Varicella-zoster virus hijacks the type I interferon response and antigen presentation pathways in matured hiPSC-derived neurospheroids. Varicella-zoster virus hijacks the type I interferon response and antigen presentation pathways in matured hiPSC-derived neurospheroids

Varicella-zoster virus (VZV) encephalitis and meningitis are potential central nervous system (CNS) complications following primary VZV infection or reactivation. With innate immune signalling, and more specifically type I interferon (IFN) signalling, being an important first line cellular defence mechanism against VZV infection, we here investigated the triggering of innate immune responses in a human CNS-like environment. For this, we established and characterised a 5-month matured hiPSC-derived neurospheroid (NSPH) model containing TuJ1+ MAP2+ NeuN+ neurons and GFAP+ S100b+ AQP4+ CD49f+ astrocytes. Immune competence of these NSPHs was demonstrated by secretion of IL-6 and CXCL10 following stimulation with a cocktail of pro-inflammatory stimuli. Subsequently, NSPHs were infected with reporter strains of VZV (eGFP-ORF23 VZV) and Sendai virus (eGFP SeV). Live cell and immunocytochemical analyses demonstrated VZV infection within NSPHs, while SeV infection was limited to the outer NSPH border. Next, a transcript-level immune profiling was performed using NanoString technology to explore innate immune signatures of virus-infected NSPHs. While SeV-infected NSPHs displayed a clear Type I IFN response, in VZV-infected NSPHs no Type I IFN response was activated. Even more, in the latter a strong suppression of genes related to IFN signalling and antigen presentation was noted. Validating these opposite immune signatures in VZV- and SeV-infected NSPHs, cytokine profiling of NSPH supernatant revealed increased secretion of IL6 and CXCL10 by SeV-infected NSPHs, but not by VZV-infected NSPHs. Similarly, immunocytochemical analysis demonstrated upregulation of type I IFN activated anti-viral proteins Mx1, IFIT2 and ISG15 in SeV-infected NSPHs, but not in VZV-infected NSPHs. Furthermore, CD74, a key part of the MHC class II antigen presentation pathway was found to be suppressed in VZV-infected NSPHs. Finally, even though VZV-infection seems to be immunologically ignored in NSPHs, its presence does result in the formation of stress granules throughout the entire NSPH. Concluding, in this study we demonstrate that 5-month matured hiPSC-derived NSPHs display functional innate immune reactivity towards SeV infection, as well as their capability to recapitulate the strong immune evasive behaviour of VZV. Subsequently, this NSPH model has the potential to study viral neuro-immune responses and evasion strategies in a human CNS-like environment. Overall design: At 7 days post stimulation/infection, NSPHs for RNA extraction were washed with ice cold PBS, snap frozen in liquid nitrogen and stored at -80°C. RNA extraction was performed on 2 pooled NSPHs of each condition (in triplicate) using the RNeasy Mini Kit (Qiagen, 74104) according to the manufacturer's protocol. The RNA samples were used for nCounter (NanoString) analysis on a nCounter® MAX Analysis System. Briefly, RNA extracts were hybridized to ± 600 unique capture/reporter pairs (50bp each) targeting 585 immune transcripts and 15 housekeeping genes, as defined in the Human Immunology nCounter® panel, as well as six positive and eight negative control probes (all from NanoString). Results were sequentially corrected for background (negative control probes), technical variation (positive control probes) and RNA content (housekeeping genes) using nSolver 4.0 (NanoString), followed by differential gene expression analysis and gene set enrichment analysis (based on GO-terms) using Omics Playground (BigOmics Analytics).