Intrastriatal B-cell administration limits infarct size after stroke in B-cell deficient mice

Abstract

Recent evidence emphasizes B-cells as a major regulatory cell type that plays an important role in limiting the pathogenic effects of ischemic stroke. The aim of the current study was to extend this initial observation to specifically examine the infiltration of regulatory B-cells and to determine if the effect of B-cells to limit the inflammatory response to cerebral ischemia is mediated by their action centrally or peripherally. Our data demonstrate the increased presence of a regulatory B-cell subset in the affected hemisphere of wild-type mice after middle cerebral artery occlusion (MCAO). We further explored the use of a novel method of stereotaxic cell delivery to bypass the blood brain barrier (BBB) and introduce CD19(+) B-cells directly into the striatum as compared to peripheral administration of B-cells. Infarct volumes after 60 minutes of MCAO and 48 hours of reperfusion were determined in B-cell deficient μMT( -/- ) mice with and without replacement of either B-cells or medium. Infarct size was significantly decreased in cerebral cortex after intrastriatal transfer of 100,000 B-cells to μMT(-/-) mice vs. controls, with a comparable effect on infarct size as obtained by 50 million B-cells transferred intraperitoneally. These findings support the hypothesis that B-cells play a protective role against ischemic brain injury, and suggest that B-cells may serve as a novel therapeutic agent for modulating the immune response in central nervous system inflammation after stroke.

Figure 1. Characterization of B-cells and B10 cells in brains of Sham MCAO and MCAO mice

(A) A relatively low percentage of CD19⁺B cells are observed in the brains of Sham MCAO and MCAO C57BL/6J mice. (B) Regulatory B10-cells (CD19⁺ CD1d^hiCD5⁺) are highly enriched in the ipsilateral (right) brain hemisphere after 60 min MCAO and 48 h reperfusion.

Figure 2. Transfer of GFP⁺ B cells from male GFP mice into the striatum of WT C57BL/6J recipient mice

Splenic CD19⁺ B cells were isolated from GFP⁺ mice, enriched using paramagnetic bead-conjugated antibodies and AutoMACS (Miltenyi Biotec) to >95% purity and injected into striatum of C57BL/6J mice (100,000 cells/2 μL). Brains of recipient mice were perfused and tissue was isolated at the indicated time points after injections for histological evaluation. Tissues were fixed with 4% buffered formalin, paraffin embedded and sectioned. Slides were analyzed by fluorescent microscopy (Olympus BX40, Japan).

Figure 3. B cells from spleens of naïve mice secrete IL-10 after activation ex vivo

CD19⁺ B cells from spleens of naïve C57BL/6J donor mice were AUTOMACS sorted, cultured with LPS/PMA/Ionomycin for 5 hours and evaluated for IL-10 secretion by flow cytometry.

Figure 4. Stereotaxic transfer of CD19⁺ B cells reduces infarct volume of μMT^−/− B-cell-deficient mice

(A) Stereotaxic transfer of 100,000 CD19⁺ B cells in 2 μL resulted in reduced infarct volume (mean ± SEM) in cortex but not striatum at 48 h of reperfusion after 60 min MCAO. Statistical analysis was performed using one-way ANOVA with post-hoc Newman-Keuls test. Infarct volume was decreased in cerebral cortex in B-cell-transferred μMT^−/− mice (n=11) compared to medium-transferred μMT^−/− mice (n=11, *p<0.05) or to control μMT^−/− mice (n=10, **p<0.01) while cortical infarct volume was comparable between control and medium-transferred μMT^−/− mice (p=0.374). No differences were seen in striatal infarct volume between the treatment groups (p=0.342). (B) Representative TTC-stained brains from B-cell-transferred, medium transferred and control μMT^−/− mice.

Figure 5. Intraperitoneal transfer of GFP⁺ CD19⁺ B-cells reduces infarct volume of μMT^−/− B-cell-deficient mice

Transfer of 50 million GFP⁺CD19⁺ B-cells resulted in reduced infarct volume (mean ± SEM) in cortex but not striatum at 48 h of reperfusion after 60 min MCAO. Statistical analysis was performed using Student’s t test. Note significant difference of cortical (*p<0.05) but not striatal (p=0.354) infarct volumes between groups receiving B-cells (n = 7) vs. untreated control mice (n = 10).