Embregts, C. W. E. | bioRxiv Science

bioRxiv2021

Replication kinetic, cell tropism and associated immune responses in SARS-CoV-2 and H5N1 virus infected human iPSC derived neural models

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with a wide variety of neurological complications. Even though SARS-CoV-2 is rarely detected in the central nervous system (CNS) or cerebrospinal fluid, evidence is accumulating that SARS-CoV-2 might enter the CNS via the olfactory nerve. However, what happens after SARS-CoV-2 enters the CNS is poorly understood. Therefore, we investigated the replication kinetics, cell tropism, and associated immune responses of SARS-CoV-2 infection in different types of neural cultures derived from human induced pluripotent stem cells (hiPSCs). SARS-CoV-2 was compared to the neurotropic and highly pathogenic H5N1 influenza A virus. SARS-CoV-2 infected a minority of individual mature neurons, without subsequent virus replication and spread, despite ACE2, TMPRSS2 and NPR1 expression in all cultures. However, this sparse infection did result in the production of type-III-interferons and IL-8. In contrast, H5N1 virus replicated and spread very efficiently in all cell types in all cultures. Taken together, our findings support the hypothesis that neurological complications might result from local immune responses triggered by virus invasion, rather than abundant SARS-CoV-2 replication in the CNS.

microbiology↗

bioRxiv2021

Street RABV induces the cholinergic anti-inflammatory pathway in human monocyte-derived macrophages by binding to nAChr-a7, and upregulates the M2-c marker CD163

Rabies virus (RABV) is able to reach the central nervous system (CNS) without triggering a strong immune response, using multiple mechanisms to evade and suppress the host immune system. After infection via a bite or scratch from a rabid animal, RABV comes into contact with macrophages, which are the first antigen-presenting cells (APCs) that are recruited to the area and play an essential role in the onset of a specific immune response. It is poorly understood how RABV affects macrophages, and if the interaction contributes to the observed immune suppression. This study was undertaken to characterize the interactions between RABV and human monocyte-derived macrophages (MDMs). We showed that street RABV does not replicate in human MDMs. Using a recombinant trimeric RABV glycoprotein (RABV-tG) we showed binding to the nicotinic acetylcholine receptor alpha 7 (nAChr [a]7) on MDMs, and confirmed the specificity using the nAChr [a]7 antagonist alpha-bungarotoxin ([a]-BTX). We found that this binding induced the cholinergic anti-inflammatory pathway (CAP), characterized by a significant decrease in tumor necrosis factor [a] (TNF-[a]) upon LPS challenge. Using confocal microscopy we found that induction of the CAP is associated with significant cytoplasmic retention of nuclear factor {kappa}B (NF-{kappa}B). Co-cultures of human MDMs exposed to street RABV and autologous T cells further revealed that the observed suppression of MDMs affects their function as T cell activators as well, as we found a significant decrease in proliferation of CD8+ T cells. Lastly, using flow cytometric analysis we observed a significant increase in expression of CD163, hinting that street RABV is able to polarize macrophages towards a M2-c anti-inflammatory phenotype. Taken together, these results show that street RABV is capable of inducing an anti-inflammatory state in human macrophages, which affects T cell proliferation. Author summaryRabies virus (RABV) is transmitted by a bite or a scratch from an infected animal. Infection leads to a lethal encephalitis and once clinical symptoms occur, there is no effective treatment available. The virus is able to travel from the initial site of infection to the central nervous system without triggering a strong immune response, using multiple mechanisms to evade and suppress the immune system. Up to present it is unclear when and where this immunosuppression is initiated, and if local immune cells are involved as well. Understanding the complete mechanisms of immunosuppression by RABV is essential for the development and improvement of effective post-exposure treatments. In this paper we studied if RABV is able to suppress human primary macrophages as these will be the first antigen-presenting cells that are recruited to the site of infection, and are known to be important in initiating an efficient immune response. We show that RABV is able to bind, but not infect, human macrophages. Binding induces an anti-inflammatory pathway, which leads to limited T cell proliferation and directs macrophages towards and anti-inflammatory state. These results show that RABV-macrophage interactions might indeed be one of the early steps in the onset of RABV-induced immunosuppression.

immunology↗