CSF1R inhibition by a small-molecule inhibitor is not microglia specific; affecting hematopoiesis and the function of macrophages

Abstract

Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a method for microglia depletion, with the assumption that it does not affect peripheral immune cells. Here, we show that CSF1R inhibition by PLX5622 indeed affects the myeloid and lymphoid compartments, causes long-term changes in bone marrow-derived macrophages by suppressing interleukin 1β, CD68, and phagocytosis but not CD208, following exposure to endotoxin, and also reduces the population of resident and interstitial macrophages of peritoneum, lung, and liver but not spleen. Thus, small-molecule CSF1R inhibition is not restricted to microglia, causing strong effects on circulating and tissue macrophages that perdure long after cessation of the treatment. Given that peripheral monocytes repopulate the central nervous system after CSF1R inhibition, these changes have practical implications for relevant experimental data.

Keywords: CNS; CSF1R; hematopoiesis; macrophages; microglia.

Fig. 1.

CSF1R inhibition affects the myeloid and lymphoid compartments of the bone marrow, spleen, and blood. Flow cytometric analysis of bone marrow cells isolated from CCR2^+/RFP::CX3CR1^+/GFP mice treated with PLX5622 for 3 wk, at different time points after inhibitor treatment cessation. (A–F) CSF1R inhibition suppresses CCR2⁺, CX3CR1⁺, CD117⁺, and CD34⁺ cells. One week after cessation of inhibitor, only macrophages recover in number, although with a lower expression of CX3CR1. (G) CSF1R inhibition does not affect CD45⁺, CD11b⁺, or Ly6C⁺ bone marrow myeloid cell populations but does suppress CD11c⁺ dendritic cells, CD4⁺ and CD8⁺ T lymphocytes, and CD115⁺, CD117⁺, and CD34⁺ hematopoietic subsets and up-regulates CD19⁺ B cells. Three weeks after cessation of CSF1R-inhibition, CX3CR1⁺, CCR2⁺, Ly6C⁺ CD3⁺, and CD8⁺ subpopulations rebound; Ly6G⁺ granulocytes, CD115⁺, and CD117⁺ cells remain suppressed; CD4⁺ T cells and CD34⁺ cells recover; and CD19⁺ B cells remain up-regulated. (H) Effects on spleen’s myeloid and lymphoid populations. Only CD19⁺ B cells remain unaffected. (I) CSF1R inhibition causes immediate suppression in the myeloid compartment and late suppression of the lymphoid compartment of the blood. n = 5 per group, mean ± SD, one-way analysis of variance with Dunnett’s correction for multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.

CSF1R inhibition affects function and survival of resident and interstitial macrophages. (A–D) Ex vivo evaluation of the function of BMDMs from CX3CR1^+/GFP mice 3 wk after cessation of CSF1R inhibitor. Macrophages from the bone marrow or spleen exhibit reduced proliferation 3 wk after cessation of CSF1R inhibition. (E–L) CSF1R inhibition suppresses IL-1β, CD68 expression, and phagocytosis of BMDMs following exposure to LPS but does not affect CD206 expression. (H) Schematic representation of the phagocytosis assay. (M and N) CSF1R inhibition causes long-term suppression of CD115 macrophage marker. (O) CSF1R-inhibitor reduces the number of resident and interstitial macrophages of the lung, liver, peritoneum, and femur but does not affect CD45⁺ CD11b⁺ CD106⁺ spleen and F4/80^lo MHCII⁺ CSF1R⁺ CD11c⁺ peritoneal macrophages. n = 5 per group, mean ± SD, Independent t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.