Coronavirus disease 2019 (COVID-19) is an unprecedented pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of 22 February 2021, the worldwide pandemic has resulted in more than 110 million cases and 2.4 million deaths. 1 Clinical investigation of COVID-19 patients has shown that a systemic cytokine storm can occur, especially in severe cases. 2 Treatment of the SARS-CoV-2-associated cytokine storm with tocilizumab 3 or anakinra 4 has been shown to immediately improve the clinical outcome in most severe and critical COVID-19 patients. These data highlight the systemic cytokine storm as an important exacerbating event in severe COVID-19; however, our understanding of the molecular mechanisms involved in the initiation of the SARS-CoV-2-associated cytokine storm is limited. In the present study, we uncovered a reasonable explanation for cytokine storm initiation through the analysis of 13 autopsy samples from severe COVID-19 patients. To investigate SARS-CoV-2-associated cytokine storm processes, we collected three control lung tissues from patients without SARS-CoV-2 infection as a control group and six lung tissues from COVID-19 patients as a patient group for RNA-seq transcriptome analysis. The clinical data of the patients are presented in the Supplementary material (Table S1). Principal component analysis (PCA) of the RNA-seq data showed clear biological differences between the control and patient group transcriptomes (Fig. S1a, b). Through differential gene expression analysis, we identified 1951 upregulated genes and 1971 downregulated genes in the patient group compared with the control group (Fig. S1c). Functional analysis of the upregulated genes revealed enrichment in functions related to ROS activation, virus infection-related signaling pathways, the HIF-1α signaling pathway, the NOD-like receptor signaling pathway, and metabolic dysregulation (Fig. S1d–f). Previous studies have suggested that cellular “danger” signals, including infection, ROS, and metabolic dysregulation, could trigger the NLRP3 inflammasome signaling pathway. 5 The data presented herein demonstrate that many cellular “danger” signals are activated in the pulmonary microenvironment of severe COVID-19 patients and indicate that NLRP3 inflammasome signaling is involved in the pathogenesis of COVID-19. Notably, we found that chemokines responsible for recruiting monocytes were significantly upregulated in the lung tissues of

COVID-19 patients (Fig. S1g), suggesting that SARS-CoV-2 infection may lead to the recruitment of monocytes to the lungs of patients. To further test this hypothesis, we used the xCell method to analyze immune infiltration in our samples. We found significantly more infiltration of monocytes, neutrophils, and plasma cells in the lung tissues of severe COVID-19 patients than in the tissues of control donors. There were no significant changes in the levels of other immune cells (Fig. 1 a). Immunohistochemical analysis also revealed that many CD14+ CD16+ double-positive proinflammatory monocytes infiltrated the alveoli of severe COVID-19 patients (Fig. 1 b), many of which transformed into CD163-positive macrophages (Fig. 1 c). Notably, CD163 staining in infiltrating macrophages was more evident in COVID-19 patients than in controls, indicating that these infiltrating proinflammatory macrophages underwent activation switching. 6 We also observed the infiltration of some CD4+ and CD8+ T cells into the alveoli of COVID-19 patients (Fig. S2a, b). We previously reported that proinflammatory monocytes play a crucial role in the SARS-CoV-2-associated cytokine storm. 7 …