Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses

Abstract

In the work presented here, we explored the influence of leptin on the kinetics of experimental autoimmune encephalomyelitis (EAE) onset, in the EAE-associated inflammatory anorexia, and in the development of pathogenic T cell responses. We found that the expression of serum leptin increased before the clinical onset of EAE in disease-susceptible C57BL/6J (H-2(b)) and SJL/J (H-2(s)) strains of mice, which are models of chronic-progressive and relapsing-remitting EAE, respectively. This increase in serum leptin correlated with disease susceptibility, reduction in food intake, and decrease in body weight. Indeed, acute starvation, which is able to prevent the increase in serum leptin, delayed disease onset and attenuated clinical symptoms by inducing a T helper 2 cytokine switch. Furthermore, immunohistochemical analysis revealed a parallel in situ production of leptin in inflammatory infiltrates and in neurons only during the acute/active phase of both chronic-progressive and relapsing-remitting EAE. We also found that leptin secretion by activated T cells sustained their proliferation in an autocrine loop, since antileptin receptor antibodies were able to inhibit the proliferative response of autoreactive T cells in vitro. Given that leptin appears to regulate EAE susceptibility, inflammatory anorexia, and pathogenic T-cell immune function, we postulate that it may offer a potential target in the treatment of multiple sclerosis.

Figures

Figure 1

Serum leptin increase precedes the acute onset of chronic-progressive EAE and correlates with disease susceptibility, body-weight loss, and food-intake inhibition in EAE-susceptible C57BL/6J wild-type mice but not in EAE-resistant leptin-deficient C57BL/6J ob/ob mice. (a) Mean clinical score (bars) and body weight (curves) of C57BL/6J wild-type littermate controls (black bars and triangles) and leptin-deficient C57BL/6J ob/ob mice (white bars and circles) after immunization with MOG35–55 peptide. Leptin-deficient mice are EAE resistant and do not lose weight after immunization, whereas wild-type controls are EAE susceptible and lose body weight. (b) Serum leptin (bars) increases before clinical onset of EAE only in wild-type controls and is undetectable in leptin-deficient mice; this increase correlates with food-intake inhibition, which is only present in wild-type animals. (c) Simple regression analysis showing a significant correlation (P = 0.0005, r = 0.89) between the change in serum leptin before and after immunization with MOG35–55 peptide (Δ indicates the increase in serum leptin) and the CDI, calculated as the sum of each daily clinical score of each single mouse (n = 10). A significant correlation was observed in wild-type control mice but not in leptin-deficient mice. One representative experiment out of two is shown. y, equation that defines this regression; R2, regression coefficient, R, correlation coefficient.

Figure 2

Serum leptin increase precedes acute onset of adoptively induced EAE and correlates with disease susceptibility, body-weight loss, and food-intake inhibition in EAE-susceptible C57BL/6J wild-type mice but not in EAE-resistant leptin-deficient C57BL/6J ob/ob mice. (a) Mean clinical score (bars) and body weight (curves) of C57BL/6J wild-type littermate controls (black bars and triangles) and leptin-deficient C57BL/6J ob/ob mice (white bars and circles) after adoptive transfer of 2.5 × 107 MOG35–55–specific CD4+ T cells. Leptin-deficient mice are EAE resistant and do not lose weight after adoptive transfer, whereas wild-type controls are EAE susceptible and lose body weight. (b) Serum leptin (bars) significantly increases before clinical onset of EAE in wild-type controls but increases very little in leptin-deficient mice; this increase correlates with food-intake inhibition, which is only present in wild-type animals. (c) Simple regression analysis showing a significant correlation (r = 0.97, P = 0.004) between the change in serum leptin before and after adoptive transfer of MOG35–55–specific CD4+ T cells (Δ indicates the increase in serum leptin) and the CDI, calculated as the sum of each daily clinical score of each single mouse (n = 5). A significant correlation was observed in wild-type control mice but not in leptin-deficient mice. One representative experiment of two is shown.

Figure 3

Serum leptin increase precedes the acute onset of relapsing-remitting EAE and correlates with disease susceptibility, body-weight loss, and food-intake inhibition in EAE-susceptible SJL/J female mice but not in EAE-resistant male mice. (a) Mean clinical score (bars) and body weight (curves) of SJL/J female mice (black bars and triangles) and male mice (white bars and circles) after immunization with PLP139–151 peptide. SJL/J male mice are EAE resistant and do not lose weight after immunization, whereas SJL/J female mice are EAE susceptible and lose body weight. (b) Serum leptin (bars) increases before clinical onset of EAE only in SJL/J female mice and is significantly lower in male mice in preimmune conditions (2080.0 ± 325.0 pg/ml in SJL/J female mice and 470.0 ± 100.0 pg/ml in SJL/J male mice, P < 0.01); the increase correlates with food-intake inhibition present only in female mice. (c) Simple regression analysis showing a significant correlation (r = 0.71, P = 0.02) between the difference in serum leptin before and after immunization with PLP139–155 peptide (Δ indicates the increase in serum leptin) and the CDI, calculated as the sum of each daily clinical score of each single mouse (n = 10). A significant correlation was observed in SJL/female mice but not in male mice. Data are accumulated and averaged from two independent experiments with similar results. y, equation that defines regression; R2, regression coefficient; R, correlation coefficient.

