CSF1R signaling is required for activation of APC co-stimulatory function
To determine whether alterations in APC populations impact antiviral immune responses, control- and PLX5622-treated mice were infected via f.p. with WNV-NY (100 PFU). After f.p. infection, WNV-infected cells traffic to draining popliteal lymph nodes (pLN) and stimulate an antiviral immune response [53]. At 4 dpi, a time-point of high serum viral loads (Fig. 2l), flow cytometric assessment revealed a trend towards decreased CD11c+CD45+ cells in the blood (Fig. 4a–d) and significantly reduced percentages and total numbers of CD11c+CD45+ cells in the pLN of PLX5622-treated compared with control-treated mice (Fig. 4k–n). There was no difference in numbers or percentages of MHCII+CD11c+CD45+ cells in either blood or pLN (Fig. 4e–g, o–q); however, the percentages of CD11c+CD45+ cells in the blood positive for co-stimulatory molecule CD80+ was significantly decreased in PLX5622- versus control-treated mice (Fig. 4h–j). In the pLN, the numbers and percentages of CD11c+CD45+ cells positive for co-stimulatory molecule CD86 were significantly reduced in PLX5622-treated mice compared with control mice (Fig. 4r–t). The impact of CSF1R antagonism on CD80 and CD86 expression appears to be tissue-dependent as the blood showed no difference in APCs expressing CD86 and the pLN showed no difference in APCs expressing CD80 (Additional file 7). Together, these data indicate that CSF1R antagonism decreases the expression of co-stimulatory B7 molecules on peripheral APC populations.
Fig. 4 CSF1R antagonism reduces response of APCs in peripheral immune compartments following peripheral infection with WNV-NY. Mice were fed PLX5622 or control chow for 2 weeks, then infected via f.p. with WNV-NY (100 PFU). At 4 dpi, leukocytes were isolated from blood (a–j) and draining popliteal LNs (k–t). a, k Representative flow cytometry plots of CD11c expression on CD45+-gated cells and b, l fluorescence minus one (FMO) gating controls. c, m Quantification of percentages and d, n total numbers of CD11c+ CD45+ cells. e, o Representative flow cytometry plots of MHCII+ expression on CD11c+CD45+ cells. f, p Quantification of percentages and g, q total numbers of MHCII+CD11c+CD45+ cells. h Representative flow cytometry plots of CD86+ expression on CD11c+CD45+ cells. i Quantification of percentages and j total numbers of CD86+CD11c+CD45+ cells. r Representative flow cytometry plots of CD80+ expression on CD11c+CD45+ cells. s Quantification of percentages and t total numbers of CD80+CD11c+CD45+ cells. For quantification panels, each symbol represents an individual control- (black) or PLX5622-treated (red) mouse, and bars indicate mean ± SEM. Data shown represent analysis from one experiment with three to five mice per group. Statistical significance was calculated using unpaired t test in all panels except d and n, which used two-way ANOVA with Sidak’s multiple comparisons test. For all data: ns, not significant at P < 0.05; *P < 0.05; **P < 0.01
Because infectious virus was detected in the spleens of mice infected i.c. with WNV-NS5-E218A (Fig. 3i), splenic APCs were analyzed using flow cytometry at 8 dpi. Results indicated no significant difference in the numbers or percentages of MHCII+CD11c+CD45+ cells in the spleens of PLX5622-treated mice compared with control-treated mice (Fig. 5a–c). However, splenic APCs collected from PLX5622-treated mice exhibited significantly reduced populations of cells expressing co-stimulatory molecules CD80 (Fig. 5d–f) and CD86 (Fig. 5g–i) compared with control-treated mice. Furthermore, splenic MHCII+CD11c+CD45+ cells showed significant reduction in the expression level of CD86 (Fig. 5l, m) and non-significant reduction of CD80 (Fig. 5j, k) in PLX5622-treated compared with control-treated mice. This correlated with a small, but significant increase in the numbers of CD4+ and CD8+ T cells, but no difference in numbers or percentages of WNV-specific NS4B tetramer staining or CD69 positivity in spleens of PLX5622-treated mice compared with control-treated mice infected i.c. with WNV-NS5-E218A (Additional file 8). Together, these data indicate that CSF1R antagonism reduces the activation of inflammatory responses in peripheral APCs even without significant depletion.
Fig. 5 CSF1R antagonism reduces co-stimulatory signal 2 on splenic APCs 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, splenic leukocytes were analyzed. a Representative flow cytometry contour plots of CD11c and MHCII expression on CD45+-gated cells and b quantification of percentages and c total numbers of CD11c+MHCII+CD45+ cells. d Representative flow cytometry plots of CD80 expression on CD11c+MHCII+CD45+ cells. e Quantification of percentages and f total numbers of CD80+CD11c+MHCII+CD45+ cells. g Representative flow cytometry plots of CD86 expression on CD11c+MHCII+CD45+ cells. h Quantification of percentages and i total numbers of CD86+CD11c+MHCII+CD45+ cells. j Representative flow cytometry histograms of CD80 expression on CD11c+MHCII+CD45+ and k quantification of MFI. l Representative flow cytometry histograms of CD86 expression on CD11c+MHCII+CD45+ and m quantification of MFI. AU, arbitrary units. For quantification panels, each symbol represents an individual control- (black) or PLX5622-treated (red) mouse, and bars indicate mean ± SEM. Data represent analysis from one experiment with three to four mice per group. Statistical significance was calculated using unpaired two-way t tests. For all data: ns, not significant at P < 0.05; *P < 0.05; **P < 0.01