Infection of Brain Pericytes Underlying Neuropathology of COVID-19 Patients

Abstract

A wide range of neurological manifestations have been associated with the development of COVID-19 following SARS-CoV-2 infection. However, the etiology of the neurological symptomatology is still largely unexplored. Here, we used state-of-the-art multiplexed immunostaining of human brains (n = 6 COVID-19, median age = 69.5 years; n = 7 control, median age = 68 years) and demonstrated that expression of the SARS-CoV-2 receptor ACE2 is restricted to a subset of neurovascular pericytes. Strikingly, neurological symptoms were exclusive to, and ubiquitous in, patients that exhibited moderate to high ACE2 expression in perivascular cells. Viral dsRNA was identified in the vascular wall and paralleled by perivascular inflammation, as signified by T cell and macrophage infiltration. Furthermore, fibrinogen leakage indicated compromised integrity of the blood-brain barrier. Notably, cerebrospinal fluid from additional 16 individuals (n = 8 COVID-19, median age = 67 years; n = 8 control, median age = 69.5 years) exhibited significantly lower levels of the pericyte marker PDGFRβ in SARS-CoV-2-infected cases, indicative of disrupted pericyte homeostasis. We conclude that pericyte infection by SARS-CoV-2 underlies virus entry into the privileged central nervous system space, as well as neurological symptomatology due to perivascular inflammation and a locally compromised blood-brain barrier.

Keywords: ACE2; COVID-19; SARS-CoV-2; blood–brain barrier; brain; infection; multiplexed IHC; pericytes; vasculature.

Figure 1

The ACE2 receptor is expressed by pericytes in murine and human brains. (A) Expression of Ace2 in cell types in the mouse brain; cell types are annotated based on the Allen Mouse Brain Atlas. (B) Representative IHC staining of perivascular ACE2 in the frontal cortex of two COVID-19 patients and one control individual. Cell nuclei are counterstained with hematoxylin (blue). (C) Representative IHC staining of perivascular ACE2 in different brain regions of the two COVID-19 patients in subfigure B. Cell nuclei are counterstained with hematoxylin (blue). (D) Clinical course of the COVID-19 patients included in the study. For each patient, appearance of symptoms, hospitalization, infection status, and progression to death are included, together with the postmortem evaluation of ACE2 immunoreactivity and thrombosis in the CNS. (E) Fourplex mIHC staining of the frontal cortex of a COVID-19 patient. The composite image depicts CD31 (endothelial cells, white), PDGFRβ (pericytes, cyan), and ACE2 (orange). Cell nuclei are counterstained with DAPI (blue). The white arrows indicate ACE2-positive signal in the abluminal side of CD31. The intensity of each individual OPAL fluorophore and the combined PDGFRβ/ACE2 and CD31/ACE2 overlays are presented in individual photomicrographs.

Figure 2

Perivascular infection by SARS-CoV-2 is paralleled by perivascular inflammation in the brain of COVID-19 patients. (A) Immunohistochemical detection of viral components in COVID-19-infected patients and non-COVID-19 controls. Cell nuclei are counterstained with hematoxylin (blue). The black arrows indicate the chromogenic deposition of the 3,3’-diaminobenzidine (DAB) substrate. (B) Representative field of a 7-plex mIHC staining panel of placental tissue infected with SARS-CoV-2. The magenta arrows indicate accumulation of viral dsRNA in correspondence of the ACE2-positive areas by the specialized epithelial layer of syncytiotrophoblast in the placenta. The intensity of each OPAL fluorophore is further presented in individual photomicrographs. (C) Immunohistochemical detection of dsRNA in the cerebral cortex of a COVID-19 patient and in a non-COVID-19 control. Cell nuclei are counterstained with hematoxylin (blue). Black arrows indicate deposition of the DAB substrate. (D) Composite mIHC image of the perivascular immune cell infiltration in the frontal cortex of a COVID-19 patient and in a control individual. The antibody panel was designed for the concomitant detection of CD34 (endothelium) and five immune cell markers: CD4 (T helper cells), CD8 (cytotoxic T lymphocytes), CD20 (B cells), CD68 (macrophages), and FOXP3 (regulatory T cells).

Figure 3

Pericyte infection leads to vascular leakage in the CNS. (A) Composite mIHC of the frontal cortex of a COVID-19 patient and a control individual. The fields highlight the fibrinogen halo surrounding leaky blood vessels following SARS-CoV-2 infection. The images depict the neurovascular unit (CD34, PDGFRβ, and ACE2), fibrinogen, viral dsRNA, and neurons. The intensity of each OPAL fluorophore is further presented in individual photomicrographs. The cyan arrows indicate fibrinogen leakage; yellow arrows highlight points of converging PDGFRβ/dsRNA staining. (B) Composite mIHC of the frontal cortex of a COVID-19 patient and a control individual. The fields focus on astrocyte priming as a readout of local neuroinflammation. The images depict the neurovascular unit (CD34, PDGFRβ, and ACE2), fibrinogen, SARS-CoV-2 spike protein, and astrocytes (GFAP). (C) Boxplot of the concentration of soluble PDGFRβ (pg/mL) in the CSF of COVID-19 patients and non-COVID-19 controls (circles: individual measurements, cross: cohort average).