Type I astrocytes and microglia induce a cytokine response in an encephalitic murine coronavirus infection

Abstract

The pathogenesis of viral infections involves an immune response by cytokines, causing a deleterious effect on organ function, in addition to tissue destruction due to viral replication. Clinical symptoms and laboratory findings of the human coronavirus disease COVID-19, caused by the novel coronavirus SARS CoV-2, indicate cytokine involvement. Our laboratory showed that an experimental murine coronavirus (MHV-A59) can be transmitted into the brain by intranasal or intracerebral exposure and that neurovirulence is mediated by cytokine secretion. In this study we investigated which cells in the brain produce cytokines, thus functioning as the brain's innate immune system. Using tissue cultures of microglia, and clonal populations of astrocytes, we found that microglia and type I astrocytes (but not types II and III), produced pro-inflammatory cytokines in response to MHV-A59 infection. A molecularly closely related, non-encephalitic strain of the virus (MHV-2) caused in vitro infection, but without cytokine induction. Furthermore, immunofluorescence and immunohistochemistry revealed that type I astrocytes and microglia have perivascular foot processes necessary for the formation of the perivascular glymphatic system, the anatomical site of the brain's innate immune system. Cytokine secretion by type I astrocytes and microglia, as part of the brain's glymphatic and innate immune system, contributes to the pathogenesis of an encephalitic coronavirus infection, and indicates the rationale for anti-cytokine therapies for COVID-19.

Keywords: Astrocytes; COVID-19; Coronavirus; Cytokines; Microglia; Mouse hepatitis virus.

Fig. 1

Murine astrocyte types in tissue cultures. Type I (A), type II (B) and type III astrocytes (C). Cell cultures were analyzed by immunofluorescence, stained with GFAP specific antibodies with red chromogen for type I and green chromogen for type II and III. Original magnification x400. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Interleukin (Il) levels in the three types of astrocytes following infections with MHV-A59 (red)), MHV-2 (yellow) and control (purple). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Interleukin (Il) levels in microglia following infections with MHV-A59 (red), MHV-2 (yellow) and control (purple). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Human brain sections depicting components of the CNS innate immune system by immunohistochemistry, GFAP antibody (A, B) and CD68 antibody (C, D), brown chromogen and hematoxylin counterstain. A. Type 1 astrocyte. x400. B. Astrocytes surrounding a blood vessel illustrating the relationship between type I astrocytic foot processes and brain blood vessels. x100. C. Microglial cells with bipolar cytoplasmic processes. x400. D. Microglial foot processes in close proximity to the endothelial cells of a brain blood vessel. x200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

An illustration of the cellular components of the blood brain barrier. In green are endothelial cells of a blood vessel surrounded by the foot processes of type I astrocytes (light blue). In red are microglia cells also extending foot processes to the endothelial cells. The glymphatic system is the virtual space between endothelial cells and foot processes of astrocytes and microglia. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)