Neutrophils predominate the immune signature of cerebral thrombi in COVID-19 stroke patients

Abstract

Coronavirus disease 2019 (COVID-19) is associated with an increased risk of thrombotic events. Ischemic stroke in COVID-19 patients entails high severity and mortality rates. Here we aimed to analyze cerebral thrombi of COVID-19 patients with large vessel occlusion (LVO) acute ischemic stroke to expose molecular evidence for SARS-CoV-2 in the thrombus and to unravel any peculiar immune-thrombotic features. We conducted a systematic pathological analysis of cerebral thrombi retrieved by endovascular thrombectomy in patients with LVO stroke infected with COVID-19 (n = 7 patients) and non-covid LVO controls (n = 23). In thrombi of COVID-19 patients, the SARS-CoV-2 docking receptor ACE2 was mainly expressed in monocytes/macrophages and showed higher expression levels compared to controls. Using polymerase chain reaction and sequencing, we detected SARS-CoV-2 Clade20A, in the thrombus of one COVID-19 patient. Comparing thrombus composition of COVID-19 and control patients, we noted no overt differences in terms of red blood cells, fibrin, neutrophil extracellular traps (NETs), von Willebrand Factor (vWF), platelets and complement complex C5b-9. However, thrombi of COVID-19 patients showed increased neutrophil density (MPO⁺ cells) and a three-fold higher Neutrophil-to-Lymphocyte Ratio (tNLR). In the ROC analysis both neutrophils and tNLR had a good discriminative ability to differentiate thrombi of COVID-19 patients from controls. In summary, cerebral thrombi of COVID-19 patients can harbor SARS-CoV2 and are characterized by an increased neutrophil number and tNLR and higher ACE2 expression. These findings suggest neutrophils as the possible culprit in COVID-19-related thrombosis.

Keywords: COVID-19; Endovascular treatment; Ischemic stroke; Neutrophils; SARS-CoV2; Thrombosis.

Fig. 1

Evaluation of ACE2 protein and endothelial cells in cerebral thrombi. A ACE2 expression in thrombi of COVID-19 patients and controls. Representative images of a thrombus of a COVID-19 and a control patient with ACE2 immunohistochemical staining (staining in brown, the inset displays a magnification of the dashed box area and arrowheads highlight some positive cells). Histogram showing the ACE2⁺ area as percentage of the total thrombus area, *p = 0.04, Mann–Whitney. B. Endothelial cells (CD34⁺ area) in cerebral thrombi of COVID-19 patients and controls. Representative images of a thrombus of a COVID-19 and control patient with CD34 immunohistochemical staining (in blue, arrowheads highlight CD34⁺ cells). Histogram of the CD34⁺ area as percentage of the total thrombus area; p = 0.86, Mann–Whitney. C. Characterization of ACE2-expressing cells. Representative images of double-immunohistochemistry with ACE2 (in either purple or blue) and either CD34 (in blue), CD68 (in brown), CD3 (in blue), or MPO (in blue). The inset displays a magnification of the dashed box area and arrowheads highlight double positive cells. A donut graph showing the percentage of cells expressing ACE2 out of the total of ACE2⁺ cells. The graphs represent the median and IQR, each dot in the scatter plot represents the thrombus of one patient (n = 7 COVID-19 patients and n = 23 controls); scale bar A- C, 50 µm

Fig. 2

SARS-CoV-2 detection within the retrieved thrombus of COVID-19 stroke patients. A. Transmission Electron Microscopy images of a cerebral thrombus of a control and B. of COVID-19 stroke patient. We did not detect evidence of SARS-CoV2 viral particles within the analyzed thrombi. The white labels in the images indicate, platelets (PLT), neutrophils (NEU), red blood cells (RBC, fibrin (fib); C. Agarose gel showing the PCR amplification of SARS-CoV2 in a thrombus of a COVID-19 stroke patient. A negative and positive control for the PCR are shown

Fig. 3

Analysis of major components of thrombi of COVID-19 and control stroke patients. A. Representative Lendrum (MSB) staining highlighting in yellow the red cell blood component in a COVID-19 and a control thrombus and quantification; p = 0.666, Mann Whitney. B. Representative images of platelets with CD61 immunohistochemical staining (in brown, and quantification; p = 0.69, Mann Whitney). C. Representative images of von Willebrand Factor (vWF in brown) immunohistochemistry and quantification; p = 0.348, Mann Whitney. D. Representative images of a COVID-19 and control thrombus with Lendrum (MSB) staining for fibrin identification (in pink) and quantification; p = 0.266, Mann Whitney. The graphs represent the median and IQR, each dot in the scatter plot represents the thrombus of one patient (n = 7 COVID-19 patients and n = 23 controls); scale bar in A–F, 100 μm

Fig. 4

Immune signature of thrombi of COVID-19 and control stroke patients. A to F. Characterization of the thrombus immune cell signature of thrombi retrieved from COVID-19 and control stroke patients. A. Neutrophil number (MPO⁺ cells) and representative images of a thrombus of a COVID-19 and control patient with MPO immunohistochemical staining (in brown; p = 0.04, Mann Whitney). B. NET content (CitH3⁺ area) and representative images of thrombi with citH3 immunohistochemical staining (in brown; % of citH3⁺ area out of total thrombus area in COVID-19 patients and controls respectively, median [IQR]; p = 0.19, Mann Whitney). C. T and B cells (CD3+ and CD20+ cells) number and representative images of thrombi with CD3 (in brown) and CD20 (in blue) immunohistochemical staining of COVID-19 and control stroke patients; p = 0.11, median [IQR], Mann Whitney). D. Histogram representing the thrombus neutrophil-to-lymphocyte ratio (tNLR, in COVID-19 and control patients; p ≤ 0.01, Mann Whitney). E. Macrophages (CD68 PGM1⁺ cells) and representative images of thrombi with CD68-PGM1 immunohistochemical staining (in brown; p = 0.12, Mann Whitney). F. Quantification of complement (C5b-C9⁺ area) and representative images of thrombi with C5b-C9 immunohistochemical staining (in brown). Scale bar inA–F, 100 μm. In the scatter plots each dot corresponds to the thrombus of one patient (n = 7 COVID-19 and n = 23 control patients)

Fig. 5

Receiver operating characteristic curves for COVID-19. A, B Receiver operating characteristic curves for COVID-19. Area under the curve (AUC) for COVID-19: AUC 0.758, confidence interval (CI) 0.583 to 0.933 for thrombus neutrophils (MPO+ cells) (A) and AUC 0.876, CI 0.719 to 1.000 for the thrombus neutrophil-to-lymphocyte ratio (B)