CSF1R antagonism limits local restimulation of antiviral CD8+ T cells during viral encephalitis

Abstract

Background: Microglia are resident macrophages of the central nervous system (CNS) locally maintained through colony-stimulating factor 1 receptor (CSF1R) signaling. Microglial depletion via CSF1R inactivation improves cognition in mouse models of neuroinflammation, but limits virologic control in the CNS of mouse models of neurotropic infections by unknown mechanisms. We hypothesize that CSF1R plays a critical role in myeloid cell responses that restrict viral replication and locally restimulate recruited antiviral T cells within the CNS.

Methods: The impact of CSF1R signaling during West Nile virus infection was assessed in vivo using a mouse model of neurotropic infection. Pharmacological inactivation of CSF1R was achieved using PLX5622 prior to infection with virulent or attenuated strains of West Nile virus (WNV), an emerging neuropathogen. The subsequent effect of CSF1R antagonism on virologic control was assessed by measuring mortality and viral titers in the CNS and peripheral organs. Immune responses were assessed by flow cytometric-based phenotypic analyses of both peripheral and CNS immune cells.

Results: Mice treated with CSF1R antagonist prior to infection exhibited higher susceptibility to lethal WNV infection and lack of virologic control in both the CNS and periphery. CSFR1 antagonism reduced B7 co-stimulatory signals on peripheral and CNS antigen-presenting cells (APCs) by depleting CNS cellular sources, which limited local reactivation of CNS-infiltrating virus-specific T cells and reduced viral clearance.

Conclusions: Our results demonstrate the impact of CSF1R antagonism on APC activation in the CNS and periphery and the importance of microglia in orchestrating the CNS immune response following neurotropic viral infection. These data will be an important consideration when assessing the benefit of CSF1R antagonism, which has been investigated as a therapeutic for neurodegenerative conditions, in which neuroinflammation is a contributing factor.

Keywords: Antiviral T cells; Colony-stimulating factor 1 receptor; Microglia; PLX5622; Viral encephalitis; West Nile virus.

Fig. 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⁺CD11b^negCD11c⁺CD45⁺, (II) CD103⁺CD11b⁺CD11c⁺CD45⁺, (III) CD103^negCD11b⁺CD11c⁺CD45⁺, and (IV) CD103^negCD11b^negCD11c⁺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 < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. CTRL: Control; PLX: PLX5622

Fig. 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 < 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001

Fig. 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 < 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001

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

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

Fig. 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⁺CD45^lo (P1), CD11b⁺CD45^hi (P2), and CD11b^negCD45^hi (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 < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 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 P2RY12^negCD45⁺ 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 < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001