PMC:6354430 / 15247-41734 JSONTXT

{"project":"2_test","denotations":[{"id":"30704498-27516055-63046404","span":{"begin":478,"end":480},"obj":"27516055"},{"id":"30704498-26232154-63046405","span":{"begin":482,"end":484},"obj":"26232154"},{"id":"30704498-11585527-63046406","span":{"begin":3914,"end":3916},"obj":"11585527"},{"id":"30704498-27337340-63046407","span":{"begin":4007,"end":4008},"obj":"27337340"},{"id":"30704498-21085181-63046408","span":{"begin":4010,"end":4012},"obj":"21085181"},{"id":"30704498-27576527-63046409","span":{"begin":5918,"end":5920},"obj":"27576527"},{"id":"30704498-24323358-63046409","span":{"begin":5918,"end":5920},"obj":"24323358"},{"id":"30704498-23000011-63046409","span":{"begin":5918,"end":5920},"obj":"23000011"},{"id":"30704498-22589727-63046410","span":{"begin":8474,"end":8476},"obj":"22589727"},{"id":"30704498-21085181-63046411","span":{"begin":8478,"end":8480},"obj":"21085181"},{"id":"30704498-17567689-63046412","span":{"begin":13231,"end":13233},"obj":"17567689"},{"id":"30704498-11559811-63046413","span":{"begin":18809,"end":18811},"obj":"11559811"},{"id":"30704498-15254203-63046414","span":{"begin":18911,"end":18913},"obj":"15254203"},{"id":"30704498-18678898-63046415","span":{"begin":18915,"end":18917},"obj":"18678898"},{"id":"30704498-16103196-63046416","span":{"begin":19026,"end":19028},"obj":"16103196"},{"id":"30704498-12424694-63046417","span":{"begin":19030,"end":19032},"obj":"12424694"},{"id":"30704498-29292385-63046418","span":{"begin":19034,"end":19036},"obj":"29292385"},{"id":"30704498-22838642-63046419","span":{"begin":22413,"end":22415},"obj":"22838642"}],"text":"Results\n\nCSF1R antagonism depletes myeloid populations within peripheral blood and CNS\nCSF1R signaling is essential for the development of mononuclear phagocytes including microglia, but pharmacological antagonism has been reported to selectively deplete microglia [48]. To examine the impact of CSF1R antagonism on both peripheral and CNS mononuclear cells, we used PLX5622, which reportedly depletes microglia in as little as 3 days and may be sustained for at least 6 weeks [37, 41]. Mice were provided either control chow or PLX5622-embedded chow for 2 weeks, after which microglia numbers were assessed by flow cytometry using the microglia-specific marker P2RY12 (Additional file 1) and by immunohistochemistry using Iba1 (Additional file 2). Quantification shows that 2 weeks of PLX5622 treatment reduced microglia in the CNS by 90–95% (Additional file 1 A–F, P, Q). Specificity of P2RY12 for CNS-resident microglia is demonstrated by lack of staining in leukocytes isolated from peripheral immune compartments (Additional file 1 G–O). Within the CNS, PLX5622 treatment significantly depleted microglia across hippocampal, cortical, and cerebellar brain regions (Additional file 2). To determine whether PLX5622 treatment depleted mononuclear cells in peripheral immune compartments, we assessed numbers of antigen-presenting cells (APCs, Fig. 1) and T cells (Additional file 3) in the spleen and blood of uninfected mice in control versus PLX5622-treated mice. PLX5622 treatment significantly reduced circulating CD11c+ (Fig. 1a–c), MHCII+CD11c+ (Fig. 1d–f), and CD11bnegCD11c+ (Fig. 1g–i) cells in the blood. In the spleen, however, there was no significant difference in populations of APCs with PLX5622 treatment (Fig. 1j–r). In addition, no significant difference in populations of T cells was detected in uninfected mice in either compartment (Additional file 3). Because of the sensitivity of microglia and APCs to CSF1R antagonism, we assessed whether CSF1R antagonism depleted CD11b+, Ly6C+, and Ly6G+ cells in the blood, spleen, and bone marrow of PLX5622-treated versus control-treated uninfected mice. Flow cytometry analysis revealed no significant differences in populations of these cells in uninfected mice (Additional file 4). Together, these data indicate that circulating APCs, but not bone marrow or splenic mononuclear cells, are decreased in the context of CSF1R antagonism.\nFig. 1 CSF1R antagonism reduces circulating APC populations in uninfected mice. Mice were fed PLX5622 chow or control chow for 2 weeks, and APCs were assessed in blood (a–i) and spleen (j–r). a, j Representative flow cytometry plots of CD11c expression on CD45+ cells. b, k Quantification of percentages and c, l total numbers of CD45+CD11c+ cells. d, m Representative flow cytometry plots of MHCII expression on CD11c+CD45+. e, n Quantification of percentages and f, o total numbers of MHCII+CD11c+CD45+ cells. g, p Representative flow cytometry plots of CD103 vs CD11b expression on CD11c+CD45+ cells. h, q Quantification of percentages and i, r total numbers of (I) CD103+CD11bnegCD11c+CD45+, (II) CD103+CD11b+CD11c+CD45+, (III) CD103negCD11b+CD11c+CD45+, and (IV) CD103negCD11bnegCD11c+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, repeated in three independent experiments. