Leptin receptor signaling in T cells is required for Th17 differentiation

Abstract

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 to 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 towards a Th17 phenotype by lepr-deficient T cells was attributed to reduced activation of the signal transducer and activator of transcription 3 (STAT3) and its downstream targets. This study establishes cell-intrinsic roles for leptin receptor signaling in the immune system and suggests that leptin signaling during T cell differentiation plays a crucial role in T cell peripheral effector function.

INTRODUCTION

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, T 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). Since the leptin receptor 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 pro-inflammatory 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(lepr) mice lack IL-17-producing CD4⁺ T cells in the intestinal lamina propria at steady state. LepR was required for naïve CD4⁺ T cells to differentiate into Th17 cells in vitro and in vivo. As a consequence, Cd4(Δlepr) mice displayed increased susceptibility to extracellular bacteria 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 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, Rag1^−/− and Stat3^fl/fl mice were purchased from the Jackson Laboratories, Cd4-Cre mice were purchased from Taconic and maintained in our facilities. 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 facilties under specific pathogen-free conditions and sentinel mice from the Rag1^−/− mouse colony were tested to be negative for Helicobacter spp. and C. rodentium. Mice were used at 7–12 weeks of age for most experiments. Animal care and experimentation were consistent with the NIH guidelines and were approved by the Institutional Animal Care and Use Committee at the Rockefeller University.

Antibodies and flow cytometry analysis

Fluorescent-dye-conjugated antibodies 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-TCR-β, 47-5961; anti-IFN-γ, 25-7311; anti-IL-22, 24-7221; anti-RORγt, 12-6981; anti-Foxp3, 17-5773). Flow cytometry data was acquired on a LSR-II flow cytometer (Becton Dickinson) and analyzed using FlowJo software package (Tree Star). Intracellular staining of Foxp3 was conducted using Foxp3 Mouse Regulatory T cell Staining Kit (eBioscience).

For flow cytometric analysis of cytokine-secreting cells, cells were incubated in the presence of 100ng/ml PMA (Sigma), 500ng/ml Ionomycin (Sigma) for 4.5h and 10μg/ml brefeldin A (BFA) (Sigma) for the last 2.5h prior staning. Cell populations were first stained with antibodies against the indicated cell surface markers, followed by permeabilization in Fix/Perm buffer, and intracellular staining in Perm/Wash buffer (BD Pharmingen).

In vitro T-cell culture

Naïve (defined as CD4⁺CD25⁻CD62^hiCD44^lo) T cells were sorted using FACS Aria cell sorter flow cytometer (Becton Dickinson) and cultured for 4.5 days in 96 well plates pre-coated with 2μg/ml of anti-CD3ε (17A2) and 1μg/ml of soluble anti-CD28 (37.51). Cells were then stimulated with indicated cytokines (10ng/ml of IL-1β, 20ng/ml of IL-6, 10ng/ml of IL-12, 10ng/ml of IL-23, 10nM of RA, 2ng/ml of TGF-β (Treg), 0.2ng/ml of TGF-β T_H17) in RPMI (Invitrogen) containing 10% FCS (Sigma), 1% L-glutamine (Gibco), 25mM HEPES (Gibco), 1% essential amino acid mixture (Gibco), 5μM β-mercapto ethanol and 1% pen-strep antibiotics (Gibco). 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 250ng/ml of mouse leptin receptor fusioned to Fc portion of immunoglobulin (LepR:Fc chimera (R&D)). For re-stimulation experiments, cells were cultured for 4.5 days as above and resuspended in new media containing the indicated cytokines for another 72h.

