IL7Rα contributes to experimental autoimmune encephalomyelitis through altered T cell responses and nonhematopoietic cell lineages

Abstract

A mutation in the IL7Rα locus has been identified as a risk factor for multiple sclerosis (MS), a neurodegenerative autoimmune disease characterized by inflammation, demyelination, and axonal damage. IL7Rα has well documented roles in lymphocyte development and homeostasis, but its involvement in disease is largely understudied. In this study, we use the experimental autoimmune encephalomyelitis (EAE) model of MS to show that a less severe form of the disease results when IL7Rα expression is largely restricted to thymic tissue in IL7RTg(IL7R-/-) mice. Compared with wild-type (WT) mice, IL7RTg(IL7R-/-) mice exhibited reduced paralysis and myelin damage that correlated with dampened effector responses, namely decreased TNF production. Furthermore, treatment of diseased WT mice with neutralizing anti-IL7Rα Ab also resulted in significant improvement of EAE. In addition, chimeric mice were generated by bone marrow transplant to limit expression of IL7Rα to cells of either hematopoietic or nonhematopoietic origin. Mice lacking IL7Rα only on hematopoietic cells develop severe EAE, suggesting that IL7Rα expression in the nonhematopoietic compartment contributes to disease. Moreover, novel IL7Rα expression was identified on astrocytes and oligodendrocytes endogenous to the CNS. Chimeric mice that lack IL7Rα only on nonhematopoietic cells also develop severe EAE, which further supports the role of IL7Rα in T cell effector function. Conversely, mice that lack IL7Rα throughout both compartments are dramatically protected from disease. Taken together, these data indicate that multiple cell types use IL7Rα signaling in the development of EAE, and inhibition of this pathway should be considered as a new therapeutic avenue for MS.

Figure 1. Lymphocyte distribution in IL7RTg^IL7R−/− mice

(A) Quantification of total cell numbers are shown for the indicated mice following flow cytometry analysis of CD4, CD8, B220, and IL7Rα expression after gating on the total lymphocyte population from the spleen. Results are expressed as the mean ± SEM with n ≥ 5 per group from 3 independent experiments. # indicates p<0.05; * indicates p<0.0005; unpaired t test. (B) Clinical course of EAE in indicated mice after induction with the MOG_35-55 peptide. Daily scores from one representative experiment are expressed as the mean ± SEM with n=4 per group. ns, indicates p>0.05; Mann Whitney t test.

Figure 2. MOG-induced EAE in IL7Rα deficient mice

(A) Clinical course of EAE in WT and IL7RTg^IL7R−/− mice after induction with the MOG_35-55 peptide. Daily scores are expressed as the mean ± SEM with n ≥ 13 per group from 3 independent experiments. * indicates p<0.0005; Mann Whitney t test. (B) Assessment of thoracic SC lesions in naïve WT (left) or 25dpi WT (center) and IL7RTg^IL7R−/−(right) mice by the myelin stain luxol fast blue with hematoxylin & eosin counterstains. Lower panels correspond to boxes drawn above. (C) Clinical course of EAE in WT mice treated with anti-IL7Rα or IgG control from days 19-37. Daily scores are expressed as the mean ± SEM with n ≥ 9 per group from two independent experiments. * indicates p<0.0001; Mann Whitney t test. (D) Assessment of thoracic SC lesions in naïve WT (left), anti-IL7Rα treated (center), or IgG treated (right) mice at 55 dpi by the myelin stain luxol fast blue with hematoxylin & eosin counterstains. Representative sections were selected to display the general differences observed over 5 mice/group. Scale bars represent 100μm.

Figure 3. IL7Rα deficiency dampens T cell effector responses in acute EAE

Quantification of total T cell numbers are shown for the spleen (A, D, G) and SC (B, E , H) of indicated mice at acute EAE (25dpi) following ex vivo stimulation and flow cytometry analysis. Total CD4 and CD8 T cell populations (A, B) were analyzed after gating on the CD45 leukocytes. Representative dot plots for TNF, IFN and IL17 expression within the CD4 (C) and CD8 (F) gates are shown. Cytokine producing CD4 (D, E) and CD8 (G, H) T cells were quantified for spleen and SC. Results are expressed as the mean ± SEM with n ≥ 7 per group from 3 independent experiments. B. *, p<0.05; **, p<0.005; ***, p<0.0005; unpaired t test.

Figure 4. IL7Rα deficiency alters T cell responses prior to EAE clinical symptoms

Quantification of total T cell numbers are shown for the spleen of indicated mice at acute EAE (25dpi) following ex vivo stimulation and flow cytometry analysis. Total CD4 and CD8 T cell populations were analyzed after gating on the CD45 leukocytes (A). Representative dot plots for TNF, IFN and IL17 expression within the CD4 (B) and CD8 (D) gates are shown. Cytokine producing CD4 (C) and CD8 (E) T cells were quantified. Results are expressed as the mean ± SEM with n = 13 per group from 3 independent experiments. *, p<0.05; **, p<0.005; ***, p<0.0005; unpaired t test.

Figure 5. Selective IL7Rα expression through bone marrow chimerism is not sufficient for EAE protection

Quantification of total cell numbers are shown for the spleen and SC of indicated mice at acute EAE (18-24 dpi) following flow cytometry analysis (B, D). Total CD45 cells were analyzed after gating on the live cell population. Total CD4 and CD8 T cell populations were analyzed after gating on the CD45 leukocytes, while Tregs were identified by CD25⁺FoxP3⁺ expression within the CD4 gate. Results are expressed as the mean ± SEM with n ≥ 8 per group from 3 independent experiments. ns, P>0.05; #, p<0.05; *, p<0.0005; ^, p<0.0001; Mann Whitney t test (A, C) and unpaired t test (B, D).

Figure 6. IL7Rαexpression on astrocytes and mature oligodendrocytes

Representative immunofluorescence staining against IL7Rα-GFP, CC1, and GFAP in thoracic SC sections taken from IL7Rα-GFP reporter mice (top 2 panels). Anti-rabbit IgG-488 staining of IL7Rα-GFP SC sections, and anti-GFP staining of WT SC sections (bottom 2 panels) were used as negative controls. Scale bars represent 50μm.