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. For all data: ns, not significant at P \u003c 0.05; *P \u003c 0.05; **P \u003c 0.01; ***P \u003c 0.001.\nCTRL: Control; PLX: PLX5622\n\nCSF1R antagonism increases susceptibility of mice to fatal WNV infection\nTo determine whether CSF1R antagonism impacts survival from WNV infection, mice were fed PLX5622-embedded chow for 2 weeks, then infected with WNV-NY [42] at either 102 or 104 PFU via footpad (f.p.) inoculation. Consistent with published data [8, 49], 30% of control-treated mice survived infection with 104 PFU and 90% of control-treated mice survived infection with 102 PFU; however, mice treated with PLX5622 universally succumbed to infection with either inoculum titer (Fig. 2a). This was associated with significantly increased weight loss (Fig. 2b) and more severe clinical score (Fig. 2c, d) in PLX5622-treated compared with control-treated mice infected with 102 PFU WNV-NY. To determine whether increased mortality caused by PLX5622 treatment is associated with increased viral replication in peripheral or CNS tissues, tissue viral loads were assessed by plaque assay, and serum viral loads were determined by quantitative RT-PCR (qRT-PCR) at 2, 4, 6, and 8 days post-infection (dpi). In the CNS, virus was first detected in the olfactory bulb (Fig. 2e), then sequentially in more caudal regions including the cortex (Fig. 2f), cerebellum (Fig. 2g), brainstem (Fig. 2h), and spinal cord (Fig. 2i). Compared with control-treated animals, PLX5622-treated mice exhibited higher viral titers in the olfactory bulb and cortex at 6 and 8 dpi and in the cerebellum, brainstem, and spinal cord at 8 dpi; however, viral titers were also significantly higher in peripheral tissues of PLX5622-treated mice including the spleen at 4 dpi (Fig. 2j), kidney at 4 and 6 dpi (Fig. 2k), and serum at 2, 4, 6, and 8 dpi (Fig. 2l). PLX5622 treatment did not cause increased BBB permeability in infected mice (Additional file 5). Once WNV-NY enters the CNS via intracranial (i.c.) inoculation, PLX5622 treatment did not affect neuronal permissivity to infection (Additional file 6). Together, these data indicate a loss of immune-mediated virologic control in both peripheral and CNS tissues in CSF1R antagonist-treated mice, consistent with prior data suggesting that CSF1R signaling plays important roles in the function of both peripheral and CNS myeloid cells [50–52].\nFig. 2 PLX5622-treated mice exhibit increased mortality, increased encephalitis score, and impaired virologic control compared with control mice following peripheral infection with WNV-NY. a–d Mice were fed chow containing PLX5622 or control chow for 2 weeks, then infected with 102 or 104 PFU via footpad infection, then monitored for (a) mortality, (b) weight loss, and (c, d) encephalitis score for up to 25 dpi. a Survival curves show a significant increase in mortality in mice treated with PLX5622 compared with control-treated mice infected with either viral inoculum dose, as calculated by log-rank (Mantel-Cox) test. b Weight was measured daily during acute illness as a measure of illness. After death, the last measured weight was carried through to the end of the experiment. Significantly greater weight loss was measured in PLX5622-treated mice compared with controls infected with 102 PFU as calculated by two-way ANOVA with matched values comparing group means without multiple comparisons. c Clinical scores of PLX5622 or control-treated mice infected with 102 PFU or d 104 PFU as indicated during acute illness. 0 = subclinical, 1 = hunched/ruffled fur, 2 = altered gate/slow movement, 3 = no movement, but responsive to stimuli, 4 = moribund, 5 = dead. e–k Tissue viral loads as measured by plaque assay at 2, 4, 6, and 8 dpi following infection with 102 PFU in control (black) or PLX5622-treated (red) mice. l Serum viral loads as measured by qRT-PCR. e–l Data are presented as scatter plots with each mouse represented by a dot with the SEM indicated by a line. Dotted lines in e–l indicate assay limit of detection. All data presented are the compilation of two independent experiments with 10 mice per group. