Figure 2: Experimental stroke induces a loss of MZ B cells. | Nature Communications

Figure 2: Experimental stroke induces a loss of MZ B cells.

From: Adrenergic-mediated loss of splenic marginal zone B cells contributes to infection susceptibility after stroke

Figure 2

(a) Immunolabelling of MZM macrophages (green, MOMA-1) with MZ B-cell markers (red, CD1d, and CD21/35) demonstrate the absence of MZ B cells (white arrows) from the MZ 2 days after MCAO in comparison to sham-operated controls. Immunolabelling to distinguish MZ (red, IgM) from follicular B cells (green, IgD) confirms this. Quantification of MZ B-cell immunolabelling outside the MOMA-1+ MZM macrophage border using CD1d (b) or CD21/35 (c) confirm significant reductions in MZ B-cell labelling after MCAO in comparison to sham-operated controls (naïve n=4, sham n=4, 1d n=4, 2d n=9, 5d n=5, 7d n=7). CD23 and CD21/35 expression on cells gated for CD45R+ labelling from a constant number of gated total events was used to distinguish MZ (B220+CD23−CD21/35hi) from follicular (B220+CD23+CD21/35int) B cells (Supplementary Fig. 1e). (d) Absolute counts of splenic MZ and follicular B cells using flow cytometry show both are significantly reduced 2 days after MCAO in comparison to sham-operated controls (naïve n=4, sham n=4, MCAO n=6). (e) MZ B-cell immunolabelling (CD1d; red) can be seen within MZ metallophillic macrophages (green, MOMA-1) 2 days after MCAO. (f) TUNEL (green) staining, a marker of cell death, alongside B cells (blue, B220) and MZ B cells (red, CD1d) shows increased death of follicular and MZ B cells 1–7 days after MCAO in comparison to sham-operated controls. (g) Quantitative analysis of the percentage of CD1d+ labelling that co-localises with TUNEL, as a measure of apoptotic MZ B cells, show increased TUNEL+ CD1d MZ B cells 1–5 days after MCAO in comparison to sham-operated controls. (h) Similar analysis of CD1d- B220+ labelling that co-localises with TUNEL, as a measure of apoptotic follicular B cells, shows a significant increase in TUNEL+ follicular B cells 2–5 days after MCAO in comparison to sham-operated controls (naïve n=4, sham n=4, 1d n=4, 2d n=9, 5d n=5, 7d n=7). Scale bars (a,f) 200 μm (e) left panel 100 μm, right panel 10 μm. Data show mean+s.d.; *P<0.05; **P<0.01; ***P<0.001; (b,c,g,h) one-way ANOVA with Bonferroni correction comparing all MCAO recovery time points to sham, (d) unpaired t-test.

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