Leptin neutralization interferes with pathogenic T cell autoreactivity in autoimmune encephalomyelitis

Abstract

Recent evidence has indicated that leptin, an adipocyte-secreted hormone belonging to the helical cytokine family, significantly influences immune and autoimmune responses. We investigate here the mechanisms by which in vivo abrogation of leptin effects protects SJL/J mice from proteolipid protein peptide PLP(139-151)-induced EAE, an animal model of MS. Blockade of leptin with anti-leptin Abs or with a soluble mouse leptin receptor chimera (ObR:Fc), either before or after onset of EAE, improved clinical score, slowed disease progression, reduced disease relapses, inhibited PLP(139-151)-specific T cell proliferation, and switched cytokine secretion toward a Th2/regulatory profile. This was also confirmed by induction of forkhead box p3 (Foxp3) expression in CD4 T cells in leptin-neutralized mice. Importantly, anti-leptin treatment induced a failure to downmodulate the cyclin-dependent kinase inhibitor p27 (p27) in autoreactive CD4 T cells. These effects were associated with increased tyrosine phosphorylation of both ERK1/2 and STAT6. Taken together, our data provide what we believe is a new molecular basis for leptin antagonism in EAE and envision novel strategies of leptin-based molecular targeting in the disease.

Figures

Figure 1

Leptin blockade during actively induced EAE with either anti-leptin Abs or ObR:Fc reduces the clinical severity of EAE. (A) Mean clinical score of SJL/J female mice treated with anti-mouse leptin Abs injected either from day –1 to day 1 or on days 8–11 (100 μg/d i.p.). Leptin blockade significantly delayed disease onset and reduced clinical score and body weight loss (see Table 1). Data are from 1 representative experiment of 3. (B) Mean clinical score of SJL/J female mice treated with mouse ObR:Fc chimera injected either from day –1 to day 1 or on days 8–11 (100 μg/d i.p.). Data are from 1 representative experiment of 3. §P = 0.01, day –1 to day 1, #P = 0.02, day –1 to day 1, and † P = 0.04, days 8–11, versus PBS or control Ig.

Figure 2

Leptin blockade during adoptively transferred EAE with either anti-leptin Abs or ObR:Fc chimera reduces the clinical severity of EAE. (A) Mean clinical score of SJL/J female mice treated with anti-mouse leptin Abs injected either from day –1 to day 1 or on days 8–11 (100 μg/d i.p.). Leptin blockade significantly reduced clinical score and body weight loss (see Table 1). Data are from 1 representative experiment of 3. #P = 0.02, day –1 to day 1, and **P = 0.001, days 8–11, versus PBS or control Ig; ‡P = 0.02, day –1 to day 1 versus days 8–11. (B) Mean clinical score of SJL/J female mice treated with mouse ObR:Fc chimera injected either from day –1 to day 1 or on days 8–11 (100 μg/d i.p.). Data are from 1 representative experiment of 3. **P = 0.001, day –1 to day 1 and days 8–11 versus control Ig.

Figure 3

In vivo leptin neutralization with anti-mouse leptin Abs in SJL/J mice inhibits DTH response and induces T cell hyporesponsiveness to PLP139–151 myelin peptide. (A) DTH reaction in leptin-neutralized and control Ig–treated mice measured as footpad swelling. Data are from 1 representative experiment of 2. (B) Proliferative response of lymph node–derived T cells against PLP139–151 was impaired after treatment with anti-mouse leptin-neutralizing Abs compared with the control Ig–treated group. Data are from 1 representative experiment of 3. (C) Anti-mouse leptin treatment did not affect polyclonal T cell proliferation induced with anti-CD3ε stimulation. #P = 0.02, day –1 to day 1 and days 8–11 versus control Ig.

Figure 4

In vivo leptin neutralization with mouse ObR:Fc chimera in SJL/J mice inhibits DTH response and induces T cell hyporesponsiveness to PLP139–151 myelin peptide. (A) DTH reaction in ObR:Fc leptin-neutralized and control Ig–treated mice measured as footpad swelling. Data are from 1 representative experiment of 2. #P = 0.02, day –1 to day 1 and days 8–11 versus control Ig. (B) Proliferative response of lymph node–derived T cells against PLP139–151 was impaired after treatment with mouse ObR:Fc chimera compared with the control Ig–treated group. Data are from 1 representative experiment of 3. **P = 0.001, day –1 to day 1, and #P = 0.02, days 8–11, versus control Ig. (C) ObR:Fc treatment did not affect polyclonal T cell proliferation induced with anti-CD3ε stimulation.