Quantitative PCR

(q)PCR was performed as previously described (11). RPL32 housekeeping gene was used to normalize samples. Primers used: Tbx21-forward 5′-ATCCTGTAATGGCTTGTGGG-3′, Tbx21-reverse 5′-TCAACCAGCACCAGACAGAG-3′; Rpl32-forward 5′-GAAACTGGCGGAAACCCA-3′, Rpl32-reverse 5′-GGATCTGGCCCTTGAACCTT-3′; FOXP3-forward 5′-CCCATCCCCAGGAGTCTTG-3′, FOXP3-reverse 5′-ACCATGACTAGGGGCACTGTA-3′; Il17a-forward 5′-TGAGAGCTGCCCCTTCACTT-3′, Il17a-reverse 5′-ACGCAGGTGCAGCCCA-3′; Rorc-forward 5′- CCGCTGAGAGGGCTTCAC-3′, Rorc-reverse 5′- TGCAGGAGTAGGCCACATTACA-3′; Hif1a-forward 5′- AAACTTCAGACTCTTTGCTTCG-3′, Hif1a-reverse 5′- CGGCGAGAACGAGAAGAA-3′.

Experimental colitis model

Colitis was induced after transfer of 5 × 10⁵ sorted naïve T cells into Rag1^−/− mice, as previously described (11). For co-transfer experiments, 2.5 × 10⁵ sorted naïve T cells from Cd4(ΔLepr) CD45.2 mice were injected together with 2.5 × 10⁵ sorted naïve 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, pilo-erection of the coat and diarrhea, and analyzed at various times after the initial transfer or when they reached 80% of their initial weight.

Citrobacter rodentium infection

Mice were infected with 2 × 10⁸ of C. rodentium per animal, as previously described (12). Bacteria were inoculated by gavage in recipient mice in a total volume of 200μl of sterile PBS. After infection, mice were followed daily for weight loss and colony forming units (CFU) in feces and liver. Mice were sacrificed and analyzed 18 days after infection.

Leptin activity by in vivo imaging

In vivo imaging of transgenic animals were performed using the Xenogen IVIS Lumina imaging system (Caliper). Anesthetised animals were injected intraperitonally with luciferin (200 μl of stock 15 mg/ml in PBS). After 15 to 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

Naïve (defined as CD4⁺CD25⁻CD62^hiCD44^lo) T cells were sorted using FACS Aria cell sorter flow cytometer (Becton Dickinson) and rested for 30 minutes at 37°C in serum free medium. Cells were then stimulated with 20ng of IL-6 for 30 minutes and protein was extracted at 4°C for 15 minutes using RIPA buffer plus Phospho Stop (Roche 04-906-837-001) and proteinase inihibitor (Calbiochem 539-134). Cell protein extract was subjected to eletrophoresis separation and transfer to PVDF membrane. The membrane was blocked for 1 hour with TBS-T 5% milk, incubated overnight with anti-phospho-STAT3 antibody (Cell Signaling Y705) and developed using secondary antibody conjugated to HRP. Anti-total STAT3 antibody (Cell Signaling 79D7) was used as a control.

Induction of Experimental Alergic Encephalomyelitis (EAE)

Female animals were immunized with 100μg of MOG peptide emulsified in CFA 1:1 mixture intradermic in the flank. Animals were inoculated 4 hour before and 2 days after immmunization with 200ng of pertussis toxin (Sigma). Animals were monitored daily for weight loss and 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.

Preparation of intraepithelial and lamina propria lymphocytes

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

Statistics

Statistical analyses were performed in GraphPad Prism software. Data was analyzed by applying one-way ANOVA or unpaired Student’s t-test whenever necessary. For analysis of histological scores non-parametric Mann- Whitney tests were used. A P value of less than 0.05 was considered significant.

RESULTS

Leptin receptor 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(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 (ILCs) (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 above 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 (not depicted). However, even after pre-analyzing 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 (not depicted). Overall, T cell populations were also relatively similar to those in Cre⁻ littermate controls in lymphoid and non-lymphoid tissues analyzed, although we found a small but significant decrease in the CD4/CD8 ratio in the mesenteric lymph nodes (Fig. 1E, 1F).

Figure 1. Lepr^fl/fl genotyping, excision validation and general analysis of Cd4(ΔLepr) mice.

