Type I/type III IFN and related factors regulate JEV infection and BBB endothelial integrity

Background: Japanese encephalitis virus (JEV) remains a predominant cause of Japanese encephalitis (JE) globally. Its Infection is usually accompanied by disrupted blood‒brain barrier (BBB) integrity and central nervous system (CNS) inflammation in a poorly understood pathogenesis. Productive JEV Infection in brain microvascular endothelial cells (BMECs) is considered the initial event of the virus in penetrating the BBB. Type I/III IFN and related factors have been described as negative regulators in CNS inflammation, whereas their role in JE remains ambiguous.

Methods: RNA-sequencing profiling (RNA-seq), real-time quantitative PCR, enzyme-linked immunosorbent assay, and Western blotting analysis were performed to analyze the gene and protein expression changes between mock- and JEV-infected hBMECs. Bioinformatic tools were used to cluster altered signaling pathway members during JEV Infection. The shRNA-mediated immune factor-knockdown hBMECs and the in vitro transwell BBB model were utilized to explore the interrelation between immune factors, as well as between immune factors and BBB endothelial integrity.

Results: RNA-Seq data of JEV-infected hBMECs identified 417, 1256, and 2748 differentially expressed genes (DEGs) at 12, 36, and 72 h post-infection (hpi), respectively. The altered genes clustered into distinct pathways in gene ontology (GO) terms and KEGG pathway enrichment analysis, including host Antiviral immune defense and endothelial cell leakage. Further investigation revealed that pattern-recognition receptors (PRRs, including TLR3, RIG-I, and MDA5) sensed JEV and initiated IRF/IFN signaling. IFNs triggered the expression of interferon-induced proteins with tetratricopeptide repeats (IFITs) via the JAK/STAT pathway. Distinct PRRs exert different functions in barrier homeostasis, while treatment with IFN (IFN-β and IFN-λ1) in hBMECs stabilizes the endothelial barrier by alleviating exogenous destruction. Despite the complex interrelationship, IFITs are considered nonessential in the IFN-mediated maintenance of hBMEC barrier integrity.

Conclusions: This research provided the first comprehensive description of the molecular mechanisms of host‒pathogen interplay in hBMECs responding to JEV invasion, in which type I/III IFN and related factors strongly correlated with regulating the hBMEC barrier and restricting JEV Infection. This might help with developing an attractive therapeutic strategy in JE.

Human brain microvascular endothelial cells; IFITs; IFNs; Japanese encephalitis virus; PRRs; RNA-Seq.