CSF1R antagonism depletes myeloid populations within peripheral blood and CNS
CSF1R 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.
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+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 < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.
CTRL: Control; PLX: PLX5622