Fig 4. CD40L deficiency causes impaired accumulation and activation of peripheral monocyte/macrophage and the brain resident microglia at the acute phase of inflammation.

On day 5 p.i., brains from MI and RSA59 infected (25000 PFUs) WT and CD40L^-/- mice were harvested for flow cytometry analysis and stained for CD45, CD11b, MHCII, CX3CR1, and CD40. The green color denotes WT, and blue indicates CD40L^-/- mice. (A) Representative flow cytometry contour plots showing percentages of overall CD45^hi and CD45^lo cell population after gating on singlets followed by live cells and absolute cell numbers from infected sets are represented in (B) comparing WT and CD40L^-/- from MI and infected mice groups. Percentages of CD45 gated cells assessed for CD45^hiCD11b⁺ (peripheral derived monocyte/macrophage) and CD45^loCD11b⁺ (brain resident microglia) from infected sets are presented in flow cytometry plots (C), and absolute numbers of MI and infected sets are graphically represented in (D) comparing WT and CD40L^-/- groups. Representative flow cytometry contour plots indicate percentages of MHCII⁺ (E), CX3CR1(G), and CD40 (I) expressing CD45^hiCD11b⁺ and CD45^loCD11b⁺ cells comparing infected WT and CD40L^-/- mice, and the absolute numbers of MI and infected sets are graphically represented in F, H, and J, respectively. Results were expressed as mean ± SEM from 3 independent biological experiments (N = 4). *Asterisk (MI WT v/s infected WT) and #hash (infected WT v/s infected CD40L-/-) represents statistical significance calculated using unpaired Student’s t-test and Welch correction, p<0.05 was considered significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.