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. For all data: ns, not significant at P \u003c 0.05; *P \u003c 0.05; **P \u003c 0.01; ****P \u003c 0.0001\nTo determine whether microglia depletion impacts innate immune mechanisms of virologic control specifically within the CNS, mice were fed control or PLX5622-embedded chow for 2 weeks, then i.c. infected with an attenuated strain of WNV (WNV-NS5-E218A) at 104 PFU. The NS5-E218A point mutation abolishes 2′-O-methyltransferase activity, which functions to methylate the 5′ cap of viral RNA and facilitate escape of IFN-induced proteins with tetratricopeptide repeats (IFIT)-mediated suppression; thus, this attenuation limits viral replication by increasing sensitivity of the virus to type I IFN-mediated innate immune mechanisms [45, 49]. Despite this attenuation, mice treated with PLX5622 were significantly more susceptible to lethal WNV-NS5-E218A infection than those receiving control chow (Fig. 3a). Weight loss was also significantly increased in PLX5622-treated mice (Fig. 3b). Consistent with this, viral titers measured by plaque assay at 2, 4, 6, and 8 dpi in the olfactory bulb (Fig. 3d), cortex (Fig. 3e), cerebellum (Fig. 3f), brainstem (Fig. 3g), and spinal cord (Fig. 3h) were significantly higher in PLX5622-treated compared in control-treated mice infected with WNV-NS5-E218A. As virus was inoculated into the cortex, it was detected there at 2 dpi, followed by spread to the olfactory bulb by 4 dpi, and cerebellum, brainstem, and spinal cord by 6 dpi. PLX5622 treatment also led to decreased proinflammatory cytokine expression in the cortex at 8 dpi (Fig. 3c) and increased neuronal apoptosis in the hippocampus and cerebellum at 6 dpi (Fig. 3l–p) after i.c. infection with WNV-NS5-E218A.\nFig. 3 CSF1R antagonism increases mortality and CNS viral burdens during i.c. infection with WNV-NS5-E218A. Mice were fed chow containing PLX5622 or control chow for 2 weeks, infected i.c. with WNV-NS5-E218A at 104 PFU, then monitored for a mortality and b weight loss for up to 25 dpi. a Survival curves show a significant increase in mortality in mice treated with PLX5622 compared with control as calculated by log-rank (Mantel-Cox) test. b Weight was measured daily during acute illness as a measure of illness. After death, the last measured weight was carried through to the end of the experiment. Significantly greater weight loss was measured in PLX5622-treated mice compared with controls as calculated by two-way ANOVA with matched values comparing group means without multiple comparisons. For a and b, data represent the compilation of two independent experiments with 10 mice per group. c At 8 dpi, cortical brain tissue was extracted and relative transcript levels in tissue homogenates were measured by SYBR qRT-PCR for indicated cytokines. Data for individual mice were normalized to Gapdh. Data are presented as mean ± SEM of three to five mice from one experiment. Statistical significance between treatment groups for each cytokine was calculated by t test. d–j Tissue viral loads were measured by plaque assay at 2, 4, 6, and 8 dpi in control (black) or PLX5622-treated (red) mice. k Serum viral loads as measured by qRT-PCR. d–k Data are presented as scatter plots with each mouse represented by a dot with the mean indicated by a line. For d–k, data represent the compilation of two-independent experiments with 10 mice per group. Dotted lines in d–k indicate assay limit of detection. l–o Representative confocal microscopic images of Tunel (red), NeuN (green), and DAPI (blue) of hippocampus (l, m) and cerebellum (n, o) of control- (l, n) or PLX5622-treated (m, o) mice infected i.c. with WNV-NS5-E218A at 6 dpi. Arrow heads point to DAPI-positive nuclei positive for both Tunel and NeuN. Asterisks denote DAPI- and Tunel-positive but NeuN-negative nuclei. p Quantification of confocal microscopic images for number of DAPI-positive nuclei positive for Tunel and NeuN in the hippocampus (Hpc), cerebellum (Cb), and brainstem (Bs). The number of Tunel+ NeuN+ nuclei per high-power field (HPF) was quantified from three to six images captured at × 40 per brain region across two separate brain sections for each of five independent mice collected in one experiment. Each symbol represents the average number for an individual mouse, with bar indicating mean ± SEM. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. For all data: ns, not significant at P \u003c 0.05; *P \u003c 0.05; **P \u003c 0.01; ****P \u003c 0.0001\nAs expected, WNV-NS5-E218A was barely detected in the kidney (Fig. 3j), and the virus was steadily cleared from the serum (Fig. 3k) in both control- and PLX5622-treated mice. However, significantly higher levels of virus were detected in the spleen of PLX5622- versus control-treated, WNV-NS5-E218A-infected mice at 4 dpi (Fig. 3i), which was consistent with data obtained during f.p. infection with WNV-NY. Overall, CSF1R antagonism leads to enhanced mortality and loss of virologic control in both the periphery and CNS regardless of the WNV strain utilized, suggesting a critical role for this molecule in antiviral immunity.\n\nCSF1R signaling is required for activation of APC co-stimulatory function\nTo 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.\nFig. 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 \u003c 0.05; *P \u003c 0.05; **P \u003c 0.01\nBecause 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.\nFig. 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 \u003c 0.05; *P \u003c 0.05; **P \u003c 0.01\n\nLoss 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\n"}