Repetitive pertussis toxin promotes development of regulatory T cells and prevents central nervous system autoimmune disease

Abstract

Bacterial and viral infections have long been implicated in pathogenesis and progression of multiple sclerosis (MS). Incidence and severity of its animal model experimental autoimmune encephalomyelitis (EAE) can be enhanced by concomitant administration of pertussis toxin (PTx), the major virulence factor of Bordetella pertussis. Its adjuvant effect at the time of immunization with myelin antigen is attributed to an unspecific activation and facilitated migration of immune cells across the blood brain barrier into the central nervous system (CNS). In order to evaluate whether recurring exposure to bacterial antigen may have a differential effect on development of CNS autoimmunity, we repetitively administered PTx prior to immunization. Mice weekly injected with PTx were largely protected from subsequent EAE induction which was reflected by a decreased proliferation and pro-inflammatory differentiation of myelin-reactive T cells. Splenocytes isolated from EAE-resistant mice predominantly produced IL-10 upon re-stimulation with PTx, while non-specific immune responses were unchanged. Longitudinal analyses revealed that repetitive exposure of mice to PTx gradually elevated serum levels for TGF-β and IL-10 which was associated with an expansion of peripheral CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg). Increased frequency of Treg persisted upon immunization and thereafter. Collectively, these data suggest a scenario in which repetitive PTx treatment protects mice from development of CNS autoimmune disease through upregulation of regulatory cytokines and expansion of CD4(+)CD25(+)FoxP3(+) Treg. Besides its therapeutic implication, this finding suggests that encounter of the immune system with microbial products may not only be part of CNS autoimmune disease pathogenesis but also of its regulation.

Figure 1. C57Bl/6 wild-type (12/group) (a–d) or MOG p35-55 TCR transgenic mice (6/group) (e, f) received weekly i.v. injections with 300 ng PTx in 200 ul of PBS or PBS alone for six months.

Splenocytes from two representative mice per group were cultured in the presence of various concentrations of pertussis toxin (PTx) (a, e), phytohemagglutinin (PHA) (b), MOG p35-55 (c, f) or MBP Ac1-11 (d) (representative for two separate experiments).

Figure 2. C57Bl/6 mice received weekly i.v. injections with 300 ng PTx in 200 ul of PBS or PBS alone.

After six months of PTx injection, mice were immunized with MOG p35-55 in CFA and injected i.v. with 300 ng PTx in PBS immediately following the immunization and 48 h later. (a) Mice were followed for clinical signs of EAE (10 mice/group, mean severity is indicated as group average +/− SEM, * indicates p<0.05). (b+c) 50 days after EAE induction, CNS tissue from all mice was analysed for histological signs of EAE. Spinal cords were isolated and representative cervical, lumbal and thoracical sections were evaluated for inflammatory lesions. (b) Indicated is the average number of lesions per slide +/− SEM in each group (* indicates p<0.05). (c) Shown are representative slides stained with H&E (upper panels) or LFB (lower panels, magnification X100), (representative for two separate experiments).

Figure 3. C57Bl/6 mice received weekly i.v. injections with 300 ng PTx in 200 µl of PBS or PBS alone.

After six months, mice were immunized with MOG p35-55 in CFA and injected i.v. with 300 ng PTx in PBS immediately following the immunization and 48 h later. 50 days after EAE induction, isolated splenocytes from 4 (control-treated group) and 6 (PTx-treated group) representative mice were cultured with anti-CD3 (0,5 µg/ml) and anti-CD28 (1 µg/ml) (a–d), MOG p35-55 (e–h) or PTx (i–l) and evaluated for proliferation (a, e, i), secretion of IFN-γ (b, f, j), TNF (c, g, k) and IL-10 (d, h, l) (* indicates p<0.05, ** p<0.001, *** p<0.0001; representative for two separate experiments).

Figure 4. C57Bl/6 mice received weekly i.v. injections with 300 ng PTx or OVA 323–339 (control) in PBS.

Serum cytokine IL-10 (a), TGF-β (b), TNF (c) and INF-γ (d) were determined at week 0-3-6-9 post PTx treatment. CD4⁺CD25⁺FoxP3⁺ Treg were analysed by flow cytometry at week 0-3-6-9 post PTx treatment (e; upper panels: indicated is the percentage of CD4⁺CD25⁺ within all CD4⁺ T cells; middle panels: FoxP3 expression of CD4⁺CD25⁺ cells; lower panels: mean FoxP3-FITC fluorescence intensity of CD4⁺CD25⁺ cells, * indicates p<0.05) (3 mice/group/time point; representative for three separate experiments).

Figure 5. C57Bl/6 mice received weekly i.v. injections with 300 ng PTx or OVA 323–339 (control) in PBS.

After 4 weeks, splenic CD4⁺ T cells were purified (3 mice/group) and co-cultured with 2×10⁴ dendritic cells and 4×10⁴ MOG p35-55 specific T cells from TCR transgenic (2D2) mice in the presence of 20 µg/ml MOG p35-55. Proliferation at the indicated ratio of MOG p35-55 specific CD4⁺ T cells (2D2)/CD4⁺ T cells from PTx-/ova-treated mice (PTx/ova) (1/1, 1/5, 1/10) is shown as percentage of the proliferative response without CD4⁺ T cells from PTx-/ova-treated mice (ratio 1/0) (a). After 10 weeks, EAE was induced with MOG p35-55 (8 mice/group). PTx pre-treatment significantly ameliorated disease severity (b). At the peak of clinical EAE severity, the frequency of CD4⁺CD25⁺FoxP3⁺ Treg and serum level of IL-10 was evaluated. Clinical benefit of PTx-pretreated mice was associated with expansion of FoxP3⁺ Treg cells (c; left panels: indicated is the percentage of CD4⁺CD25⁺ within all CD4⁺ T cells; right upper panel: FoxP3 expression of CD4⁺CD25⁺ cells; right lower panel: mean FoxP3-FITC fluorescence intensity of CD4⁺CD25⁺ cells) and elevated serum levels for IL-10 (d) (*indicates p<0.05, ** indicates p<0.001; representative for two separate experiments).