(A) PCR for plox-flanked lepr allele in the ear tissue from wild-type (+/+), 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 wild-type homozygous (Cd4(ΔLepr)) mice. (C) PCR for lepr-excised allele in sorted CD4⁺ T cells and B cells from the peripheral blood of Cd4(ΔLepr). (D) Body weight of Cd4(ΔLepr) mice and litter mate control at 7 (left) and 12 (right) weeks 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 Lepr^fl/fl (Ctrl) and Cd4(ΔLepr) mice. Pooled data are from at least three independent experiments (n=4–5 per group/experiment, error bars=SEM). * p<0.05. Primers for genotyping; LepR-forward 5′-TCTAGCCCTCCAGCACTGGAC-3′, LepR-reverse1 5′-GTCACCTAGGTTAATGTATTC-3′. Primer for excision validation; LepR-reverse2 5′-GCAATTCATATCAAAACGCC-3′.

To address whether lepR is involved in the physiological regulation of CD4⁺ helper T cell differentiation in the gut, we analyzed Foxp3-expressing regulatory T (Treg) cells and IL-17A-producing Th17 cells in the intestinal lamina propria (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⁺ Treg cells 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 about 50% reduction in the frequency of IL-17A-producing CD4⁺ T cells in the small intestine LP of Cd4(Δlepr) mice (Fig. 2A). The above data suggest a cell-intrinsic role for the leptin receptor in effector CD4⁺ T cell differentiation.

Figure 2. Leptin receptor signaling is required for Th17 cell differentiation.

(A) Frequency of Foxp3, IFN-γ and IL-17A-expresing cells among CD4⁺ T cells in the lamina propria (LP) of Lepr^fl/fl (control) or Cd4-Cre⁺Lepr^fl/fl (Cd4(ΔLepr)) mice. Plots are representative of two to three independent experiments (n=4–8 per group). (B–H) Sorted naïve CD4⁺ T cells from db/db and wild-type (B, C) or from Lepr^fl/fl (control) and Cd4(ΔLepr) mice (D–H) mice were cultured with plate-bound anti-CD3ε and soluble anti-CD28 in the presence of the indicated cytokines. (B) Expression of intracellular IL-17A. (C, F) Expression of intracellular RORγt. Numbers indicate mean fluorescence intensity (MFI) of RORγt⁺ cells for each group. (D) Expression of intracellular IFNγ and Foxp3 by cells cultured with Th1- or Treg-polarizing conditions, respectively. Bar graphs depict mean ± 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 ± 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 hours of culture with Th17-polarizing conditions. Representative data (mean ± SEM of technical replicates) from at least five independent experiments. (H) Expression of intracellular IL-17A, IFN-γ and IL-22 by CD4⁺ T cells cultured under TGF-β+IL-6 conditions (in the presence or not of recombinant mouse lepR Fc chimera) with serum-free media. (I) Sorted naïve CD4⁺ T cells from Lepr^fl/fl (control), Ox40(ΔLepr) or Cd4(ΔStat3) mice were cultured as in H in the presence or not of leptin. (H, I) Representative data (mean ± SEM of technical replicates) from three independent experiments.

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 naïve CD4⁺ T cells isolated from db/db mice, both under “non-pathogenic” (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 T helper 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 naïve CD4⁺ T cells from Cd4(Δlepr) or Cre⁻ littermate control mice. Cd4(Δlepr)-derived T cells differentiated towards Th1 and Treg cells as efficiently as control Cre⁻ cells (Fig. 2D). However, differentiation towards either non-pathogenic or pathogenic Th17 cells was severely reduced (around 80%) in Cd4(Δlepr) cells (Fig. 2E, F). 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). Additionally, blocking leptin-lepR interaction using a recombinant leptin receptor/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 towards 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 (not depicted). The above 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-β, IL-1β, IL-6, IL-21 and IL-23 mediated signaling, and several transcription factors, such as RORα, RORγt and STAT3 (1, 2). Leptin receptor 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 hours post 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, STAT3 phosphorylation (pSTAT3), analyzed by flow cytometry, was significantly impaired in Cd4(Δlepr)-derived cells (Fig. 3B). Western-blot analysis confirmed an impairment in pSTAT3 in Cd4(Δlepr)-derived cells after 4 days under Th17 differentiating conditions (Fig. 3C). Similarly, sorted naïve CD4⁺ T, but not B cells from Cd4(Δlepr) mice showed reduced pSTAT3 30 minutes 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 above data suggest that the leptin receptor 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 (18, 19) mediated transcription, while 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 naïve 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 due to their reduced pSTAT3 upregulation, Cd4(Δlepr)-derived T cells could be skewed towards 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 naïve 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 naïve cells (Fig. 3G). These results indicate that Cd4(Δlepr) CD4⁺ T cells fail to produce the appropriate pSTAT3 levels to undergo complete Th17 differentiation, resulting in alternative helper differentiation towards Treg and Th1 programs.

