Cytokine storm and histopathological findings in 60 cases of COVID-19-related death: from viral load research to immunohistochemical quantification of major players IL-1β, IL-6, IL-15 and TNF-α

Abstract

This study involves the histological analysis of samples taken during autopsies in cases of COVID-19 related death to evaluate the inflammatory cytokine response and the tissue localization of the virus in various organs. In all the selected cases, SARS-CoV-2 RT-PCR on swabs collected from the upper (nasopharynx and oropharynx) and/or the lower respiratory (trachea and primary bronchi) tracts were positive. Tissue localization of SARS-CoV-2 was detected using antibodies against the nucleoprotein and the spike protein. Overall, we tested the hypothesis that the overexpression of proinflammatory cytokines plays an important role in the development of COVID-19-associated pneumonia by estimating the expression of multiple cytokines (IL-1β, IL-6, IL-10, IL-15, TNF-α, and MCP-1), inflammatory cells (CD4, CD8, CD20, and CD45), and fibrinogen. Immunohistochemical staining showed that endothelial cells expressed IL-1β in lung samples obtained from the COVID-19 group (p < 0.001). Similarly, alveolar capillary endothelial cells showed strong and diffuse immunoreactivity for IL-6 and IL-15 in the COVID-19 group (p < 0.001). TNF-α showed a higher immunoreactivity in the COVID-19 group than in the control group (p < 0.001). CD8 + T cells where more numerous in the lung samples obtained from the COVID-19 group (p < 0.001). Current evidence suggests that a cytokine storm is the major cause of acute respiratory distress syndrome (ARDS) and multiple organ failure and is consistently linked with fatal outcomes.

Keywords: Autopsy; COVID-19 related-death; Cytokines; Immunohistochemistry; Pneumonia; RT-PCR; SARS-CoV-2.

Fig. 1

A Lung tissue showed edema, early stage DAD with hyaline membranes (green arrows) and microvascular thrombi (yellow arrows) (H&E, × 40); B Lung: capillary congestion, and microvascular thrombi (black arrows) (H&E, × 60)

Fig. 2

Statistically significant difference in the group of COVID-19-related death (grey columns) compared to the control group (black columns) for the following cytokines: IL-1β, IL-6, IL-15, TNF-α, MCP1, CD4, CD8, CD20, CD45, fibrinogen: NS: p > 0.05; **: p < 0.01; ***: p < 0.001

Fig. 3

A, B Immunohistochemical reaction of IL-1β in group of COVID-19-related death showed a wide endothelial expression and positivity (brown reactions indicated with black arrows) in lung samples (× 60, × 100); Insert in (a): control case (× 60); C, D IL-6 showed a strong (black arrows) and diffusely positive reactions in capillary-alveolar endothelial cells in the COVID-19 group, expressed by brown dots in the endothelial cells than the negative control case (× 80, × 40); Insert in (c): control case (× 60)

Fig. 4

A, B Immunohistochemical reaction of IL-15 in group of COVID-19-related death showed a showed a strong positive reaction (arrows) in capillary-alveolar endothelial cells in lung samples (× 100, × 60); insert in (a): control case (× 60); C, D TNF-α showed a diffusely positive reaction in capillary-alveolar endothelial cells in the COVID-19 group, expressed by brown dots in the endothelial cells than the negative control case (× 100, × 60); insert in (c): control case (× 60)

Fig. 5

A Immunohistochemical reaction to CD4 + T cells demonstrated a greater positivity in lungs from control group (Group 2) than in COVID-19 group (Group 1); B (× 60, × 80); C, D CD8 + T cells were more numerous adjacent to the alveolar epithelial lining in the COVID-19 group than in the control group expressed by brown reactions in the endothelial cells than the negative control case (× 80, × 80)

Fig. 6

A Heart: immunohistochemistry demonstrated strong reactions (arrows) into the myocardiocytes (× 100); B, C Liver cells colonized by numerous viral particles (arrows); diffuse positivity (arrows) to SARS-CoV-2 antigen [nucleocapsid (black arrows) and spike (red arrows)] into the spleen (× 100)

Fig. 7

A, B Lung: transmission electron microscopy demonstrated viral particles into the endothelial cells (× 6300, × 10,000; bar 500 nm); C, D Glomerular endothelial cells colonized by numerous viral particles (× 40,000, × 50,000; bar 100 nm)