Figure 4

Starvation at priming with the encephalitogenic peptide of SJL/J females reduces the clinical severity of EAE by inducing a Th2 cytokine switch, reversible by recombinant leptin administration. (a) Mean clinical score (bars) and body weight (curves) of SJL/J female mice starved for 48 hours (hatched bars and circles), ad libitum–fed controls (black bars and triangles), and leptin-treated female mice (gray bars and squares). Starvation delayed disease onset and reduced clinical score and body-weight loss during the acute phase of the disease. Leptin replacement during the 48-hour starvation reversed the starvation-induced blunting of the disease, so that the EAE course was similar to that observed in the ad libitum–fed group. (b) Proliferative response of lymph node–derived T cells against PLP139–151 is impaired after 48 hours of starvation when compared with the control group. (c and d) Starvation for 48 hours reduced IFN-γ secretion but increased IL-4 production. (e) Addition of recombinant leptin to T-cell cultures is able to partially restore the capacity of T cells from starved mice to secrete IFN-γ. One representative experiment out of two is shown.

Figure 5

Lymph node and CNS expression of leptin during acute/active EAE. (a) Leptin expression in SJL/J female mouse adipose tissue used as positive control. (b and c) Expression of leptin in T cells and macrophages in a draining lymph node from SJL/J female mice after immunization with PLP139–151. (d) Leptin was not expressed in the brain of C57BL/6J ob/ob mice after immunization with MOG35–55 peptide (n = 4). (e and f) Expression of leptin in inflammatory infiltrates (white square) and in choroid plexus (arrow) during the acute phase of EAE in C57BL/J6 WT mice (n = 4). (g) Leptin was not expressed in the brain of SJL/J male mice after immunization with PLP139–151 peptide (n = 6). (h and i) Leptin expression in inflammatory lesions in the acute phase of EAE in SJL/J female mice (n = 6). (j) Cerebellum of SJL/J male mice did not express leptin after immunization with PLP139–151 peptide, whereas in k and l leptin was expressed in inflammatory infiltrates (white square) and choroid plexus (arrow) of SJL/J females. (m) Spinal cord C57BL/J6 ob/ob mice immunized with MOG35–55 peptide did not express leptin. (n and o) Expression of leptin in neurons (white square in n) and two inflammatory infiltrates around blood vessels (arrows in n) detectable during the acute phase of EAE in C57BL/6J WT mice spinal cord. (p–r) Leptin expression was revealed in T cells present in inflammatory infiltrates of the brain, cerebellum, and spinal cord (arrows) of C57BL/J6 WT mice after adoptive transfer, but it was not detectable in the CNS of C57BL/6J ob/ob mice after adoptive transfer (not shown). The white squares in b, e, h, k, and n represent the zone of higher magnification shown in c, f, i, l, and o, respectively. Magnifications, ×200 (b, d, e, g, h, j, k, and m); ×300 (a, l, and n); and ×400 (c, f, i, o, and p–r).

Figure 6

Simple regression analysis between the number of leptin-positive cells in active-EAE lesions and the inflammatory score in EAE-susceptible mice, and in vitro leptin secretion by activated CD4+ T cells. (a and b) In both chronic-progressive and relapsing-remitting EAE, there is a statistically significant positive correlation between the number of leptin-positive cells in active EAE lesions and the CNS inflammatory score. (c) PLP139–151–specific CD4+ T cells, derived from immunized mice (see Methods), secrete on PLP139–151 72-hour in vitro stimulation a small but consistent amount of immunoreactive leptin in the supernatants, when compared to unstimulated T cells (387.5 ± 96.01 pg/ml vs. 32.5 ± 5.6 pg/ml, respectively; P = 0.01).

Figure 7

Antileptin receptor (anti-ObR) blocking antibodies inhibit antigen-specific and non-antigen-specific T-cell activation. (a) The anti-PLP139–151 proliferative response of lymph node–derived T lymphocytes from immunized SJL/J female mice is completely inhibited by the addition to cell cultures of anti-ObR. (b–d) Anti-ObR partially inhibits the proliferative response of T cells from immunized SJL/J female mice toward polyclonal mitogenic stimuli such as concanavalin A, anti-CD3ε, and MLR against irradiated allogeneic splenocytes from C57BL/6J mice (see Methods). (e and f) The addition of anti-ObR antibody to concanavalin A– or anti-CD3ε–activated spleen-derived T cells, obtained from leptin receptor mutant C57BL/Ks db/db mice, is not able to affect their proliferative response as compared with the control antibody. One representative experiment out of four is shown.