Figure 3. Impaired IL-6-induced STAT3 phosphorylation in Cd4(Δlepr) T cells.

(A–C, F, G) Sorted naïve CD4⁺ T cells from Lepr^fl/fl (control) or Cd4(ΔLepr) mice were cultured with plate-bound anti-CD3ε and soluble anti-CD28 in the presence of the indicated cytokines. (A) Expression of IL-6Rα and IL-23R was analyzed after 48 hours. Numbers indicate MFI for each group. Flow cytometry (B) and western blot (C) for total and phosphorylated STAT3. Numbers indicate MFI for each group. Wild-type CD4⁺ T cells differentiated under Th1 skewing conditions (IL-12 + anti-IFN-γ) were used as negative control for p-STAT3 staining (neg). Representative data from at least three independent experiments. (D) Western blot for total and phosphorylated STAT3 in sorted CD4⁺ T and B cells from Lepr^fl/fl (control) or Cd4(ΔLepr) mice after 30 minutes incubation with IL-6. (E) Frequency of Foxp3-expressing cells from Stat3^fl/fl (control) or Cd4(ΔStat3) mice cultured as in A in the presence of TGFβ ± IL-6. Representative data (mean ± SEM of technical replicates) from two independent experiments. (F) Frequency of IFN-γ-producing and T-bet-expressing cells. (G) Frequency of Foxp3-expressing cells (left) and expression of mRNA (right) for Foxp3. Graphs depict mean ± SEM of biological replicates pooled from five independent experiments. * p<0.05.

Cd4(Δlepr) mice show impaired extracellular bacteria clearance and intestinal Th17 differentiation

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, Citrobacter 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 pSTAT3 under Th17 conditions. Additionally, Cd4(Δlepr) mice showed roughly 100 fold increase in C. rodentium colony-forming units (CFU) recovered from the liver when compared to Cre⁻ littermate controls (Fig. 4C). Analysis of cytokine production by intestinal CD4⁺ T cells at days 10 and 18-post infection showed a drastic reduction in the levels of IL-17 and IL-22, but not IFN-γ, in Cd4(Δlepr) mice (Fig. 4D, E). Similar to what was observed after in vitro Th17 differentiation, intestinal RORγt⁺ T cell pools were significantly reduced in Cd4(Δlepr) mice, while 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 deficiency CD4⁺ dendritic cells contributed significantly to the phenotype (Fig 4G, H). The above results indicate that in vivo protective Th17/Th22 responses to pathogens require lepR expression by T cells.

Figure 4. Leptin receptor signaling is required for protective and inflammatory intestinal Th17 responses.

(A–F) Mice were orally infected with C. rodentium and analyzed 10 and 18 days post-infection (p.i.). (A, C–F) Data from infection Lepr^fl/fl (control) and Cd4(ΔLepr) mice. (B) Data from infected Stat3^fl/fl (control) and Cd4(ΔStat3) mice. (G, H) Data from infected Lepr^fl/fl (control) and Cd11c(ΔStat3) mice. Colony-forming-unit (CFU) of C. rodentium from fecal pellets throughout the infection (A, B, G) or liver (C) at day 18 p.i. Pooled data from five (A, C–F) or from two (B) independent experiments, n=3–5 per group/experiment (error bars=SEM). * p<0.05. (D) Expression of IL-17A, IL-22 and IFN-γ or (E) RORγt and T-bet by CD4⁺ T cells in the large intestine lamina propria (LPL) and epithelial (IEL) compartments 10 days post infection. In E, numbers indicate MFI of RORγt⁺ or T-bet⁺ cells for each group. (F, H) Frequency of IL-17A and IFN-γ-producing cells among CD4⁺ T cells in the LPL 18 days post infection. (I, J) Data from non-infected (control) and infected Leptin-luciferase transgenic animals. Leptin-luciferase transgenic animals were infected with 2×10⁹ CFU of Citrobacter rodentium intragastrically and luciferase activity mesured 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 ± SEM).

