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Infection with flaviviruses causes mild to severe diseases, including viral hemorrhagic fever, vascular shock syndrome, and viral encephalitis. Several animal models explore the pathogenesis of viral encephalitis, as shown by neuron destruction due to neurotoxicity after viral infection. While neuronal cells are injuries caused by inflammatory cytokine production following microglial/macrophage activation, the blockade of inflammatory cytokines can reduce neurotoxicity to improve the survival rate. This study investigated the involvement of macrophage phenotypes in facilitating CNS inflammation and neurotoxicity during flavivirus infection, including the Japanese encephalitis virus, dengue virus (DENV), and Zika virus. Mice infected with different flaviviruses presented encephalitis-like symptoms, including limbic seizure and paralysis. Histology indicated that brain lesions were identified in the hippocampus and surrounded by mononuclear cells. In those regions, both the infiltrated macrophages and resident microglia were significantly increased. RNA-seq analysis showed the gene profile shifting toward type 1 macrophage (M1) polarization, while M1 markers validated this phenomenon. Pharmacologically blocking C-C chemokine receptor 2 and tumor necrosis factor-α partly retarded DENV-induced M1 polarization. In summary, flavivirus infection, such as JEV and DENV, promoted type 1 macrophage polarization in the brain associated with encephalitic severity.

Background: Phorbol 12-myristate 13-acetate (PMA)-induced differentiation of human monocytic THP-1 cells is an experimental model for preparing resting macrophages (M 0 ) for cell polarization toward the different functional specializations of macrophages. Methods: In this study, we examined the expression of immune checkpoints by using flow cytometry following multicolor staining. The blockade of immune checkpoint by using neutralizing antibodies was performed to assess their role in PMA-induced THP-1-differentiated macrophages. Results: Upon the inducible macrophage differentiation caused by PMA, increased expression levels of CD11b and CD68 were measured and characterized according to their adherent phenotype accompanied by the generation of cellular complexity. While the cell growth rate was abolished post-differentiation, some cells underwent cell death. Notably, we found increases in the expression of programmed cell death protein 1, also known as PD-1 (CD279), and its ligand PD-L1 (CD274), mainly in differentiated M 0 (CD68 + CD11b + ) macrophages. However, neutralizing PD-L1/PD-1 neither blocked THP-1 cell differentiation toward macrophages nor inhibited macrophage polarization in M 1 and M 2 . In specializing macrophages, a decrease both in CD274 and CD279 was found in M 2 . Conclusion: These results revealed the inducible expression of PD-L1/PD-1 in PMAinduced THP-1-differentiated M 0 macrophages followed by a decrease in M 2 macrophages.