PLX5622 Reduces Disease Severity in Lethal CNS Infection by Off-Target Inhibition of Peripheral Inflammatory Monocyte Production

Abstract

PLX5622 is a CSF-1R inhibitor and microglia-depleting reagent, widely used to investigate the biology of this central nervous system (CNS)-resident myeloid population, but the indirect or off-target effects of this agent remain largely unexplored. In a murine model of severe neuroinflammation induced by West Nile virus encephalitis (WNE), we showed PLX5622 efficiently depleted both microglia and a sub-population of border-associated macrophages in the CNS. However, PLX5622 also significantly depleted mature Ly6C^hi monocytes in the bone marrow (BM), inhibiting their proliferation and lethal recruitment into the infected brain, reducing neuroinflammation and clinical disease scores. Notably, in addition, BM dendritic cell subsets, plasmacytoid DC and classical DC, were depleted differentially in infected and uninfected mice. Confirming its protective effect in WNE, cessation of PLX5622 treatment exacerbated disease scores and was associated with robust repopulation of microglia, rebound BM monopoiesis and markedly increased inflammatory monocyte infiltration into the CNS. Monoclonal anti-CSF-1R antibody blockade late in WNE also impeded BM monocyte proliferation and recruitment to the brain, suggesting that the protective effect of PLX5622 is via the inhibition of CSF-1R, rather than other kinase targets. Importantly, BrdU incorporation in PLX5622-treated mice, suggest remaining microglia proliferate independently of CSF-1 in WNE. Our study uncovers significantly broader effects of PLX5622 on the myeloid lineage beyond microglia depletion, advising caution in the interpretation of PLX5622 data as microglia-specific. However, this work also strikingly demonstrates the unexpected therapeutic potential of this molecule in CNS viral infection, as well as other monocyte-mediated diseases.

Keywords: CNS infection; CSF-1R antagonism; West Nile virus-induced encephalitis; microglia; microglia depletion; monocyte-derived cells; monocyte-mediated inflammation; neuroinflammation.

Figure 1

PLX5622-mediated microglia depletion is protective in WNE and reversed upon cessation of treatment. (A) Schematic of experimental design. Mice were fed control chow (AIN-76A, Ctrl) or PLX5622-formulated chow until sacrifice at dpi 5 (or day 26) or dpi 7 (or day 28) (PLX) or fed PLX5622-formulated chow until dpi 0 (or day 21) and then fed control chow (AIN-76A) until dpi 7 (PLX-Ctrl). (B) Number of microglia in the brain of mock-infected and infected Ctrl and PLX mice at dpi 5 and 7 and infected PLX-Ctrl mice at dpi 7. (C) Percent of microglia depleted in PLX5622-treated and mock-infected or infected mice at dpi 5 and 7. (D, E) Weight (g) of chow consumed (D) and percent of weight lost (E) by mock-infected and infected mice culled at dpi 5 or 7. (F) Disease score of Ctrl, PLX-Ctrl and PLX mice at WNV dpi 7. (G, H) Number and percent of BrdU⁺ microglia in mock-infected and infected Ctrl and PLX mice at dpi 5 and 7 and infected PLX-Ctrl mice at dpi 7. Data is presented as mean ± SEM from two independent experiments with at least three mice per group.

Figure 2

Increasing numbers of microglia are required to reduce viral load late in WNV-infection. (A) Table showing experimental groups. (B, C) PFU of WNV in brains of PLX and Ctrl mice at dpi 5 (B) and 7 (C). (D, E) Expression of Wnv as determined by qPCR in brains of infected Ctrl and PLX mice at dpi 5 (D) and 7 (E) and PLX-Ctrl at dpi 7 (E). Wnv was normalized to the housekeeping gene, Rpl13a. (F) Correlation analysis between the expression of Wnv and the number of microglia in PLX mice at dpi 7. Data is presented as mean ± SEM from one (plaque assay) or two (qPCR data) independent experiments with at least three mice per group.

