Neurovascular injury with complement activation and inflammation in COVID-19

Abstract

The underlying mechanisms by which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to acute and long-term neurological manifestations remains obscure. We aimed to characterize the neuropathological changes in patients with coronavirus disease 2019 and determine the underlying pathophysiological mechanisms. In this autopsy study of the brain, we characterized the vascular pathology, the neuroinflammatory changes and cellular and humoral immune responses by immunohistochemistry. All patients died during the first wave of the pandemic from March to July 2020. All patients were adults who died after a short duration of the infection, some had died suddenly with minimal respiratory involvement. Infection with SARS-CoV-2 was confirmed on ante-mortem or post-mortem testing. Descriptive analysis of the pathological changes and quantitative analyses of the infiltrates and vascular changes were performed. All patients had multifocal vascular damage as determined by leakage of serum proteins into the brain parenchyma. This was accompanied by widespread endothelial cell activation. Platelet aggregates and microthrombi were found adherent to the endothelial cells along vascular lumina. Immune complexes with activation of the classical complement pathway were found on the endothelial cells and platelets. Perivascular infiltrates consisted of predominantly macrophages and some CD8+ T cells. Only rare CD4+ T cells and CD20+ B cells were present. Astrogliosis was also prominent in the perivascular regions. Microglial nodules were predominant in the hindbrain, which were associated with focal neuronal loss and neuronophagia. Antibody-mediated cytotoxicity directed against the endothelial cells is the most likely initiating event that leads to vascular leakage, platelet aggregation, neuroinflammation and neuronal injury. Therapeutic modalities directed against immune complexes should be considered.

Keywords: COVID-19; SARS-CoV-2; complement deposition; neuroinflammation; neurovascular injury.

Figure 1

Microvascular injury and thrombus formation. Immunostaining for (A and B) fibrinogen, (C and D) CD61 for activated platelets, (E and F) vWF and (G and H) PECAM-1 shows absence or minimal staining in the non-COVID-19 brain but in the COVID-19 patients, there was (B) perivascular leakage of fibrinogen, (D) platelet aggregates, (F) thrombi and (H) increased PECAM-1 on endothelial cells. Significantly greater (I) leakage of fibrinogen (**P = 0.0011, ****P < 0.0001) and (J) blood vessels with platelet aggregates were present in the COVID-19 brains (****P < 0.0001). (K) PECAM-1 was significantly increased in the brains of COVID-19 patients compared to non-COVID-19 controls (****P < 0.0001). (L–N) Dots represent the average of individual values in the brain regions that make up the forebrain or hindbrain of a patient. The forebrain includes the cerebrum, basal ganglia and thalamus. The hindbrain contains the pons, medulla and cerebellum. (L) Strong fibrinogen deposition was equally distributed in different regions of the brain, while weak deposition was more prevalent in the hindbrain compared to the forebrain (*P = 0.04). (M) There were more thrombi in the hindbrain compared to the forebrain of the COVID-19 patients (*P = 0.04). (N) PECAM-1 immunostaining was equally distributed in different brain regions of the COVID-19 patients. Data represents mean ± SEM. Scale bars = 100 µm.

Figure 2

Complement activation and immune complexes. Immunostaining for (A and B) C1q, (C and D) C4d, (E and F) IgG, and (G and H) IgM shows minimal staining in the brains of non-COVID-19 controls and extensive deposition on endothelial cells in the blood vessels of COVID-19 patients. (I–V) Multiplex immunostaining (I) minimal PECAM-1 on endothelial cells and lack of deposition of (J) C1q, (K) C4d, (L) C5b-9, (M) IgG, and (N) IgM in a non-COVID-19 brain tissue. (O) is a composite of each of the markers. In a COVID-19 tissue, there is (P) increased PECAM-1 in endothelial cells and deposition of (Q) C1q, (R) C4d, (S) C5b-9, (T) IgG and (U) IgM. (V) shows co-localization of these markers on the endothelial cells. Scale bars = 50 µm.

