IL-11 Induces Encephalitogenic Th17 Cells in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis

Abstract

IL-11⁺CD4⁺ cells accumulate in the cerebrospinal fluid of patients with early relapsing-remitting multiple sclerosis (MS) and in active brain MS lesions. Mouse studies have confirmed a causal role of IL-11 in the exacerbation of relapsing-remitting experimental autoimmune encephalomyelitis (RREAE). Administration of IL-11 at the time of clinical onset of RREAE induced an acute exacerbation and increased clinical scores, which persisted during the entire course of the disease. IL-11 increased the numbers of spinal cord inflammatory foci, as well as the numbers of peripheral and CNS-infiltrating IL-17⁺CD4⁺ cells and IL-17A serum levels. Ag recall assays revealed that IL-11 induces IL-17A⁺, GM-CSF⁺, and IL-21⁺CD4⁺ myelin Ag-reactive cells. Passive transfer of these encephalitogenic CD4⁺ T cells induced severe RREAE with IL-17A⁺CCR6⁺ CD4⁺ and B cell accumulation within the CNS. Furthermore, passive transfer of nonmanipulated CNS-derived mononuclear cells from mice with RREAE after a single dose of IL-11 induced severe RREAE with increased accumulation of IL-17A⁺ and CCR6⁺ CD4⁺ cells within the CNS. These results suggest that IL-11 might serve as a biomarker of early autoimmune response and a selective therapeutic target for patients with early relapsing-remitting MS.

FIGURE 1.

IL-11⁺CD4⁺ and Th17 cells are enriched in the CSF from RRMS patients in comparison with the matched blood samples. (A) PBMCs and CSF cells were separated from blood and CSF samples from six untreated RRMS patients. The cells were stimulated with PMA and ionomycin for intracellular staining. The percentage of cells expressing the indicated cytokines was determined in gated CD4⁺ T cells. Each symbol represents an individual donor, and horizontal bars represent mean value. Statistical analysis was performed using a two-tailed paired Student t test. (B) Representative staining of the indicated cytokines in the gated CD4⁺ cells from one out of six untreated RRMS patients.

FIGURE 2.

Immunohistochemistry studies of the active brain MS lesions reveal accumulation of IL-11⁺CD4⁺ cells in the inflammatory infiltrates. (A) Active brain MS lesion tissue from five patients obtained by biopsy was used for CD4, IL-11, and IL-17A staining. Presented is an average percentage of IL-11⁺ and IL-17A⁺ CD4⁺ cells ± SD in the myelinated and demyelinated tissue. (B) Representative staining from one out of five patients. Cell nuclei were visualized in DAPI channel (blue), CD4 in Cy3 channel (green), and IL-11 and IL-17A in Cy5 (red). Scale bars, 1 μm (H&E and LFB panels).

FIGURE 3.

IL-11 induces STAT3 phosphorylation and RORγτ expression. (A) Spleen cells from nine naive SJL/J mice were cultured in the absence or presence of IL-11 (100 ng/ml), followed by intracellular staining for IL-17A and surface staining for CCR6. The percentage of cells expressing each molecule was determined in gated CD4⁺ T cells. Each symbol represents one mouse; horizontal bars represent mean values. Statistical analysis was performed using a two-tailed paired Student t test. (B) Spleen cells were separated from three naive SJL/J mice and incubated in the absence or presence of IL-11 (10, 50, 100 ng/ml) and αIL-11 mAb. Cell lysates were prepared following 30 min of culture and used for Western blotting to detect total and pSTAT3, STAT3, RORγτ, and β-actin expression.

FIGURE 4.

IL-11 induces worsening of the clinical disease course in RREAE. (A) Twelve female 8- to 12-wk-old SJL/J mice were immunized with 80 μg/mouse of PLP_139–151 peptide. Starting from day 12 postimmunization, six mice per group received i.p. injections of recombinant mouse IL-11 (0.5 μg/mouse) or control vehicle daily for 10 d. Clinical scores were assigned daily for 70 d. Presented are mean clinical scores ± SD. Statistical analysis was performed using a two-tailed Student t test. (B) On day 70 postimmunization, mice were sacrificed, and the brain and spinal columns were removed and fixed. The paraffin sections were stained with Luxol fast blue and H&E. Parenchymal inflammatory foci (>10 mononuclear cells) were quantified by a blinded neuropathologist, and results are presented as mean ± SD per group. Statistical analysis was performed using a two-tailed Student t test. (C) Representative sections demonstrate more meningeal and parenchymal inflammation and larger areas of demyelination in the IL-11–treated versus the control mouse spinal cord. Luxol fast blue and H&E stain. Scale bar, 50 μm.

FIGURE 5.

