Distinguishing the effects of systemic CSF1R inhibition by PLX3397 on microglia and peripheral immune cells.

Microglia, the primary immune cells of the central nervous system (CNS), are derived from the yolk sac and populate the brain during development. Once microglia migrate to the CNS, they are self-renewing and require CSF1R signaling for their maintenance. Pexidartinib (PLX3397, PLX), a small molecule inhibitor of the CSF1R, has been shown to effectively deplete microglia since microglial maintenance is CSF1R-dependent. There have, however, been several conflicting reports that have shown the potential off-target effects of PLX on peripheral immune cells particularly those of lymphoid origin. Given this controversy in the use of the PLX family of drugs, it has become important to ascertain to what extent PLX affects the peripheral immune profile in lymphoid (spleen, and bone marrow) and non-lymphoid (kidney, lungs, and heart) organs. PLX3397 chow treatment at 660 mg/kg for 7 days significantly reduced CD45+ macrophages, CX3CR1-GFP cells, CD11b+CD45intermediate cells, and P2RY12 expression in the brain. However, there were minimal effects on peripheral immune cells from both lymphoid and non-lymphoid organs except in the heart where there was a significant decrease in CD3+ cells, inflammatory and patrolling monocytes, and CD11b+Ly6G+ neutrophils. We then stimulated the immune system with 1 mg/kg of LPS which resulted in a significant reduction in the number of innate immune cells. In this context, PLX did not alter the cytokine profile in the serum and the brain of naïve mice but did so in the LPS-stimulated group resulting in a significant reduction in TNFα, IL-1α, IFN-γ and IL-1ß. Furthermore, PLX did not alter locomotor activity in the open field test suggesting that microglia do not contribute to LPS-induced sickness behavior. Our results provide an assessment of immune cell populations with PLX3397 treatment on brain, lymphoid and non-lymphoid organs without and during LPS treatment that can serve as a resource for understanding consequences of such approaches.

Mitoxantrone modulates a heparan sulfate-spike complex to inhibit SARS-CoV-2 infection.

Spike-mediated entry of SARS-CoV-2 into human airway epithelial cells is an attractive therapeutic target for COVID-19. In addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of spike with a downstream receptor to promote viral entry. Here, we show that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a heparan sulfate-spike complex to compromise the fusogenic function of spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar heparan sulfate-binding activities but with reduced affinity for DNA topoisomerases may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era.