Infection with Mycobacterium bovis BCG diverts traffic of myelin oligodendroglial glycoprotein autoantigen-specific T cells away from the central nervous system and ameliorates experimental autoimmune encephalomyelitis

Abstract

Infectious agents have been proposed to influence susceptibility to autoimmune diseases such as multiple sclerosis. We induced a Th1-mediated central nervous system (CNS) autoimmune disease, experimental autoimmune encephalomyelitis (EAE) in mice with an ongoing infection with Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) to study this possibility. C57BL/6 mice infected with live BCG for 6 weeks were immunized with myelin oligodendroglial glycoprotein peptide (MOG(35-55)) to induce EAE. The clinical severity of EAE was reduced in BCG-infected mice in a BCG dose-dependent manner. Inflammatory-cell infiltration and demyelination of the spinal cord were significantly lessened in BCG-infected animals compared with uninfected EAE controls. ELISPOT and gamma interferon intracellular cytokine analysis of the frequency of antigen-specific CD4(+) T cells in the CNS and in BCG-induced granulomas and adoptive transfer of MOG(35-55)-specific green fluorescent protein-expressing cells into BCG-infected animals indicated that nervous tissue-specific (MOG(35-55)) CD4(+) T cells accumulate in the BCG-induced granuloma sites. These data suggest a novel mechanism for infection-mediated modulation of autoimmunity. We demonstrate that redirected trafficking of activated CNS antigen-specific CD4(+) T cells to local inflammatory sites induced by BCG infection modulates the initiation and progression of a Th1-mediated CNS autoimmune disease.

FIG. 1.

Infection with M. bovis BCG delays onset and lessens the clinical severity of EAE in a dose-dependent manner. (A and B) Four to 5-week-old female C57BL/6 mice (see Table 1 for numbers of animals) were infected (circles) with 3.4 × 10⁷ CFU (A) or 2.8 × 10⁶ CFU (B) of M. bovis BCG 6 weeks prior to induction of EAE with MOG_35-55 peptide in CFA in three separate experiments. Age- and sex-matched C57BL/6 mice were left uninfected (diamonds) prior to EAE induction. The data (mean clinical score ± standard error of the mean) demonstrate delay in and protection from EAE in both low-dose and high-dose infections, with higher-dose infections providing more protection. (C) Spinal cords were extruded from spinal columns by insufflation at day 15 following EAE induction in control uninfected and BCG-infected C57BL/6 mice. Formalin-fixed tissue was sectionedand stained with either H&E or luxol fast blue myelin stain. Uninfected mice demonstrate typical mononuclear cell infiltrates with corresponding areas of demyelination (arrowheads). Spinal cords of BCG-infected animals retain a normal myelin-staining pattern and show no evidence of infiltrates.

FIG. 2.

Quantitation and activation status of peripheral and CNS lymphocytes in control EAE and BCG-infected EAE. Splenocytes and brain-infiltrating cells were isolated from BCG-infected C57BL/6 mice or age matched uninfected mice at day 15 after EAE induction. (A) Cells were quantitated by trypan blue exclusion. Results represent mean numbers of cells isolated from spleens and brains of three animals per group, ± standard errors of the means. Statistical significance was calculated by Student's t test. *, P < 0.01; **, P < 0.001. (B) FACS analysis of CD4 gated T lymphocytes isolated from spleens and brains of BCG-infected and uninfected mice induced for EAE at day 15 after EAE induction. Histograms of brain lymphocytes and splenocytes demonstrate a similar postactivated phenotype (CD44^high LFA-1^high CD25^high) in BCG-infected and uninfected groups. Brain weights for these animals were within a very tight range, i.e., 0.42 ± 0.03 g/brain (mean ± standard deviation). All plots are representative of three animals per group.

FIG. 3.

Intracellular IFN-γ staining of cells isolated at day 15 after EAE induction demonstrates that MOG antigen-specific IFN-γ production is localized differentially in BCG-infected and uninfected mice. Brains and spleens were removed from perfused uninfected and BCG-infected mice at day 15 after EAE induction. Cells were isolated and incubated overnight with MOG_35-55, phorbol myristate acetate and ionomycin, or medium (untreated) and Golgistop protein transport inhibitor. The cells were surface stained with CD4-CYCR, Mac-1-Cy5 and CD8b-FITC; permeabilized; and stained with either isotype-PE or IFN-γ-PE. Dot plots represent CD8-negative and Mac-1-negative lymphocyte gated events. Uninfected mice induced for EAE demonstrate MOG-specific IFN-γ-producing T cells in the brain that are absent from the BCG-infected animals. Conversely, more MOG-specific T cells remain in the periphery (spleen) in the infected mice. Numbers represent percentages of CD4⁺ cells that stained for intracellular IFN-γ. Plots are representative of three mice per group.

FIG. 4.

IFN-γ-producing, MOG antigen-specific T cells localize to granulomas in BCG-infected mice and to brains of control uninfected mice after EAE induction. (A) ELISPOT data from control uninfected and BCG-infected mice at 15 days after EAE induction demonstrate the presence of MOG antigen-specific IFN-γ-producing T cells localized to BCG granulomas. (B) MOG antigen-specific IFN-γ-producing T cells are localized to the brains of uninfected mice, not BCG-infected mice. The wells shown are representative of triplicates. Counts represent the mean ± standard deviation from one of three experiments with similar results. Granuloma cells were plated at 5 × 10⁵/well. Brain cells were plated at 10³/well.

FIG. 5.

When MOG antigen-specific GFP expressing T cells are adoptively transferred into BCG-infected C57BL/6 mice, they localize to liver granulomas. EAE was induced in BCG-infected mice by adoptive transfer of GFP-expressing MOG-specific T cells. Livers were removed and frozen sections were prepared at day 30 after EAE induction. Adjacent sections were examined by confocal microscopy (unstained) (A) or stained with H&E (B). Granulomas that were identified in the H&E sections were found in corresponding locations in the frozen confocal sections, and large numbers of GFP-expressing T cells (white in the confocal images) were localized to the granulomas and absent from surrounding parenchyma.