West Nile virus (WNV) and Japanese encephalitis virus (JEV) are emerging mosquito-borne flaviviruses that cause encephalitis globally. Interleukin-6 (IL-6) upregulation is associated with viral infections in mice and humans, implying that it may influence the outcome of the disease. In this study, we investigated the role of IL-6 during neurotropic flavivirus infections. We found that IL-6 neutralization in human neuronal cells and its deletion in primary mouse cells led to increased flavivirus titers, suggesting that IL-6 inhibits flavivirus replication in vitro. In vivo experiments using IL-6 knockout mice (IL-6^−/−) showed higher mortality and viral titers in the periphery and brain, revealing IL-6's protective role against WNV and JEV infections. We also examined how IL-6 modulates immune responses. We observed reduced cytokine levels in the infected human neuronal cells with neutralized IL-6 and in the serum of IL-6^−/− mice despite high viral load. We further detected downregulated type I interferon response in the IL-6^−/− mice.

Our subsequent research involves comprehensive transcriptome analysis of WNV-infected murine brains to elucidate the function of IL-6. We used RNA-seq to compare IL-6^−/− mice with wild-type (WT) mice, revealing key genes with differential expressions in IL-6^−/− mice, primarily related to inflammation, cell death, and interferon pathways. IL-6 deficiency in mice weakened interferon-stimulated gene (ISG) activation, potentially compromising the antiviral response. These mice exhibited upregulated proinflammatory cytokine and chemokine genes but downregulated immunoregulatory genes. Ingenuity Pathway Analysis (IPA) highlighted increased immune cell recruitment and inflammatory pathways in IL-6^−/− mice, along with decreased expressions of immune checkpoint regulators and NF-κB and MAPK pathway suppressors. An examination of differentially expressed pattern recognition receptors and neuroinflammation-associated genes in IL-6^−/− mice brains corroborated these findings by revealing an upregulation of antigen presentation, immune cell activation, leukocyte recruitment, and NF-κB pathway, but downregulation of genes crucial for stimulating interferon and antiviral responses. Overall, this study demonstrates that the absence of IL-6 increases the severity of WNV or JEV infection both in vitro and in vivo and sheds light on global immune pathway modulation by IL-6 during a neurotropic flavivirus infection.