Novel insight into the underlying dysregulation mechanisms of immune cell-to-cell communication by analyzing multitissue single-cell atlas of two COVID-19 patients.

How does SARS-CoV-2 cause lung microenvironment disturbance and inflammatory storm is still obscure. We here performed the single-cell transcriptome sequencing from lung, blood, and bone marrow of two dead COVID-19 patients and detected the cellular communication among them. Our results demonstrated that SARS-CoV-2 infection increase the frequency of cellular communication between alveolar type I cells (AT1) or alveolar type II cells (AT2) and myeloid cells triggering immune activation and inflammation microenvironment and then induce the disorder of fibroblasts, club, and ciliated cells, which may cause increased pulmonary fibrosis and mucus accumulation. Further study showed that the increase of T cells in the lungs may be mainly recruited by myeloid cells through ligands/receptors (e.g., ANXA1/FPR1, C5AR1/RPS19, and CCL5/CCR1). Interestingly, we also found that certain ligands/receptors (e.g., ANXA1/FPR1, CD74/COPA, CXCLs/CXCRs, ALOX5/ALOX5AP, CCL5/CCR1) are significantly activated and shared among lungs, blood and bone marrow of COVID-19 patients, implying that the dysregulation of ligands/receptors may lead to immune cell's activation, migration, and the inflammatory storm in different tissues of COVID-19 patients. Collectively, our study revealed a possible mechanism by which the disorder of cell communication caused by SARS-CoV-2 infection results in the lung inflammatory microenvironment and systemic immune responses across tissues in COVID-19 patients.

COVID-19-associated monocytic encephalitis (CAME): histological and proteomic evidence from autopsy.

Severe neurological symptoms are associated with Coronavirus disease 2019 (COVID-19). However, the morphologic features, pathological nature and their potential mechanisms in patient brains have not been revealed despite evidence of neurotropic infection. In this study, neuropathological damages and infiltrating inflammatory cells were quantitatively evaluated by immunohistochemical staining, ultrastructural examination under electron microscopy, and an image threshold method, in postmortem brains from nine critically ill COVID-19 patients and nine age-matched cadavers of healthy individuals. Differentially expressed proteins were identified by quantitative proteomic assays. Histopathological findings included neurophagocytosis, microglia nodules, satellite phenomena, extensive edema, focal hemorrhage, and infarction, as well as infiltrating mononuclear cells. Immunostaining of COVID-19 brains revealed extensive activation of both microglia and astrocytes, severe damage of the blood-brain barrier (BBB) and various degrees of perivascular infiltration by predominantly CD14+/CD16+/CD141+/CCR7+/CD11c+ monocytes and occasionally CD4+/CD8+ T lymphocytes. Quantitative proteomic assays combined with bioinformatics analysis identified upregulated proteins predominantly involved in immune responses, autophagy and cellular metabolism in COVID-19 patient brains compared with control brains. Proteins involved in brain development, neuroprotection, and extracellular matrix proteins of the basement membrane were downregulated, potentially caused by the activation of transforming growth factor ß receptor and vascular endothelial growth factor signaling pathways. Thus, our results define histopathological and molecular profiles of COVID-19-associated monocytic encephalitis (CAME) and suggest potential therapeutic targets.

Neutrophil infiltration and myocarditis in patients with severe COVID-19: A post-mortem study

Aims To investigate cardiac pathology in critically ill patients with coronavirus disease 2019 (COVID-19) and identify associations between pathological changes and clinical characteristics. Methods The present autopsy cohort study included hearts from 26 deceased patients hospitalized in intensive care units due to COVID-19, and was conducted at four sites in Wuhan, China. Cases were divided into a neutrophil infiltration group and a no-neutrophil group based on the presence or absence of histopathologically identified neutrophilic infiltrates. Results Among the 26 patients, histopathological examination identified active myocarditis in four patients. All patients with myocarditis exhibited extensive accompanying neutrophil infiltration, and all patients without myocarditis did not. The neutrophil infiltration group exhibited significantly higher rates of detection of interleukin-6 (100 vs. 4.6%) and tumor necrosis factor-alpha (100 vs. 31.8%) than the no-neutrophil group (both p < 0.05). On admission, four patients with neutrophil infiltration in myocardium had significantly higher baseline levels of aspartate aminotransferase, D dimer, and high-sensitivity C reactive protein than the other 22 patients (all p < 0.05). During hospitalization, patients with neutrophil infiltration had significantly higher maximum creatine kinase-MB (median 280.0 IU/L vs. 38.7 IU/L, p = 0.04) and higher troponin I (median 1.112 ng/ml vs. 0.220 ng/ml, p = 0.56) than patients without neutrophil infiltration. Conclusion Active myocarditis was frequently associated with neutrophil infiltration in the hearts of deceased patients with severe COVID-19. Patients with neutrophil-infiltrated myocarditis had a series of severely abnormal laboratory test results on admission, and high maximum creatine kinase-MB during hospitalization. The role of neutrophils in severe heart injury and systemic conditions in patients with COVID-19 should be emphasized.