IL-11 induces Th17 cell response in RREAE. (A) SJL/J mice were immunized with PLP_139–151 peptide. On day 12 postimmunization, six mice per group were i.p. injected with recombinant mouse IL-11 (0.5 μg/mouse) or vehicle control. After 16 h, the mice were sacrificed and tissues were harvested. The cells were stimulated with PMA and ionomycin for intracellular staining, and the percentage of cells expressing each molecule was determined in gated CD4⁺ T cells. (B) Representative staining. (C) The percentage of CCR6-expressing cells in gated IL-17A⁺CD4⁺ cells. (D) The concentration of IL-17A in serum samples was measured by ELISA. (E) On day 56 postimmunization, six mice per group received recombinant mouse IL-11 (0.5 μg/mouse) or control vehicle. After 16 h, the mice were sacrificed. The percentage of IL-17A⁺ cells was determined in gated CD4⁺ T cells from the PBMCs, spleen, and spinal cord inflammatory infiltrates. The percentage of ICAM-1⁺ cells was determined in gated CD4⁺ T cells from brain cell infiltrates. (F) The gene expression of IL-11 in LN and IL-17A in brain inflammatory infiltrates was detected by RT-PCR. (G) The concentration of IL-17A in the serum samples was measured by ELISA. (H) The percentages of ICAM-1⁺ cells were determined in gated IL-11⁺CD4⁺ T cells from the brain and spinal cord cell infiltrates. The percentage of CCR6⁺ cells was determined in IL-11⁺CD4⁺ T cells from the spinal cord infiltrates. The results are presented as mean ± SD per group. Statistical analysis was performed using a two-tailed paired Student t test. Each experiment was performed once.

FIGURE 6.

IL-11 induced encephalitogenic CD4⁺ cells. Spleen and LN cells were separated from PLP_139–151-immunized SJL mice on day 11 postimmunization. Cells were cultured with PLP peptides (20 μg/ml) in the absence or presence of IL-11 (100 ng/ml) and the control Th-17–polarizing cytokine IL-23 (20 ng/ml) for 7 d. (A) Cells were restimulated with PMA/ionomycin for intracellular staining. The percentages of the cells expressing each molecule in gated CD4⁺ T cells (n = 12), (B) CD8⁺ cells (n = 6), and (C) CD19⁺ cells (n = 6) were determined by flow cytometry. Each symbol represents one mouse, and horizontal bars represent mean values. Statistical analysis was performed using repeated measures ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7.

Passive transfer of IL-11–stimulated CD4⁺ cells induces severe RREAE. (A) CD4⁺ T cells were isolated from the cultures described in Fig. 6. A total of 20 × 10⁶ CD4⁺ T cells per recipient were injected into naive SJL/J mice. Clinical scores of the six recipient mice per group were assigned daily for 70 d. Statistical analysis was performed using a repeated measures ANOVA. (B) On day 70 post–passive transfer, mice were sacrificed, and the brain and spinal cords were stained with Luxol fast blue and H&E. Meningeal inflammatory foci (>10 mononuclear cells) were quantified by a blinded neuropathologist, and statistical analysis was performed using a two-tailed Student t test. (C and D) Single-cell solutions were obtained from recipient mice sacrificed at day 14 post–passive transfer and incubated with PMA/ionomycin for intracellular staining. The percentages of cells expressing indicated markers in gated CD4⁺ cells, (E–G) viable cells, and (H) gated CD19⁺ B cells were determined by flow cytometry. (I) In mice sacrificed at day 70 post–passive transfer, the percentage of cells expressing the indicated markers in the viable cells and (J) gated CD4⁺ cells was determined by flow cytometry. The results represent mean ± SD values per group. Statistical analysis was performed using paired t test. *p < 0.5.

FIGURE 8.

IL-11 enhances the disease severity induced by passive transfer of nonmanipulated CNS mononuclear cells from mice with RREAE. (A) SJL/J mice that were immunized with PLP_139–151 received either IL-11 (0.5 μg) or control 1× PBS injection on day 11 postimmunization. The mice were sacrificed after 16 h. A total of 2 × 10⁶ mononuclear cells from the CNS infiltrate were isolated using a Percoll gradient and transferred to recipient mice via i.p. injection. Clinical scores of six recipient mice per group were assigned daily for 70 d. (B) On day 70 after passive transfer, CNS-infiltrating mononuclear cells of the recipient mice were isolated by Percoll gradient. Cells were restimulated with PMA/ionomycin for intracellular staining. The percentages of CD4⁺ cells expressing each molecule were determined by flow cytometry. The results represent mean ± SD values. Statistical analysis was performed using repeated measures paired t test. Each experiment was performed once. *p < 0.05.