West Nile virus is a globally occurring, neurotropic mosquito-borne virus of the Flaviviridae family, alongside dengue and Zika virus. During murine WNV encephalitis, bone marrow(BM)-derived Ly6C^hi inflammatory monocyte infiltration of the central nervous system (CNS) causes lethal immunopathology. Recently, NK cell-derived IFNγ has been shown to prime Ly6C^hi monocytes in the BM prior to their emigration during anti-parasitic responses in C57BL/6 mice. Despite the belief that WNV-induced MHC-I upregulation inhibits NK cell activity, NK cells infiltrate into the CNS in high numbers, suggesting involvement in disease.

To determine the role of NK cells in WNV encephalitis, we used high dimensional flow and mass cytometry (CyTOF) to characterize NK cells in the BM and CNS. In addition to traditional analysis methods, we used viSNE and SPADE computational approaches.

Surprisingly, most NK cells in the brain were terminally mature, expressed several activation markers and produced cytotoxic Granzyme B. However, 75% of NK cells in the brain also expressed inhibitory markers, suggesting suppression by local presence of virus. In contrast, in the BM, only 25% of NK cells expressed inhibitory markers. Furthermore, NK cells were the main IFNγ-producing cell in the BM and depletion of either NK cells or IFNγ, reduced the output of Ly6C^hi monocytes. To confirm the role of NK cell-derived IFNγ in Ly6C^hi monocyte egress from the BM, we compared brain infiltration in C57BL/6 and SJL/J mice, since the latter have reduced NK cells numbers and IFNγ responses, which directly correlated to reduced numbers of Ly6C^hi monocytes in the CNS.

In conclusion, despite their apparent lack of explicit anti-viral activity in the infected CNS, IFNγ-producing NK cells in the BM play a pivotal role in Ly6C^hi monocyte mobilisation, and subsequent immunopathology in WNV encephalitis. This reveals NK cells as a potential therapeutic target in WNV encephalitis.