VCAM1/VLA4 interaction mediates Ly6Clow monocyte recruitment to the brain in a TNFR signaling dependent manner during fungal infection
Fig 5
TNFR signaling is crucial for monocyte recruitment to the brain by enhancing monocyte VLA4 expression.
(A) The level of TNF-α mRNA in the brain of mice (n = 5 per time point) before and after i.v. infection with 20x106 C. neoformans. (B) IVM analysis of monocyte recruitment to brain postcapillary venules of TNFR-/- mice (n = 5) as compared to WT mice (n = 5) 24 h after i.v. infection with 20x106 C. neoformans. The infected mice were i.v. injected with 2 μg AF647-anti-CX3CR1 mAb to label monocytes 5 min before imaging. Left panel: representative images, right panel: quantification of monocytes. (C) Flow cytometry determination of the numbers of Ly6Chi and Ly6Clow monocytes and neutrophils in the brain of TNFR-/- and WT mice (n = 5 per group) 24 h (upper panel) and 48 h (lower panel) after i.v. infection with 20x106 C. neoformans. (D) The gating strategy for brain endothelial cells. Endothelial cells were defined as CD45-CD11b-CD31+ population which demonstrated expression of CD102. (E) Flow cytometry determination of the percentage of brain endothelial cells expressing VCAM1 in WT and TNFR-/- mice (n = 4 per group) 24 h after infection of 20x106 C. neoformans as compared to naïve mice. Left panel: representative plots, right panel: quantification. (F) Flow cytometry analysis of the expression of VLA4 on Ly6Clow monocytes from the brain of WT and TNFR-/- mice (n = 5 per group) 24 h after infection with 20x106 C. neoformans as compared to naïve mice. Left panel: representative histograms, right panel: quantification. (G) Isolated monocytes from WT and TNFR-/- mice (CD45.2 background) were stained by lipophilic dye CellVue and PKH26 respectively and mixed at 1:1 ratio. The mixed monocytes (2x106) were transferred into CD45.1 recipient mice (n = 5 mice) and the mice were i.v. infected with 20x106 C. neoformans for 24 h. Flow cytometry was performed to analyze the recruitment of adoptively transferred monocytes to the brain (left, gated on CD45.2+CD45.1- donor monocytes). The quantification of recruited donor monocytes was shown in the right panel. Scale bar: 10 μm. Data are expressed as mean ± SEM and representative of 2 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.