Figure 3

PLX5622 reduces leukocyte influx into WNV-infected brains. (A) tSNE plot clustered on CD45⁺ brain cells from dpi 5 PLX and Ctrl mice. (B) Stacked bar graph showing the number of brain cells in PLX and Ctrl mice at dpi 5. (C, D) Number of myeloid (C) or lymphoid (D) cells in brains of mice culled at dpi 5. (E) tSNE plot clustered on CD45⁺ brain cells from PLX and Ctrl mice at dpi 7. (F) Stacked bar graph showing the number of brain cells at dpi 7. (G, H) Number of myeloid (G) or lymphoid (H) cells at dpi 7 in brains of PLX, PLX-Ctrl and Ctrl mice. Data is presented as mean ± SEM from one (dpi 5 data) or two (dpi 7 data) independent experiments with at least three mice per group.

Figure 4

PLX5622 reduces cytokine production and the neuroinflammatory response to WNV infection. (A, B) Expression of select cytokines in brains of Ctrl and PLX mice at dpi 5 (A) and Ctrl, PLX-Ctrl and PLX mice at dpi 7 (B), as determined by qPCR. Gene expression was normalized to the housekeeping gene, Rpl13a. Data is presented as mean ± SEM from one (dpi 5 data) or two (dpi 7 data) independent experiments with at least three mice per group.

Figure 5

PLX5622 inhibits BM monocyte production. (A, B) Number of infiltrating brain cells (A) and microglia (B) at dpi 7 in mouse groups fed low-dose PLX5622: Ctrl, PLX^lo-Ctrl and PLX^lo . (C) Correlation between the frequency of BrdU⁺ mature BM monocytes out of CD45⁺ BM cells and the number of CNS infiltrating Ly6C^hi macrophages at dpi 7. (D) Correlation between the number of mature BM monocytes and the number of CNS infiltrating Ly6C^hi macrophages at dpi 7. (E–G) Number of myeloid cells in the BM of mock- and WNV-infected mice (E), mock-infected PLX and Ctrl mice (F) and infected PLX, PLX-Ctrl and Ctrl mice (G). (H) tSNE plot clustered on BM cells from PLX, PLX-Ctrl or Ctrl mice at dpi 7. (I) tSNE plots showing the expression of BrdU and CD115 in/on BM cells from PLX, PLX-Ctrl or Ctrl mice at dpi 7. (J–L) Number and frequency of BrdU⁺ mature BM monocytes in mock and infected mice (J), mock-infected PLX and Ctrl mice (K) and infected PLX, PLX-Ctrl and Ctrl mice (L). Data is presented as mean ± SEM from one (A, B, F, K) or three (E, G, H–J, L) independent experiment with at least three mice per group.

Figure 6

Treatment with anti-CSF-1R, like PLX5622, reduces CNS infiltration and BM monocyte production. (A) Schematic of experimental workflow. Mice were infected with WNV and treated with an isotype control monoclonal blocking antibody or anti-CSF-1R at either 1) dpi 0, 2, 4 and 6 or 2) dpi 5 and 6. (B) Number of myeloid cells in brains of mice treated with an isotype control or anti-CSF-1R at WNV dpi 7. (C) Number of myeloid cells in infected (dpi 7) BMs from mice treated with an isotype control or anti-CSF-1R. (D, E) Number (D) and percent (E) of BrdU⁺ myeloid cells in infected (dpi 7) BMs from mice treated with an isotype control or anti-CSF-1R. Data is presented as mean ± SEM from two independent experiment with at least six mice per group.

Figure 7

Anti-CSF-1R does not affect monocyte trafficking into the CNS. (A) Schematic of experimental workflow. Mice were infected and treated with an isotype control monoclonal blocking antibody or anti-CSF-1R. Two hours prior to tissue collection animals were injected with an intravenous dye. (B) Number of myeloid and lymphoid populations in brains of mice infected with WNV and treated with an isotype control antibody or anti-CSF-1R. (C) Number of PKH26⁺ Ly6C^hi macrophages infiltrating the WNV-infected brain per minute at dpi 7. (D) Percent of PKH26⁺ Ly6C^hi macrophages in WNV-infected brains at dpi 7. (E, F) Number (E) and percent (F) of myeloid cells in infected (dpi 7) BMs from mice treated with an isotype control or anti-CSF-1R. (G, H) Number (G) and percent (H) of BrdU⁺ myeloid cells in infected (dpi 7) BMs from mice treated with an isotype control or anti-CSF-1R. Data is presented as mean ± SEM from one independent experiment with at least three mice per group.