Oestrogen-mediated protection of experimental autoimmune encephalomyelitis in the absence of Foxp3+ regulatory T cells implicates compensatory pathways including regulatory B cells

Abstract

Oestrogen (17β-oestradiol, E₂) is a highly effective treatment for experimental autoimmune encephalomyelitis (EAE) that may potentiate Foxp3+ regulatory T (Treg) cells, which in turn limit the expansion of encephalitogenic T-cell specificities. To determine if Treg cells constitute the major non-redundant protective pathway for E₂, we evaluated E₂ protection of EAE after targeted deletion of Foxp3 expression in Foxp3-DTR mice. Unexpectedly, E₂-treated Foxp3-deficient mice were completely protected against clinical and histological myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide-induced EAE before succumbing to diphtheria toxin-induced mortality. This finding indicated the presence of alternative E₂-dependent EAE-protective pathways that could compensate for the lack of Treg cells. Further investigation revealed that E₂ treatment inhibited proliferation and expression of CCL2 and CXCL2, but enhanced secretion of interleukin-10 (IL-10) and IL-13 by MOG-35-55-specific spleen cells. These changes occurred concomitantly with increased expression of several chemokines and receptors, including CXCL13 and CXCR5, and the negative co-activation molecules, PD-L1 and B7.2, by B cells and dendritic cells. Furthermore, E₂ treatment resulted in higher percentages of spleen and lymph node T cells expressing IL-17, interferon-γ and tumour necrosis factor-α, but with lower expression of CCR6, suggesting sequestration of MOG-35-55 peptide-specific T cells in peripheral immune organs. Taken together, these data suggest that E₂-induced mechanisms that provide protection against EAE in the absence of Foxp3+ Treg cells include induction of regulatory B cells and peripheral sequestration of encephalitogenic T cells.

Figure 1

Diphtheria toxin (DT) treatment effectively depletes Foxp3⁺ regulatory T (Treg) cells. Wild-type (WT) C57BL/6 and Foxp3-DTR mice were treated with or without 1 μg diphtheria toxin (DT) on days 0, 1 and 3 relative to onset of experimental autoimmune encephalomyelitis (EAE). Spleen (a) and lymph node (b) were harvested on day 5 after DT treatment and Foxp3 expression was measured by flow cytometry using specific antibodies.

Figure 2

Oestrogen (E₂) protects mice from developing experimental autoimmune encephalomyelitis (EAE) in the absence of Foxp3⁺ regulatory T (Treg) cells. Foxp3-DTR mice were implanted with 2.5 mg 60-day release 17β-oestradiol pellets or placebo pellets 1 week before immunization with myelin oligodendrocyte glycoprotein (MOG) -35-55 peptide in complete Freund's adjuvant and then injected with 1 μg diphtheria toxin (DT) on days 0, 1 and 3 relative to disease onset. Mice were monitored for EAE. Data represent the mean of four experiments.

Figure 3

Histopathological evaluation of spinal cords from placebo Foxp3-DTR + diphtheria toxin (DT) (a,b) and oestrogen (E₂)-treated (c,d) Foxp3-DTR + DT mice. Spinal cords (shown at a 10 × magnification) from Foxp3-DTR + DT mice exhibit massive leucocyte infiltration (a) as shown by haematoxylin & eosin staining, whereas E₂-treated spinal cords lack infiltrating cells (c). Also, demyelination was prevented in E₂-treated Foxp3-DTR mice (d). The inset panels represent the sections at a 20 × magnification. Arrows denote foci of inflammation. Representative images of three experiments are shown.

Figure 4

Expression of programmed death 1 ligand 1 (PD-L1), CCR6 and activation markers on splenic B cells. Foxp3-DTR mice were administered three doses of diphtheria toxin (DT) and splenocytes from placebo and oestrogen (E₂) -treated mice were analysed by flow cytometry on day 4–5 for expression of (a) PD-L1, CCR6, Gr-1 and (b) B7.1 (CD80) and B7.2 (CD86) on specific cell types. Cells were gated on CD19 and CD11c to determine expression of B7.1 and B7.2. One representative experiment of four is shown with a total of 10 placebo-treated and E₂-implanted Foxp3-DTR mice.

Figure 5

Oestrogen (E₂) treatment of experimental autoimmune encephalomyelitis (EAE) significantly inhibits proliferation of spleen cells. Spleens were collected from Foxp3-DTR mice with EAE treated or not with 2.5 mg E₂. Cells were plated on a 96-well microtitre plate and cultured with 10 or 50 μg/ml of myelin oligodendrocyte glycoprotein (MOG) -35-55 peptide for 72 hr, the last 18 hr in the presence of [³H]-thymidine. Stimulation indices were determined by calculating the ratio of antigen-specific counts/min to medium alone counts/min. Foxp3-DTR group without and with E₂ implants showed a background of 600 and 1000 counts/min, respectively. Representative data from a total of three experiments are shown.

Figure 6

T helper type 1 and type 17 intracellular cytokine levels are elevated in spleen and lymph nodes after oestrogen (E₂) treatment. At the end of three diphtheria toxin (DT) treatments, (a) spleen and (b) lymph node cells from placebo and E₂-treated Foxp3-DTR mice were cultured in the presence of a leucocyte activation cocktail and then stained for intracellular accumulation of interleukin-17 (IL-17), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) on T cells. Data shown include cells pooled from a total of three placebo and three E₂-treated Foxp3-DTR mice and experiments were repeated twice.

Figure 7

Splenocytes (a) and lymph node cells (b) from placebo-treated and oestrogen (E₂) -treated Foxp3-DTR + DT mice were cultured in the presence of 25 μg/ml myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide for 48 hr. Culture supernatants were assayed for secreted levels of cytokines by the Luminex bead array. One representative experiment of three separate experiments is shown.

Figure 8

Messenger RNA expression of chemokines and chemokine receptors in spleens of oestrogen (E₂)-treated Foxp3-DTR mice. Spleen cells from placebo treated Foxp3-DTR + diphtheria toxin (DT) and E₂-treated Foxp3-DTR + DT mice were harvested at the end of three DT treatments, mRNA was synthesized and real-time PCR performed using Universal Taqman master mix and specific primers. One representative experiment of three separate experiments is shown. *P < 0.01.