PMC:6354430 / 33801-41733 JSONTXT

{"project":"2_test","denotations":[{"id":"30704498-11559811-63046413","span":{"begin":255,"end":257},"obj":"11559811"},{"id":"30704498-15254203-63046414","span":{"begin":357,"end":359},"obj":"15254203"},{"id":"30704498-18678898-63046415","span":{"begin":361,"end":363},"obj":"18678898"},{"id":"30704498-16103196-63046416","span":{"begin":472,"end":474},"obj":"16103196"},{"id":"30704498-12424694-63046417","span":{"begin":476,"end":478},"obj":"12424694"},{"id":"30704498-29292385-63046418","span":{"begin":480,"end":482},"obj":"29292385"},{"id":"30704498-22838642-63046419","span":{"begin":3859,"end":3861},"obj":"22838642"}],"text":"Loss of cellular sources of co-stimulatory signals in the CNS results in decreased T cell reactivation\nMicroglia are hypothesized to act as immediate responders to CNS pathogens as resident APCs that coordinate intra-parenchymal adaptive immune response [54]. Previous studies indicated that clearance of WNV in the CNS requires antiviral CD8+ T cells [19, 32, 55], and that microglia, astrocytes, and neurons express chemokines that attract mononuclear cells to the CNS [21, 56, 57]. To determine the impact of CSF1R antagonism on recruitment of these cells to the CNS, leukocytes isolated from the cortices of WNV-NS5-E218A-infected mice were assessed by flow cytometry at 8 dpi. As expected, CD45loCD11b+ (P1) population, which is primarily microglia [58], remained significantly depleted in the PLX5622-treated compared with control-treated mice (Fig. 6a–c). The population of CD45hiCD11b+ (P2), which is primarily infiltrating monocytes/macrophages [58], were non-significantly reduced in the PLX5622-treated compared with control-treated mice. However, the number of CD45hiCD11bneg (P3) lymphocyte population [58] were non-significantly elevated in PLX5622-treated compared with control mice, though the percentage of this population was significantly elevated (Fig. 6a–c). This increase was largely due to elevated numbers of CD8+ T cell infiltration in PLX5622-treated mice (Fig. 6d–f). Though there was no significant difference in the percentage of CD8+ T cells specific for the WNV immunodominant peptide between PLX5622 and control-treated mice, there were significantly more WNV-specific CD8+ T cells isolated from PLX5622 compared with control-treated cortex (Fig. 6g–i). Despite the increased infiltration of WNV-specific CD8+ T cells in PLX5622-treated mice, the percentages of cells expressing early activation marker CD69 and their level of expression were significantly reduced in PLX5622-treated compared with control-treated mice (Fig. 6j–m). Additionally, the percentage of WNV-specific CD8+ T cells positive for CD160 as well as the level of CD160 expression, which is specifically expressed on highly activated CD8+ T cells [59], were reduced in PLX5622-treated compared with control-treated mice. However, the numbers of NS4B+CD8+ cells positive for either CD69 or CD160 were non-significantly elevated in PLX5622-treated compared with control-treated cortices (Fig. 6l, p) due to the increased numbers of NS4B+CD8+ cells.\nFig. 6 CSF1R antagonism does not hinder antiviral T cell recruitment to the CNS, but recruited T cells lack full activation during i.c. infection with WNV-NS5-E218A. Mice were fed PLX5622 chow or control chow for 2 weeks, then infected i.c. with WNV-NS5-E218A (104 PFU). At 8 dpi, leukocytes were isolated from the cortex and analyzed by flow cytometry. a Representative flow cytometry plots of CD45+-gated cells analyzed by CD11b vs CD45 expression, identifying three populations of cells: CD11b+CD45lo (P1), CD11b+CD45hi (P2), and CD11bnegCD45hi (P3). b Quantification of percentages and c number of cells in each gate. d Representative flow cytometry plots of CD4 vs CD8 expression and e quantification of percentages and f total numbers of CD4+ vs CD8+ cells within P3 gate. g Representative flow cytometry plots of NS4B+WNV-specific tetramer staining and h quantification of percentages and i total number of NS4B+CD8+CD45+ cells. j Representative flow cytometry plots of CD69 expression on NS4B+CD8+CD45+ cells and k quantification of percentages, l total numbers, and m MFI of CD69+NS4B+CD8+CD45+ cells. n Representative flow cytometry plots of CD160 expression on NS4B+CD8+CD45+ cells and o quantification