Spatial region-resolved proteome map reveals mechanism of COVID-19-associated heart injury.

Direct myocardial and vascular injuries due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-driven inflammation is the leading cause of acute cardiac injury associated with coronavirus disease 2019 (COVID-19). However, in-depth knowledge of the injury characteristics of the heart affected by inflammation is lacking. In this study, using a quantitative spatial proteomics strategy that combines comparative anatomy, laser-capture microdissection, and histological examination, we establish a region-resolved proteome map of the myocardia and microvessels with obvious inflammatory cells from hearts of patients with COVID-19. A series of molecular dysfunctions of myocardia and microvessels is observed in different cardiac regions. The myocardia and microvessels of the left atrial are the most susceptible to virus infection and inflammatory storm, suggesting more attention should be paid to the lesion and treatment of these two parts. These results can guide in improving clinical treatments for cardiovascular diseases associated with COVID-19.

Ligand/Receptor-mediated Cell Communication Alteration Causes the Lung Inflammation Microenvironment and the Redistribution of Inter-tissue Immune Cells in COVID-19 Patients (preprint)

How SARS-CoV-2 causes disturbances of the lung microenvironment and systemic immune response remains a mystery. Here, we first analyze detailedly paired single-cell transcriptome data of the lungs, blood and bone marrow of two patients who died of COVID-19. Second, our results demonstrate that SARS-CoV-2 infection significantly increases the cellular communication frequency between AT1/AT2 cells and highly inflammatory myeloid cells, and induces the pulmonary inflammation microenvironment, and drives the disorder of fibroblasts, club and ciliated cells, thereby causing the increase of pulmonary fibrosis and mucus accumulation. Third, our works reveal that the increase of the lung T cell infiltration is mainly recruited by myeloid cells through certain ligands/receptors (ANXA1/FPR1, C5AR1/RPS19 and CCL5/CCR1), rather than AT1/AT2. Fourth, we find that some ligands and receptors such as ANXA1/FPR1, CD74/COPA, CXCLs/CXCRs, ALOX5/ALOX5AP, CCL5/CCR1, are significantly activated and shared among patients’ lungs, blood and bone marrow, implying that dysregulated ligands and receptors may cause the migration, redistribution and the inflammatory storm of immune cells in different tissues. Overall, our study reveals a latent mechanism by which the disorders of ligands and receptors caused by SARS-CoV-2 infection drive cell communication alteration, the pulmonary inflammatory microenvironment and systemic immune responses across tissues in COVID-19 patients.

A single-cell transcriptomic landscape of the lungs of patients with COVID-19.

The lung is the primary organ targeted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making respiratory failure a leading coronavirus disease 2019 (COVID-19)-related mortality. However, our cellular and molecular understanding of how SARS-CoV-2 infection drives lung pathology is limited. Here we constructed multi-omics and single-nucleus transcriptomic atlases of the lungs of patients with COVID-19, which integrate histological, transcriptomic and proteomic analyses. Our work reveals the molecular basis of pathological hallmarks associated with SARS-CoV-2 infection in different lung and infiltrating immune cell populations. We report molecular fingerprints of hyperinflammation, alveolar epithelial cell exhaustion, vascular changes and fibrosis, and identify parenchymal lung senescence as a molecular state of COVID-19 pathology. Moreover, our data suggest that FOXO3A suppression is a potential mechanism underlying the fibroblast-to-myofibroblast transition associated with COVID-19 pulmonary fibrosis. Our work depicts a comprehensive cellular and molecular atlas of the lungs of patients with COVID-19 and provides insights into SARS-CoV-2-related pulmonary injury, facilitating the identification of biomarkers and development of symptomatic treatments.

A cohort autopsy study defines COVID-19 systemic pathogenesis.

