IL-10-producing B-cells limit CNS inflammation and infarct volume in experimental stroke

Abstract

Clinical stroke induces inflammatory processes leading to cerebral injury. IL-10 expression is elevated during major CNS diseases and limits inflammation in the brain. Recent evidence demonstrated that absence of B-cells led to larger infarct volumes and increased numbers of activated T-cells, monocytes and microglial cells in the brain, thus implicating a regulatory role of B-cell subpopulations in limiting CNS damage from stroke. The aim of this study was to determine whether the IL-10-producing regulatory B-cell subset can limit CNS inflammation and reduce infarct volume following ischemic stroke in B-cell deficient (μMT(-/-)) mice. Five million IL-10-producing B-cells were obtained from IL-10-GFP reporter mice and transferred i.v. to μMT(-/-)mice. After 24 h following this transfer, recipients were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by 48 h of reperfusion. Compared to vehicle-treated controls, the IL-10(+) B-cell-replenished μMT(-/-)mice had reduced infarct volume and fewer infiltrating activated T-cells and monocytes in the affected brain hemisphere. These effects in CNS were accompanied by significant increases in regulatory T-cells and expression of the co-inhibitory receptor, PD-1, with a significant reduction in the proinflammatory milieu in the periphery. These novel observations provide the first proof of both immunoregulatory and protective functions of IL-10-secreting B-cells in MCAO that potentially could impart significant benefit for stroke patients in the clinic.

Figure 1. Adoptive transfer of IL-10-GFP⁺ B-cells to µMT^−/− mice reduces ischemic infarct size and experimental stroke outcome

A. Schematic representation of enrichment of IL-10⁺ GFP⁺ CD19⁺ B-cells from spleens of IL-10 GFP reporter mice, cultured in the presence of 1µg/ml LPS for 48 hours and adoptive transfer of 5 million IL-10-GFP⁺ B-cells into µMT^−/− mice, via i.v. injections. B. Intravenous transfer of 5 million IL-10-GFP⁺ B-cells reduced infarct volume in µMT^−/− B-cell deficient mice 48 hours following 60 minutes of middle cerebral artery occlusion (MCAO) compared to intravenous transfer of RPMI vehicle (no cells). *p≤0.05; **p≤0.01 (Student t-test). C. Representative 2,3,5 triphenyltetetrazolium chloride stained cerebral sections 48 hours following 60 minutes of MCAO. Localization of the ischemic lesion differed between µMT^−/− mice receiving intravenous IL-10-GFP⁺ B-cells or RPMI vehicle (no cells). D. Purified B-cells obtained from IL-10 GFP reporter mice were further characterized for regulatory B-cell sub-populations (B10, B1a & T2-MZ) after 48 hours of culture in the presence of LPS by flow cytometry.

Figure 2. IL-10 GFP⁺ B-cells reduce cerebral cell infiltration post-MCAO

Forty-eight hours after MCAO, mononuclear cells were isolated from brains of RPMI and IL-10 GFP⁺ B-cell recipient mice and were analyzed for A. Total cell count via hemocytometer. Values represent mean numbers (±SEM) of indicated cell subsets from 10–11 mice per group, from 3 separate experiments. B. CD3⁺ T-cells. C. CD11b⁺CD45^high activated microglia (MG)/monocytes, and D.CD11b⁺CD45^low resting MG obtained from the non-ischemic (left) and ischemic (right) hemispheres of µMT^−/− recipient mice. B–D, Values represent mean numbers (±SEM) of indicated cell subsets, gated on live leukocytes (by PI exclusion), from 4–5 mice per group, from at least 2 separate experiments. Statistical analysis was performed with ANOVA followed by Neuman-Kuels post hoc test. Significant differences between sample means are indicated (*p≤0.05; **p≤ 0.01; ***p≤ 0.001).

Figure 3. IL-10-GFP⁺ B-cells reduce cerebral inflammatory responses post-MCAO

Determinations of: A. total TNF-α production by CD45⁺ cells; B. TNF-α⁺CD3⁺ T-cells; and C. TNF-α⁺CD11b⁺ cells quantified in the nonischemic (left) and ischemic (right) hemispheres from µMT^−/− mice transferred with medium or IL-10-GFP⁺ B-cells after 48 hours of MCAO. Values represent mean numbers (±SEM) of indicated cell subsets from 4–5 mice of each group, from at least 2 separate experiments. Statistical analysis was performed with ANOVA followed by Neuman-Kuels post hoc test. Significant differences between sample means are indicated (*p≤0.05).

Figure 4. IL-10-GFP⁺ B-cells rescue MCAO-induced splenic atrophy in recipient µMT^−/− mice

Forty-eight hours after MCAO, mononuclear cells were isolated from spleens of RPMI or IL-10-GFP⁺ B-cell recipient µMT^−/− mice and were analyzed for: A. Total cell count via hemocytometer. Values represent mean numbers (±SEM) of indicated cell subsets from 10–11 mice in each group, from at least 3 separate experiments; B. CD3⁺ T-cells and CD11b⁺ monocytes; and C. CD4⁺ and CD8⁺ T-cell populations. Values represent mean numbers (±SEM) of indicated cell subsets, gated on live leukocytes (by PI exclusion), from 4–5 mice of each group, from 2 separate experiments. Statistical analysis was performed with Student’s t-test to compare between RPMI and IL-10-GFP⁺ B-cell recipient mice. Significant differences between sample means are indicated (*p≤0.05).

Figure 5. IL-10-GFP⁺ B-cells inhibit activation and suppress enhanced pro-inflammatory states of the T-cells and monocytes in the periphery of recipient mice

Forty-eight hours after MCAO, mononuclear cells were isolated from spleens of RPMI or IL-10-GFP⁺ B-cell recipient µMT^−/− mice and were analyzed for: A. and B. expression of activation markers, CD69 and CD44 on gated CD4⁺ T-cells; C. expression of MHC class II by CD11b⁺ monocytes, D. TNF-α⁺ and IFN-γ⁺ CD3⁺ T-cells and E. TNF-α⁺CD11b⁺ monocytes. Values represent mean numbers (±SEM) of indicated cell subsets from 4–5 mice of each group, from 2 separate experiments. Statistical analysis was performed with Student's t-test to compare between RPMI and IL-10-GFP⁺ B-cell recipient mice. Significant differences between sample means are indicated (*p≤0.05 and **p≤0.01).

Figure 6. Regulatory IL-10-GFP⁺ B-cells enhance regulatory T-cell populations and PD-1 expression in the periphery of recipient mice after MCAO

Splenocytes from RPMI and IL-10-GFP⁺ B-cell transferred µMT^−/− recipient mice were harvested 48 hours after MCAO and assessed for expression of: A and B. FoxP3⁺CD4⁺ T-cells and CD8⁺CD122⁺ T-cells; C. PD-1-expressing CD4⁺ T-cells and CD11b⁺ monocytes. Data are representative of 2 independent experiments with spleens processed from 4–5 individual mice (mean ± SEM). Significant differences between the groups (* p≤0.05) were determined using Student’s t-test.