of percentages, p total numbers, and q MFI of CD160+NS4B+CD8+CD45+ cells. MFI quantifications are shown as arbitrary units (AU) by normalizing each sample to the MFI of FMO negative control value [77]. For quantification panels, each symbol represents an individual control- (black) or PLX5622-treated (red) mouse, and bars indicate mean ± SEM. Data presented are the compilation of two independent experiments with seven to nine mice per group. Statistical significance was calculated using unpaired t tests with Welch’s correction. For all data: ns, not significant at P \u003c 0.05; *P \u003c 0.05; **P \u003c 0.01; ***P \u003c 0.001; ****P \u003c 0.0001\nBecause numbers of infiltrating WNV-specific CD8+ T cells derived from the CNS of PLX5622-treated animals were increased, we hypothesized that their lack of local reactivation may be the result of loss of co-stimulatory signals and/or inflammatory cytokines (Fig. 3c) in the CNS. In order to distinguish resident microglia from infiltrating monocytes/macrophages, CNS myeloid cells were examined for expression of the microglia-specific marker P2RY12 (Fig. 7a). Infiltrating monocytes/macrophages were then identified via gating of P2RY12negCD11b+ cells (Fig. 7b). Populations of both CNS-derived P2RY12+CD45+ microglia (P1, Fig. 7a) and CD11b+P2RY12negCD45+ monocytes/macrophages (P2, Fig. 7b) were reduced in PLX5622-treated compared with control-treated mice (Fig. 7i, j). While no differences in the overall number of cells expressing MHCII were observed in P2RY12+CD45+, a significant reduction of MHCII+CD11b+P2RY12negCD45+ was seen (Fig. 7c, d, l). This corresponded with a relative increase in the percentage of MHCII+ cells in both P2RY12+CD45+ and CD11b+P2RY12negCD45+ populations due to the decreased numbers of total P2RY12+CD45+ and CD11b+P2RY12negCD45+ cells in the cortices of PLX5622-treated mice compared with control-treated mice (Fig. 7k). However, the numbers of P2RY12+CD45+ cells expressing B7 co-stimulatory signals CD80 and CD86 were significantly reduced in the CNS of PLX5622-treated mice compared with control-treated animals (Fig. 7e–h, m–p). The percentage of remaining P2RY12+CD45+ cells expressing CD80 or CD86 were not reduced (Fig. 7m, o); however, the percentage of remaining CD11b+P2RY12negCD45+ cells expressing CD86 was significantly reduced (Fig. 7o), similar to results seen in peripheral APCs (Figs. 4, 5) Together, these data suggest that resident microglia and infiltrating macrophages are important sources of CD80 and CD86 in the CNS, and PLX5622 treatment depletes these cellular sources of co-stimulatory molecules.\nFig. 7 Microglia supply co-stimulatory signal 2 for T cell activation in the WNV-NS5-E218A-infected CNS. Mice were fed PLX5622 chow or control chow for 2 weeks, then i.c. infected with WNV-NS5-E218A (104 PFU). At 8 dpi, leukocytes were analyzed from the cortex. a Representative flow cytometry contour plots of P2RY12 expression on CD45+-gated cells to identify resident microglia (P1). b Representative flow cytometry plots of CD11b expression on P2RY12negCD45+ cells to identify infiltrating macrophages (P2). c, d Representative flow cytometry plots of MHCII expression on c P1 microglia and d P2 macrophages. e, f Representative flow cytometry plots of CD80 expression on e P1 microglia and f P2 macrophages. g, h Representative flow cytometry dot plots of CD86 expression on g P1 microglia and h P2 macrophages. i, j Quantification of flow cytometry analysis of i percentages and j total numbers of P1- vs P2-gated cells. k Quantification of percentages and l total numbers of MHCII+CD45+ cells within each P1 and P2 population. m Quantification of percentages and n total number of CD80+CD45+ cells within each P1 and P2 population. o Quantification of percentages and p total numbers of CD86+CD45+ cells within each P1 and P2 population. For quantification panels, each symbol represents an individual control- (black) or PLX5622-treated (red) mouse, and bars indicate mean ± SEM. Data presented are the compilation of two independent experiments with seven to nine mice per group. Statistical significance was calculated using multiple t tests with Welch’s correction. For all data: ns, not significant at P \u003c 0.05; *P \u003c 0.05; **P \u003c 0.01; ***P \u003c 0.001; ****P \u003c 0.0001"}