Severe COVID-19 disease caused by SARS-CoV-2 is frequently accompanied by dysfunction of the lungs and extrapulmonary organs. However, the organotropism of SARS-CoV-2 and the port of virus entry for systemic dissemination remain largely unknown. We profiled 26 COVID-19 autopsy cases from four cohorts in Wuhan, China, and determined the systemic distribution of SARS-CoV-2. SARS-CoV-2 was detected in the lungs and multiple extrapulmonary organs of critically ill COVID-19 patients up to 67 days after symptom onset. Based on organotropism and pathological features of the patients, COVID-19 was divided into viral intrapulmonary and systemic subtypes. In patients with systemic viral distribution, SARS-CoV-2 was detected in monocytes, macrophages, and vascular endothelia at blood-air barrier, blood-testis barrier, and filtration barrier. Critically ill patients with long disease duration showed decreased pulmonary cell proliferation, reduced viral RNA, and marked fibrosis in the lungs. Permanent SARS-CoV-2 presence and tissue injuries in the lungs and extrapulmonary organs suggest direct viral invasion as a mechanism of pathogenicity in critically ill patients. SARS-CoV-2 may hijack monocytes, macrophages, and vascular endothelia at physiological barriers as the ports of entry for systemic dissemination. Our study thus delineates systemic pathological features of SARS-CoV-2 infection, which sheds light on the development of novel COVID-19 treatment.

SARS-CoV-2 spike protein dictates syncytium-mediated lymphocyte elimination.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is highly contagious and causes lymphocytopenia, but the underlying mechanisms are poorly understood. We demonstrate here that heterotypic cell-in-cell structures with lymphocytes inside multinucleate syncytia are prevalent in the lung tissues of coronavirus disease 2019 (COVID-19) patients. These unique cellular structures are a direct result of SARS-CoV-2 infection, as the expression of the SARS-CoV-2 spike glycoprotein is sufficient to induce a rapid (~45.1 nm/s) membrane fusion to produce syncytium, which could readily internalize multiple lines of lymphocytes to form typical cell-in-cell structures, remarkably leading to the death of internalized cells. This membrane fusion is dictated by a bi-arginine motif within the polybasic S1/S2 cleavage site, which is frequently present in the surface glycoprotein of most highly contagious viruses. Moreover, candidate anti-viral drugs could efficiently inhibit spike glycoprotein processing, membrane fusion, and cell-in-cell formation. Together, we delineate a molecular and cellular rationale for SARS-CoV-2 pathogenesis and identify novel targets for COVID-19 therapy.

Pathological changes in the lungs and lymphatic organs of 12 COVID-19 autopsy cases.

Systematic autopsy and comprehensive pathological analyses of COVID-19 decedents should provide insights into the disease characteristics and facilitate the development of novel therapeutics. In this study, we report the autopsy findings from the lungs and lymphatic organs of 12 COVID-19 decedents-findings that evaluated histopathological changes, immune cell signature and inflammatory factor expression in the lungs, spleen and lymph nodes. Here we show that the major pulmonary alterations included diffuse alveolar damage, interstitial fibrosis and exudative inflammation featured with extensive serous and fibrin exudates, macrophage infiltration and abundant production of inflammatory factors (IL-6, IP-10, TNFα and IL-1ß). The spleen and hilar lymph nodes contained lesions with tissue structure disruption and immune cell dysregulation, including lymphopenia and macrophage accumulation. These findings provide pathological evidence that links injuries of the lungs and lymphatic organs with the fatal systematic respiratory and immune malfunction in critically ill COVID-19 patients.

Retrospective Study of Clinical Features of COVID-19 in Inpatients and Their Association with Disease Severity.

BACKGROUND The aim of this study was to analyze the clinical features and laboratory indices of patients with coronavirus disease (COVID-19) and explore their association with the severity of the disease. MATERIAL AND METHODS A total of 61 patients with COVID-19 were divided into groups with common symptoms and with severe diseases, and clinical data were collected to analyze and compare the differences between them. RESULTS In patients with severe COVID-19, compared with the common group, lymphocyte count and albumin levels were lower, and aspartate aminotransferase (AST), blood urea, blood creatinine, lactate dehydrogenase (LDH), and C-reactive protein (CRP) levels, and prothrombin time (PT) were elevated (all P<0.05). The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), mean platelet volume-to-lymphocyte ratio (MPVLR), and C-reactive protein-to-albumin ratio (CAR) were significantly elevated in the severe group compared with the group with common symptoms; however, the lymphocyte-to-monocyte ratio (LMR) was significantly reduced (P<0.05). Univariate logistic regression showed that lower lymphocyte count, prolonged PT, elevated CRP and LDH levels, and elevated NLR, PLR, MPVLR, and CAR were risk factors for COVID-19 severity (P<0.05). Multivariate logistic regression showed that elevated CRP levels (odds ratio [OR], 0.028; 95% confidence interval [CI]: 0.002-0.526; P=0.017), prolonged PT (OR, 0.014; 95% CI: 0.001-0.341; P=0.09), and an MPVLR >8.9 (OR, 0.026; 95% CI: 0.002-0.349; P=0.006) were independent risk factors for COVID-19 severity. CONCLUSIONS Elevated CRP and prolonged PT, and an MPVLR >8.9 were independent risk factors for COVID-19 severity.