Figure 5

In vivo leptin neutralization with anti-mouse leptin Abs inhibits IFN-γ production and induces the secretion of IL-4 and IL-10 regulatory cytokines. (A and B) IFN-γ secretion of lymph node–derived T cells was inhibited by anti-leptin treatment when T cells were stimulated with the myelin antigen PLP139–151 (A) and by anti-CD3ε (B). (C and D) IL-4 secretion of lymph node–derived T cells was enhanced by anti-leptin treatment when T cells were stimulated with the myelin antigen PLP139–151 (C) and by anti-CD3ε (D). (E and F) IL-10 secretion of lymph node–derived T cells was markedly increased by anti-leptin treatment when T cells were stimulated with the myelin antigen PLP139–151 (E) and by anti-CD3ε (F). (A, C, and E) #P = 0.02, day –1 to day 1 and days 8–11, and **P = 0.001, days 8–11, versus control Ig. Data are from 1 representative experiment of 3. (B, D, and F) #P = 0.02, **P = 0.001, *P = 0.002, † P = 0.04 versus control Ig.

Figure 6

In vivo leptin neutralization with ObR:Fc inhibits IFN-γ production and induces the secretion of IL-4 and IL-10 regulatory cytokines. (A and B) IFN-γ secretion of lymph node–derived T cells was inhibited by ObR:Fc treatment when T cells were stimulated with the myelin antigen PLP139–151 (A) and by anti-CD3ε (B). (C and D) IL-4 secretion of lymph node–derived T cells was enhanced by ObR:Fc treatment when T cells were stimulated with the myelin antigen PLP139–151 (C) and by anti-CD3ε (D). (E and F) IL-10 secretion of lymph node–derived T cells was markedly increased by ObR:Fc treatment when T cells were stimulated with the myelin antigen PLP139–151 (E) and by anti-CD3ε (F). Data are from 1 representative experiment of 3. (A) **P = 0.001, day –1 to day 1 and days 8–11 versus control Ig. (C) **P = 0.001, day –1 to day 1, and † P = 0.04, days 8–11, versus control Ig. (E) **P = 0.001, days 8–11, and #P = 0.02, day –1 to day 1, versus control Ig. (B, D, and F) #P = 0.02, *P = 0.002, † P = 0.04 versus control Ig.

Figure 7

Increased expression of Foxp3 in CD4+ T cells induced by leptin neutralization in mice with EAE. (A) Western blot analysis for Foxp3 on purified CD4+ T cells obtained from SJL/J mice immunized with PLP139–151 revealed significant increase of the expression of this molecule after leptin neutralization with ObR:Fc. (B) Results are presented as Foxp3 protein level normalized to tubulin expression. Data are from 1 representative experiment of 3.

Figure 8

Leptin neutralization suppresses ICAM-1 and OX-40 expression on CD4+ cells but upregulates VLA-4 in mice with EAE. (A) Flow cytometric analysis of cell-surface ICAM-1 molecules on CD4+ T cells from ObR:Fc-treated mice and controls (left) and mean fluorescence intensity (MFI) from 3 independent experiments (right). (B) OX-40 surface expression was also reduced by ObR:Fc treatment. (C) VLA-4 expression was enhanced by ObR:Fc treatment, particularly on CD4+ T cells at high intensity. #P = 0.02, *P = 0.002, † P = 0.04 versus control Ig.

Figure 9

Leptin neutralization determines the failure to downmodulate the anergy factor p27Kip-1 and is associated with sustained phosphorylation of ERK1/2 and STAT6. (A and B) Western blot analysis for p27Kip-1 and tubulin on purified CD4+ T cells obtained from SJL/J mice immunized with PLP139–151. Ex vivo analysis revealed high levels of p27Kip-1 in resting CD4+ T cells from naive mice; this phenomenon was accompanied by a strong downmodulation of p27Kip-1 in control Ig mice that developed classical EAE. Conversely, ObR:Fc treatment either from day –1 to day 1 or on days 8–11 caused a failure to downmodulate p27Kip-1, resulting in massive p27Kip-1 accumulation. (C and D) Ex vivo analysis revealed very low levels of phosphorylation of ERK1/2 in resting CD4+ T cells from naive mice. Conversely, control Ig–treated mice with EAE showed an increase in ERK1/2 phosphorylation. Leptin neutralization either from day –1 to day 1 or on days 8–11 induced sustained phosphorylation of the ERK1/2 molecule compared with control Ig–treated mice. (E and F) Ex vivo analysis revealed low levels of STAT6 phosphorylation in resting CD4+ T cells from naive mice; conversely, control Ig–treated mice with EAE showed a modest increase in STAT6 phosphorylation. Leptin neutralization also induced marked phosphorylation of STAT6. For each panel, 1 representative experiment of 5 is shown.