IL-23 modulated myelin-specific T cells induce EAE via an IFNγ driven, IL-17 independent pathway

Abstract

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) mediated by myelin-reactive CD4(+) T cells. An unresolved issue that has important clinical implications concerns the cytokines produced by myelin-reactive T cells that determine their pathogenicity. Initially, IL-12 polarized, IFNγ producing Th1 cells were thought to be essential for the development of EAE. More recently, IL-23 polarized, IL-17 producing Th17 cells have been highlighted as critical encephalitogenic effectors. There is growing evidence that parallel autoimmune pathways can result in common clinical and histopathological endpoints. In the current study, we describe a form of EAE induced by the transfer of IL-23 modulated CD4(+) T cells into IL-17 receptor (IL-17R) deficient hosts. We found that IL-23 stimulates myelin-reactive T cells to produce both IFNγ and IL-17. Surprisingly, in this model the development of EAE is IFNγ dependent. Our findings illustrate a novel mechanism by which IL-23 promotes encephalitogenicity and they further expand the spectrum of autoreactive T cells capable of mediating inflammatory demyelinating disease of the CNS.

Figure 1. EAE mediated by IL-23 polarized cells is independent of IL-17 signaling

Lymph nodes were harvested from WT mice that had been immunized 10 days earlier with MOG_35–55+CFA. (A) Whole lymph node cells were stimulated with MOG_35–55 and IL-23 for 4 days. Afterwards CD4⁺ T cells were isolated and transferred to either WT or IL-17R −/− naïve recipients (5×10⁶ cells/host i.p.). (B) WT (left) or IL-17R −/− (right) mice that received Th17 polarized CD4⁺ T cells were sacrificed at peak disease. CNS specimens were harvested, fixed, sectioned, and stained with H&E. Upper panels, magnification 4×; lower panels, 40×. Bar 50µm. Data are representative of 4 independent experiments with 5–7 mice/group.

Figure 2. IL-23 induces IFNγ production by CD4⁺ T cells

(A) Lymph node cells were harvested from MOG immunized C57BL/6 mice and polarized in vitro under Th17 or Th0 conditions. Following a 4 day incubation, CD4⁺ T cells were purified and restimulated with MOG_35–55 and naïve T-depleted splenocytes in ELISPOT assays. (B) CD4 T cells were isolated from the draining lymph nodes of MOG-immunized mice and stimulated under Th17 or Th0 conditions. At 96 hours, RNA was isolated from the cells in each group and reverse transcribed into cDNA. IL-17 and IFNγ transcripts were analyzed by qPCR and normalized to the geometric mean of GAPDH, βactin, and HPRT. The data shown represent the ratio of normalized cytokine mRNA expression in Th17 over Th0 CD4⁺ T cells. Data are representative of 2–3 independent experiments. * P<.0001 Th17 versus Th0

Figure 3. IFNγ contributes to EAE mediated by IL-23 polarized cells

Lymph nodes were harvested from WT or IFNγ −/− mice that had been immunized 10 days earlier with MOG_35–55+CFA. Whole lymph node cells were stimulated with MOG_35–55 and IL-23 for 4 days before 5×10⁶ purified CD4⁺ T cells were transferred to naive IL-17R −/− (A) or wild-type (B) hosts. (C) WT or IFNγ −/− donor cells were challenged with antigen and IL-23 before transfer to WT or IL-17R −/− hosts. Mice were sacrificed at peak disease. CNS specimens were fixed, sectioned, and stained with H&E. In some cases, the same sections was photographed at low (4×) and high (40×) magnification. Bar, 50µm. Data are representative of 3 independent experiments with 5 mice/group.

Figure 4. Gene expression in donor CD4⁺ T cells and peak EAE

(A) RNA was isolated from spinal cords of mice at peak EAE (n=3/ group). qPCR was performed to measure expression of a panel of proinflammatory molecules. Transcript levels were normalized to the geometric mean of GAPDH, βactin, and HPRT. Data are shown as fold change over naïve spinal cords. (B) RNA was isolated from WT of IFNγ−/− MOG-specific CD4⁺ T cells after polarization under Th17 or Th0 polarizing conditions. qPCR was performed, and data was normalized to the geometric mean of GAPDH, βactin, and HPRT. Data is shown as the ratio of normalized mRNA expression in Th17 over Th0 CD4⁺ T cells.