Treatment with IL-10 producing B cells in combination with E2 ameliorates EAE severity and decreases CNS inflammation in B cell-deficient mice - PubMed (original) (raw)

Treatment with IL-10 producing B cells in combination with E2 ameliorates EAE severity and decreases CNS inflammation in B cell-deficient mice

Jun Zhang et al. Metab Brain Dis. 2015 Oct.

Abstract

Clinical improvement during pregnancy in multiple sclerosis (MS) patients suggests that sex hormones exert potent regulatory effects on autoimmune function. Our previous studies demonstrated that estrogen- (17β-estradiol; E2) mediated protection against experimental autoimmune encephalomyelitis (EAE), a mouse model for MS, hinges on the B cells, leading to elevated numbers of IL-10 secreting CD1d(hi)CD5(+) B regulatory cells (Bregs) in wild type mice. Our data show that co-administration of E2 and IL-10(+) B cells ameliorates EAE disease severity and limits CNS infiltrating leukocytes in B cell deficient mice. Additionally, treatment with E2 and Bregs reduces demyelination and dramatically decreases the proportion of CD11b(+)CD45(hi) activated microglia/macrophages found in the CNS of immunized animals compared to vehicle, E2 or Breg cells alone. Furthermore, mice given E2 and Bregs exhibit increased numbers of peripheral programmed death-1 positive CD4(+)Foxp3(+) regulatory T cells (Tregs) and up-regulation of programmed death receptor-ligand-1 and CD80 expression on monocytes. Our study suggests IL-10 producing Bregs have powerful therapeutic potential as an agent against EAE when augmented with E2 treatment.

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Conflict of interest statement

Conflict of interest

The authors declare that they have no conflict of interest.

Figures

Fig. 1

Fig. 1. IL-10 producing B cells and E2 ameliorate EAE and decrease spinal cord inflammation in µMT−/− mice

Spinal cords from each group were collected 24 days post-immunization, fixed in PFA, and embedded in paraffin for sectioning. Ten µm transverse sections from different regions of the spinal cord from each of the groups were stained with Hematoxylin & Eosin to enumerate infiltrating leukocytes (Fig. 1a) and with Luxol Fast Blue to visualize extent of demyelination (Fig. 1b). Arrows denote foci of inflammation. Magnification was 50× and 200× (inset). Sections are representative of 2 experiments (n = 3 mice/group/experiment).

Fig. 2

Fig. 2. IL-10 producing B cells and E2 treatment reduce infiltration of leukocytes into the CNS of µMT−/− mice

Mononuclear cells were isolated from CNS tissues and total live cell numbers from spinal cords (Fig. 2a) and brains (Fig. 2b) were counted on a hemocytometer. Data shown are representative of 4 independent experiments consisting of 6 mice per group (mean ± SEM). Statistical analysis was performed with ANOVA followed by Newman-Kuels post hoc test. Significant differences between sample means are indicated (*p≤0.05; **p≤0.01; ***p≤0.001)

Fig. 3

Fig. 3. Administration of IL-10 producing B cells in combination with E2 treatment decreased activation of microglia/macrophages in the CNS

CD11b+CD45hi activated microglia/macrophages (Fig. 3a, 3b) and CD11b+CD45low resting microglia/macrophages (Fig. 3c, 3d) were determined in individual spinal cords and brains. Data shown are representative of 4 independent experiments consisting of 6 mice per group (mean ± SEM). Statistical analysis was performed with ANOVA followed by Newman-Kuels post hoc test. Significant differences between sample means are indicated (*p≤0.05; **p≤0.01; ***p≤0.001)

Fig. 4

Fig. 4. Lower infiltration of proinflammatory immune cells in the CNS with IL-10 producing B cells plus E2 treatment

Frequencies of CD4+ (Fig. 4a, 4b) and CD8+ T cells (Fig. 4c, 4d) were determined in individual spinal cords and brains and indicate the percentages of total gated live leukocytes (n=6). Data are representative of 4 independent experiments consisting of 6 mice per group (mean ± SEM). Statistical analysis was performed with ANOVA followed by Newman-Kuels post hoc test. Significant differences between sample means are indicated (*p≤0.05; **p≤0.01; ***p≤0.001)

Fig. 5

Fig. 5. Activation and pro-inflammatory state of CD11b+ monocytes

Expression of the monocyte activation markers I-Ab (Fig. 5a) and CD80 (Fig. 5b) were determined on CD11b+ splenocytes from the various treatment groups. Fig. 5c: TNF-α producing CD11b+ monocytes. Data are representative of 4 independent experiments consisting of 6 mice per group (mean ± SEM). Statistical analysis was performed with ANOVA followed by Newman-Kuels post hoc test. Significant differences between sample means are indicated (*p≤0.05; **p≤0.01; ***p≤0.001)

Fig. 6

Fig. 6. PD-1/PD-L1 pathway molecules are up-regulated in µMT−/− mice that receive IL-10 producing B cells and E2

PD-L1 expression was assessed on splenic CD11b+ monocytes (Fig. 6a); CD122, on splenic CD8+ T cells (Fig. 6b); Foxp3, on splenic CD4+ T cells (Fig. 6c) and PD-1, on splenic CD4+Foxp3+ regulatory T cells (Fig. 6d). Data are representative from 4 independent experiments consisting of 6 mice per group (mean ± SEM). Statistical analysis was performed with ANOVA followed by Newman-Kuels post hoc test. Significant differences between sample means are indicated (*p≤0.05; **p≤0.01; ***p≤0.001)

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