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 to non-infected control animals in any of the time-points analyzed (Fig. 4I, J). These data suggests 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 co-transferred wild-type CD45.1⁺ and Cd4(Δlepr) CD45.2⁺ sorted naïve CD4⁺ T cells into Rag1^−/− host mice at a 1:1 ratio. Eight weeks post-transfer, 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⁺ to 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, since BrDU incorporation was similar to WT CD45.1 cells (Fig. 5B). Consistent with these data, naïve 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, D). 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 leptin receptor signaling in the modulation of intestinal Th17 differentiation and protective function.

Figure 5. Leptin receptor signaling in CD4⁺ T cells is required for transfer colitis development.

(A–D) Sorted naïve CD4⁺ T cells from wild-type CD45.1⁺ and Cd4(ΔLepr) CD45.2⁺ mice were co-transferred at 1:1 ratio (A, B) or single-transferred (C–E) to Rag1^−/− host mice and analyzed 40–60 days after transfer. (A) Frequency of CD45.1⁺ and CD45.2⁺ donor T cells and (B) BrdU-positive cells recovered from spleen, mesenteric lymph node (mLN) and LPL of recipient mice. Animals were injected with BrdU 18 hours before analysis. (C) Body weight of recipient animals after transfer of indicated cells. (D) Hematoxylin and eosin staining of the proximal colon of recipient mice. Original magnification, 20x. (E) Expression of IL-17 and IFN-γ 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.

Cd4(Δlepr) mice are resistant to EAE development

To evaluate whether pathogenic Th17 responses were also impaired in Cd4(Δlepr) beyond the intestine, we used a 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 in to the spinal cord and consequently IL-17-producing and IFN-γproducing CD4⁺ T cells were reduced (roughly a 5-fold reduction) in Cd4(Δlepr) mice (Fig. 6B, C). Consistently, the frequency of RORγt⁺ CD4⁺ T cells was two to ten-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⁺ Treg cells in the cellular infiltrate of Cd4(Δlepr) mice (Fig. 6D).

Figure 6. Leptin receptor signaling in CD4⁺ T cells is required for EAE development.

(A–D) Lepr^fl/fl (control) and Cd4(ΔLepr) mice were immunized with MOG peptide for EAE induction and analyzed 14 days post immunization. (A) Body weight (left) and disease progression (right). (B) Total leukocyte cell number in the spleen, draining lymph nodes and spinal cord. (C) Total CD4⁺ T cell number (left) and IL-17 or IFN-γ-producing CD4⁺ T cell number (right) in the spinal cord. (D) Frequency of RORγt or Foxp3-expressing cells among CD4⁺ T cells in the spinal cord and draining lymph nodes (dLN). Representative data from three independent experiments (n=3–5 per group; error bars=SEM). * p<0.05.

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

DISCUSSION

In this study we demonstrated a T cell-intrinsic requirement for leptin receptor 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, since they were observed in both “pathogenic” and “non-pathogenic” 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-mTOR 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, is 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 lamina propria. A surprising aspect of experiments shown here 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 in order 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 leptin receptor pathway in the regulation of Th17 differentiation (9, 14, 15). Additionally, 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 leptin receptor, the db/db mutation results in virtually no expression of leprb (33). Collectively, those observations suggest that lepRβ is the main leptin receptor 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, leptin receptor isoform (34) (not depicted). Nonetheless, the fact that results performed in db/db mice (15) showed a less pronounced defect in Th17 differentiation than the one described here 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 above for IL-6R signaling.

Our findings indicate that leptin receptor signaling regulates Th17 responses independently of obesity-induced metabolic defects, suggesting a novel physiological checkpoint for helper T 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.