhibited a trend toward higher frequency of IFN-γ–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(Δlepr) 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-β–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 collagen-induced 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-β, 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 RORγt 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(Δlepr) or Cre⁻ littermate control mice. Cd4(Δlepr)-derived T cells differentiated toward Th1 and Treg cells as efficiently as control Cre⁻ cells (Fig. 2D). However, differentiation toward either nonpathogenic or pathogenic Th17 cells was severely reduced (around 80%) in Cd4(Δlepr) cells (Fig. 2E, 2F). Consistently, rorc, hif1a, and il17a mRNA levels were also reduced in Th17-differentiated Cd4(Δlepr)-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 serum-free 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 lepr^fl/fl with Ox40-Cre mice, restricting Cre expression to activated/memory CD4⁺ T cells (11). We found that Ox40(Δlepr)-derived cells were also impaired in Th17 differentiation (Fig. 2I). Exogenous leptin enhanced RORγt⁺ expression in T cells from Cre⁻ control cells but had no effect in cells from Ox40(Δlepr) mice (Fig. 2I). As expected (2), no Th17 differentiation was observed in STAT3 conditional knockout mice [Cd4(Δstat3)], and exogenous leptin had no effect on Cd4(Δstat3) CD4⁺ T cells (Fig. 2I). ELISAs for secreted IL-17A and IL-22 corroborated the RORγt 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.

Complete Th17 differentiation is a stepwise process that involves multiple cytokine signaling pathways, such as TGF-β–, IL-1β–, IL-6–, IL-21–, and IL-23–mediated signaling, and several transcription factors, such as RORα, RORγt, 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(Δlepr) mice, we analyzed both upstream and downstream molecules involved in Th17 differentiation. At 48 h after initial activation, both IL-6Rα and IL-23R expression levels were slightly but reproducibly reduced in Cd4(Δlepr) cells under Th17 differentiating conditions (Fig. 3A). In addition, p-STAT3, analyzed by flow cytometry, was significantly impaired in Cd4(Δlepr)-derived cells (Fig. 3B). Western blot analysis confirmed an impairment in p-STAT3 in Cd4(Δlepr)-derived cells after 4 d under Th17 differentiating conditions (Fig. 3C). Similarly, sorted naive CD4⁺ T, but not B, cells from Cd4(Δlepr) 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 IL-6Rα–mediated STAT3 expression and phosphorylation. Overall, these results indicate that the observed defects in Th17 differentiation in Cd4(Δlepr) 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(Δstat3) mice readily expressed Foxp3 when cultured with TGF-β even in the presence of IL-6 (Fig. 3E). We therefore reasoned that because of their reduced p-STAT3 upregulation, Cd4(Δlepr)-derived T cells could be skewed toward Th1 and Treg programs when stimulated under Th17 conditions. Although Th1 differentiation was unaltered in Cd4(Δlepr), we observed that Th1-related effector molecules such as IFN-γ and T-bet were upregulated in naive CD4⁺ T from Cd4(Δlepr) mice differentiated under Th17 conditions (Fig. 3F). Furthermore, Foxp3 expression was also increased in Cd4(Δlepr) T cells differentiated under TGF-β–containing Th17 conditions from naive cells (Fig. 3G). These results indicate that Cd4(Δlepr) 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.

Next, we asked whether local intestinal responses to infection were affected in Cd4(Δlepr) mice. We used the murine analog of enteropathogenic E. coli infection, C. rodentium, which requires IL-17/IL-22–producing T cells for clearance (12, 21). Cd4(Δlepr) 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(Δstat3) mice (Fig. 4B), further supporting the conclusion that the Cd4(Δlepr) phenotype is a consequence of impaired p-STAT3 under Th17 conditions. In addition, Cd4(Δlepr) mice showed roughly 100-fold increase in C. rodentium CFUs recovered from the liver when compared with Cre⁻ 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-γ, in Cd4(Δlepr) mice (Fig. 4D, 4E). Similar to what was observed after in vitro Th17 differentiation, intestinal RORγt⁺ T cell pools were significantly reduced in Cd4(Δlepr) mice, although we did not observe differential T-bet expression between the groups (Fig. 4F). In contrast, conditional deletion of LepR in CD11c-expressing cells [Cd11c(Δlepr)] 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(Δlepr) 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(Δlepr) CD45.2⁺ sorted naive CD4⁺ T cells into Rag1^−/− host mice at a 1:1 ratio. Eight weeks posttransfer, Cd4(Δlepr) 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(Δlepr) 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(Δlepr) animals, contrary to WT controls, were unable to trigger colitis when adoptively transferred into Rag1^−/− host mice (Fig. 5C, 5D). Finally, even with impaired survival of transferred cells, Cd4(Δlepr)-derived cells clearly lacked a Th17 phenotype while showing normal or enhanced IFN-γ production (Fig. 5E), in line with the in vitro findings. These results establish a cell-intrinsic role for LepR signaling in the modulation of intestinal Th17 differentiation and protective function.

To evaluate whether pathogenic Th17 responses were also impaired in Cd4(Δlepr) beyond the intestine, we used an MOG-induced model of experimental autoimmune encephalomyelitis (EAE), which depends on RORγt (26), IL-6Rα, and IL-23R signaling, as well as on STAT3 expression by CD4⁺ T cells (27). We found that Cd4(Δlepr) 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-γ–producing CD4⁺ T cells were reduced (roughly a 5-fold reduction) in Cd4(Δlepr) mice (Fig. 6B, 6C). Consistently, the frequency of RORγt⁺ CD4⁺ T cells was 2- to 10-fold diminished in the draining lymph nodes and in the spinal cord of Cd4(Δlepr) mice, respectively (Fig. 6D). In this model, we also observed increased frequency of Foxp3⁺ Tregs in the cellular infiltrate of Cd4(Δlepr) mice (Fig. 6D).

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

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, RORγt 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-γ, TNF-α, 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 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(Δlepr)-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-6Rα/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(Δlepr) 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 phenotype in Obra^−/− 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(Δlepr) 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 IL-6R 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.

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 lepr^fl/fl strain, and members of the Friedman Laboratory (The Rockefeller University), particularly Z. Li, for providing the Obra^−/− 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.

The authors have no financial conflicts of interest.