Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis

Abstract

Although the effects of commensal bacteria on intestinal immune development seem to be profound, it remains speculative whether the gut microbiota influences extraintestinal biological functions. Multiple sclerosis (MS) is a devastating autoimmune disease leading to progressive deterioration of neurological function. Although the cause of MS is unknown, microorganisms seem to be important for the onset and/or progression of disease. However, it is unclear how microbial colonization, either symbiotic or infectious, affects autoimmunity. Herein, we investigate a role for the microbiota during the induction of experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Mice maintained under germ-free conditions develop significantly attenuated EAE compared with conventionally colonized mice. Germ-free animals, induced for EAE, produce lower levels of the proinflammatory cytokines IFN-γ and IL-17A in both the intestine and spinal cord but display a reciprocal increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Mechanistically, we show that gut dendritic cells from germ-free animals are reduced in the ability to stimulate proinflammatory T cell responses. Intestinal colonization with segmented filamentous bacteria (SFB) is known to promote IL-17 production in the gut; here, we show that SFBs also induced IL-17A-producing CD4(+) T cells (Th17) in the CNS. Remarkably, germ-free animals harboring SFBs alone developed EAE, showing that gut bacteria can affect neurologic inflammation. These findings reveal that the intestinal microbiota profoundly impacts the balance between pro- and antiinflammatory immune responses during EAE and suggest that modulation of gut bacteria may provide therapeutic targets for extraintestinal inflammatory diseases such as MS.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

GF mice develop less severe EAE than SPF mice. (A) Clinical EAE scores of SPF (dark symbols) and GF (light symbols) mice immunized with MOG peptide in CFA plus pertussis toxin. Data are representative of three independent experiments. Symbols represent the mean ± SD at each time point. (B) Proportions of SPF and GF mice that develop various degrees of EAE. Results are combined from four independent experiments, with a total number of animals per group of 20 for SPF and 22 for GF mice. (C) H&E (Upper) and myelin basic protein (Lower) staining of the spinal cord of SPF and GF mice at the onset of EAE on day 12. Each panel is representative of tissue analyzed from at least four mice. (D) EAE score of conventional Rag−/− recipient mice (n = 4) that were reconstituted with in vitro primed CD4+ cells from SPF (black symbols) or GF (gray symbols) mice immunized with MOG/CFA and pertussis toxin. Data are representative of three independent experiments. Symbols represent the mean ± SD at each time point.

Fig. 2.

Inflammation is attenuated in GF mice on EAE induction. (A) Lymphocytes were harvested from draining LN of treated SPF and GF mice at 8 d after immunization with MOG/CFA. Intracellular staining of CD4+ T cell for IFNγ and IL-17A after 3 d in vitro culture with MOG peptide, restimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin for the last 5 h. Numbers in each quadrant indicate percentage of cytokine-positive CD4+ T cells. (B) Mean ± SD of the CD4+IFNγ+ T cell subsets from A. Data are representative of three independent experiments with at least four mice per group. (C) Real-time PCR of the transcription factor RORγt in cells isolated by Percoll gradient from spinal cords of SPF and GF mice at the onset of disease (day 8). Each symbol represents a single mouse. (D) IFNγ and IL-17A cytokine ELISA from cells harvested from draining LN of SPF and GF mice 8 d after immunization with MOG/CFA and cultured for 3 d in vitro with MOG peptide. Data are representative of three independent experiments, with mean ± SD of samples run in triplicate.

Fig. 3.

Increase in CD25+Foxp3+ Treg cells after induction of EAE in GF mice. Intracellular staining of CD4+CD25+Foxp3+ T cells cultured for 3 d with MOG peptide, restimulated with PMA/ionomycin for the last 5 h. Lymphocytes were harvested from draining lymph nodes (LN) and spleens of SPF and GF mice at 8 d p.i. (A and B) or 15 d p.i. (C and D) after i.v. immunization with MOG/CFA. In A and C, numbers in each quadrant indicate percentage of positive cells. In B and D, results are shown for mean ± SD of the CD4+ subsets of the cells from draining LN and spleen on day 8 and day 15 postimmunization. Data are representative of three independent experiments with at least four mice per group.

Fig. 4.

DCs from GF animals are defective in inducing Th17 and Th1 responses from CD4+ T cells. Purified CD4+ T cells from MOG-Tg mice were cultured with MLN DCs from SPF or GF mice in the absence or presence of MOG peptide and IL-2. At day 3 and day 5 of culture, IL-17A (A) and IFNγ (B) cytokines were measured by ELISA. Data are representative of two independent experiments, with mean ± SD of samples run in triplicate. *P < 0.05; **P < 0.005.

Fig. 5.

SFBs promote proinflammatory T cell responses outside the gut during EAE. (A) Clinical EAE scores of SPF, GF, and GF-SFB colonized mice immunized with MOG-peptide in CFA plus pertussis toxin. GF mice were colonized with SFB for 3 wk before MOG/CFA immunization (GF-SFB). Data are representative of two independent experiments with at least four mice per group. Symbols represent the mean ± SD at each time point. (B) Representative FC plots of IL-17A– and IFNγ-producing CD4+ T cells from LPLs of spinal cords and small intestines of SPF, GF, and GF-SFB colonized mice at day 15 after EAE induction. Numbers in each quadrant indicate percentage of cytokine-positive CD4+ T cells. Data are representative of two independent experiments with at least four mice per group. (C) Representative FC plots of IL-17A– and Foxp3-producing CD4+ T cells from LPLs of spinal cords and small intestines of SPF, GF, and GF-SFB colonized mice at day 15 after EAE induction. Numbers in each quadrant indicate percentage of positive CD4+ T cells. Data are representative of two independent experiments with at least four mice per group.