Figure 3

Characterization of inflammatory infiltrates. The perivascular region shows infiltration of (A) CD68⁺ macrophages, (B) CD3⁺ T cells, (C) CD4⁺ T cells, (D) CD8⁺ T cells and (E) a few CD20⁺ B cells. (F) Increased amounts of CD68⁺ and CD3⁺ cells were present in COVID-19 cases compared to non-COVID-19 controls (****P < 0.0001). The predominant inflammatory response is mediated by macrophages with a lesser contribution from T-cell populations (nearly 20-fold difference). (G) CD68⁺ cells were present in greater numbers in the hindbrain compared to the forebrain of the COVID-19 patients (***P = 0.0007). Dots represent the average of individual values in the brain regions that make up the forebrain or hindbrain of a patient. The forebrain includes the cerebrum, basal ganglia and thalamus. The hindbrain contains the pons, medulla and cerebellum. (H) CD3⁺ and CD8⁺ T cells were significantly increased in COVID-19 cases compared to non-COVID-19 controls (*P = 0.04, ***P = 0.0003, ****P < 0.0001). CD8⁺ T cells were present in relatively higher numbers than CD4⁺ T cells. Only rare CD4⁺ T cells and CD20⁺ B cells were present. All lymphatic cell types were predominantly in the perivascular (PV) regions with few cells in the parenchyma (PC). Data represents mean ± SEM. Scale bars = 100 µm.

Figure 4

Microglial nodules and neuronal injury. Microglial nodules, clusters of microglia, surrounding neurons in the grey matter were present. (A) CD68⁺ cells were found in clusters in the cerebellum of case 9. (B) Double labelling of CD68 (brown) and calbindin (red) in the cerebellum of Case 9 shows multifocal loss of neuronal processes. (C–F) Clusters of CD68⁺ microglia surrounding neurons were present in the (C) hippocampal CA1 region of Case 2, (D) thalamus of Case 8, (E) pons of Case 7 and (F) solitary nucleus of the medulla. (G) The number of foci of neuronophagia was significantly increased in the brains of COVID-19 patients compared to non-COVID-19 controls (****P < 0.0001). (H) There were significantly more foci of neuronophagia in the hindbrain compared to the forebrain of the COVID-19 patients (**P = 0.0071). Dots represent the average of individual values in the brain regions that make up the forebrain or hindbrain of a patient. The forebrain includes the cerebrum, basal ganglia and thalamus. The hindbrain contains the pons, medulla and cerebellum. Data represents mean ± SEM. Scale bars = 50 µm.

Figure 5

Spatial gene expression profiling. Gene expression data of the brainstem obtained with the NanoString GeoMx platform was analysed by DEA, PCA and IPA. (A–D) Gene expression profiling performed on all regions of interest (ROIs) was analysed. (A) Heat map of the top 1000 genes in terms of median absolute deviation across samples. Complete-linkage hierarchical clustering with a Euclidean distance function was used to generate dendrograms. Column values were annotated based on COVID patient (red) or control (yellow) status. Z-scores were scaled and centred based on row values and the histogram at the top left indicates no significant outliers or overly influential genes. (B) The volcano plot shows the relationship between shrunken LFCs and P-values from our DEA of patients versus controls. Red dots indicate differentially expressed genes with an LFC > 1 and P < 0.05. (C) PCA plot of regularized log (rlog) transformed counts highlighting patient and control samples. (D) Loading plot of the top seven features in terms of loadings from principal component 2, which separated patients from controls. Gene expression profiling performed on (E) PECAM-1 rich and (F) CD45-rich regions of interest were analysed and plotted in volcano plots. (G) IPA output table shows a list of differentially regulated pathways in the all, PECAM-1-rich and CD45-rich regions of interest of the COVID-19 brainstem. Upregulation of a pathway is represented by shades of orange and downregulation in blue.

Figure 6

Neurovascular injury with complement activation and inflammation in COVID-19. (A) Correlation matrix of the various cell type markers. Pearson’s correlation coefficient (r) between two pairs of variables is shown in the heat map. The correlation coefficients are represented in terms of the changes of the intensity of red/blue colour, as shown in the colour bar. (B) The proposed cascade of events to explain the neuropathological findings is as follows: C1 binds to the IgG and IgM antibodies and activates the classic complement pathway. The end product of this cascade, C5b-9 binds to endothelial cells and causes endothelial cell damage. This leads to activation of endothelial cells and increased PECAM-1 and vWF release, resulting in platelet aggregation and thrombus formation. Simultaneously, there is leakage of serum proteins into the perivascular space, which leads to an influx of monocytes and T lymphocytes into the parenchyma. Monocytes differentiate to macrophages and there is activation of microglia and astrocytes in the brain parenchyma. This leads to neuronal injury and neuronophagia.