Studies on the pathogenesis of West Nile virus encephalitis in a human cerebral organoid model and immortalized cell lines

Abstract

West Nile Virus (WNV), an arbovirus on the rise in Europe, can cause severe neurological disease, including encephalitis, in humans. At the time of writing, neither a protective vaccine nor specific treatment are available, making it crucial to further understand the underlying mechanisms of WNV encephalitis. Astrocytes and microglia are known to play an important role in the antiviral defense of the central nervous system (CNS). Aside from protective functions, they have been suspected to contribute to WNV pathology. Understanding the role of astrocytes and microglia is of great importance in laying the foundation for new treatment approaches. For this, 2D immortal cell lines were investigated for their susceptibility and response to WNV infection. As human cerebral organoids are of increasing interest in viral research and proven to be a versatile 3D model in studies on viral pathogenesis, a WNV infection model in human cerebral organoids, including mature neurons, astrocytes, and microglia-like cells, was established. In 2D, astrocytes recapitulated aspects of lower infectability in comparison to a neuron-like control cell line, which is in line with observations in patients and other in vitro approaches. While microglia were more permissive to infection than previously reported in the literature, their capacity to become activated and produce an inflammatory response highlighted their potential contribution to further understanding partial aspects of the role of microglia in WNV encephalitis. The host response observed in the transcriptome hinted at a versatile role of astrocytes and microglia in WNV encephalitis, providing a multitude of potentially involved cytokines, chemokines, and growth factors. Specifically, a local interferon response was observed, with type I interferon beta (IFN-β) in astrocytes and type III interferons as well as numerous antiviral interferon-induced genes in both astrocytes and microglia. Furthermore, astrocyte-derived interleukin 1 beta (IL-1β) could be involved in microglia activation and thereby contribute to both defense or pathology in WNV encephalitis. Moreover, the observed increase in interleukin 1 alpha (IL-1α) was previously linked to the loss of important functions in astrocytes. Additionally, an inflammatory response could be observed on the protein level by the release of pro-inflammatory interleukin 6 (IL-6) by astrocytes and C-X-C motif chemokine 10 (CXCL10) by both astrocytes and microglia. WNV infection of human cerebral organoids resulted in robust infection but, in contrast to consistent viral kinetics reported in the immortal cell lines, various courses of infection could be observed in a time frame of acute infection until 14 days post (dpi). Furthermore, a diverse response was revealed, characterized by the release of pro-inflammatory cytokines, chemokines, and other biomarkers. Interestingly, the observed increase of IL-6 and tumor necrosis factor alpha (TNF-α) may imply a cross-talk between astrocytes and microglia, promoting activation and potential cytotoxic effects. Moreover, the strong increase reported in CXCL10 was previously connected to neuronal damage, however, in this context, TNF-α has been previously observed to provide regulatory function, which is in line with the absence of a cytopathic effect (CPE) over the course of the experiment. Lastly, subgrouping of the organoids by their morphology, differentiating between organoids with and without choroid plexus structures, and the observed course of infection, differentiating between a peak in viral titer until 4 dpi and later until 14 dpi, revealed not only more homologous kinetics within the subgroups but also showed differences in the release of soluble markers. Overall, this study illustrated the potential of human cerebral organoids as a model to study WNV infection in a complex system, allowing for cell-cell interaction. In conclusion, the knowledge provided by this study includes diverse potential factors of interest to further illuminate the role of astrocytes and microglia in WNV encephalitis and demonstrated the potential of human cerebral organoids to further contribute to research on various aspects of this infectious disease.