CCR5 ameliorates Japanese encephalitis via dictating the equilibrium of regulatory CD4⁺Foxp3⁺ T and IL-17⁺CD4⁺ Th17 cells

Background: CCR5 is a CC chemokine receptor involved in the migration of effector leukocytes including macrophages, NK, and T cells into inflamed tissues. Also, the role of CCR5 in CD4⁺Foxp3⁺ regulatory T cell (Treg) homing has recently begun to grab attention. Japanese encephalitis (JE) is defined as severe neuroinflammation of the central nervous system (CNS) following infection with mosquito-borne flavivirus JE virus. However, the potential contribution of CCR5 to JE progression via mediating CD4⁺Foxp3⁺ Treg homing has not been investigated. Methods: Infected wild-type (Ccr5^+/+) and CCR5-deficient (Ccr5 ^/ ) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, NK- and JEV-specific T cell responses were analyzed. Adoptive transfer of CCR5⁺CD4⁺Foxp3⁺ Tregs was used to evaluate the role of Tregs in JE progression. Results: CCR5 ablation exacerbated JE without altering viral burden in the extraneural and CNS tissues, as manifested by increased CNS infiltration of Ly-6C^hi monocytes and Ly-6G^hi granulocytes. Compared to Ccr5^+/+ mice, Ccr5 ^/ mice unexpectedly showed increased responses of IFN-γ⁺NK and CD8⁺ T cells in the spleen, but not CD4⁺ T cells. More interestingly, CCR5-ablation resulted in a skewed response to IL-17⁺CD4⁺ Th17 cells and correspondingly reduced CD4⁺Foxp3⁺ Tregs in the spleen and brain, which was closely associated with exacerbated JE. Our results also revealed that adoptive transfer of sorted CCR5⁺CD4⁺Foxp3⁺ Tregs into Ccr5 ^/ mice could ameliorate JE progression without apparently altering the viral burden and CNS infiltration of IL-17⁺CD4⁺ Th17 cells, myeloid-derived Ly-6C^hi monocytes and Ly-6G^hi granulocytes. Instead, adoptive transfer of CCR5⁺CD4⁺Foxp3⁺ Tregs into Ccr5 ^/ mice resulted in increased expression of anti-inflammatory cytokines (IL-10 and TGF-β) in the spleen and brain, and transferred CCR5⁺ Tregs were found to produce IL-10. Conclusions: CCR5 regulates JE progression via governing timely and appropriate CNS infiltration of CD4⁺Foxp3⁺ Tregs, thereby facilitating host survival. Therefore, this critical and extended role of CCR5 in JE raises possible safety concerns regarding the use of CCR5 antagonists in human immunodeficiency virus (HIV)-infected individuals who inhabit regions in which both HIV and flaviviruses, such as JEV and West Nile virus, are endemic.