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1.
Clin Immunol ; 237: 108991, 2022 04.
Article in English | MEDLINE | ID: covidwho-1866980

ABSTRACT

Many studies have been performed in severe COVID-19 on immune cells in the circulation and on cells obtained by bronchoalveolar lavage. Most studies have tended to provide relative information rather than a quantitative view, and it is a combination of approaches by various groups that is helping the field build a picture of the mechanisms that drive severe lung disease. Approaches employed to date have not revealed information on lung parenchymal T cell subsets in severe COVID-19. Therefore, we sought to examine early and late T cell subset alterations in the lungs and draining lymph nodes in severe COVID-19 using a rapid autopsy protocol and quantitative imaging approaches. Here, we have established that cytotoxic CD4+ T cells (CD4 + CTLs) increase in the lungs, draining lymph nodes and blood as COVID-19 progresses. CD4 + CTLs are prominently expanded in the lung parenchyma in severe COVID-19. In contrast CD8+ T cells are not prominent, exhibit increased PD-1 expression, and no obvious increase is seen in the number of Granzyme B+ CD8+ T cells in the lung parenchyma in severe COVID-19. Based on quantitative evidence for re-activation in the lung milieu, CD4 + CTLs may be as likely to drive viral clearance as CD8+ T cells and may also be contributors to lung inflammation and eventually to fibrosis in severe COVID-19.


Subject(s)
CD4-Positive T-Lymphocytes , COVID-19 , CD8-Positive T-Lymphocytes , Humans , Lung , T-Lymphocyte Subsets , T-Lymphocytes, Cytotoxic
2.
Pulm Circ ; 12(2): e12071, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1772839

ABSTRACT

The pathobiology of in situ pulmonary thrombosis in acute respiratory distress syndrome (ARDS) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is incompletely characterized. In human pulmonary artery endothelial cells (HPAECs), hypoxia increases neural precursor cell expressed, developmentally downregulated 9 (NEDD9) and induces expression of a prothrombotic NEDD9 peptide (N9P) on the extracellular plasma membrane surface. We hypothesized that the SARS-CoV-2-ARDS pathophenotype involves increased pulmonary endothelial N9P. Paraffin-embedded autopsy lung specimens were acquired from patients with SARS-CoV-2-​​​​​​ARDS (n = 13), ARDS from other causes (n = 10), and organ donor controls (n = 5). Immunofluorescence characterized the expression of N9P, fibrin, and transcription factor 12 (TCF12), a putative binding target of SARS-CoV-2 and known transcriptional regulator of NEDD9. We performed RNA-sequencing on normal HPAECs treated with normoxia or hypoxia (0.2% O2) for 24 h. Immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) profiled protein-protein interactions involving N9P relevant to thrombus stabilization. Hypoxia increased TCF12 messenger RNA significantly compared to normoxia in HPAECs in vitro (+1.19-fold, p = 0.001; false discovery rate = 0.005), and pulmonary endothelial TCF12 expression was increased threefold in SARS-CoV-2-ARDS versus donor control lungs (p < 0.001). Compared to donor controls, pulmonary endothelial N9P-fibrin colocalization was increased in situ in non-SARS-CoV-2-ARDS and SARS-CoV-2-ARDS decedents (3.7 ± 1.2 vs. 10.3 ± 3.2 and 21.8 ± 4.0 arb. units, p < 0.001). However, total pulmonary endothelial N9P was increased significantly only in SARS-CoV-2-ARDS versus donor controls (15 ± 4.2 vs. 6.3 ± 0.9 arb. units, p < 0.001). In HPAEC plasma membrane isolates, IP-LC-MS identified a novel protein-protein interaction between NEDD9 and the ß3-subunit of the αvß3-integrin, which regulates fibrin anchoring to endothelial cells. In conclusion, lethal SARS-CoV-2-ARDS is associated with increased pulmonary endothelial N9P expression and N9P-fibrin colocalization in situ. Further investigation is needed to determine the pathogenetic and potential therapeutic relevance of N9P to the thrombotic pathophenotype of SARS-CoV-2-ARDS.

3.
PubMed; 2020.
Preprint in English | PubMed | ID: ppcovidwho-292772

ABSTRACT

Humoral responses in COVID-19 disease are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined postmortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers, a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+TFH cell differentiation together with an increase in T-bet+TH1 cells and aberrant extra-follicular TNF-a accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections and suggest that achieving herd immunity through natural infection may be difficult. Funding: This work was supported by NIH U19 AI110495 to SP, NIH R01 AI146779 to AGS, NIH R01AI137057 and DP2DA042422 to DL, BMH was supported by NIGMS T32 GM007753, TMC was supported by T32 AI007245. Funding for these studies from the Massachusetts Consortium of Pathogen Readiness, the Mark and Lisa Schwartz Foundation and Enid Schwartz is also acknowledged. Conflict of Interest: None. Ethical Approval: This study was performed with the approval of the Institutional Review Boards at the Massachusetts General Hospital and the Brigham and Women's Hospital.

4.
Angiogenesis ; 25(2): 225-240, 2022 May.
Article in English | MEDLINE | ID: covidwho-1491183

ABSTRACT

Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a hyperinflammatory state typified by elevated circulating pro-inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID-19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS-CoV-2 binding receptor ACE2. Herein we describe SARS-CoV-2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID-19, demonstrating both heterogeneous ACE2 expression and endothelial damage. In primary endothelial cell cultures, we show that SARS-CoV-2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon-alpha (IFNα) or -beta(ß)-two of the main anti-viral cytokines induced in severe SARS-CoV-2 infection-but not significantly by other cytokines (including interleukin 6 and interferon γ/λ). Our findings suggest that the stereotypical anti-viral interferon response may paradoxically facilitate the propagation of COVID-19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/ß in the treatment of patients with COVID-19.

5.
J Clin Microbiol ; 58(8)2020 07 23.
Article in English | MEDLINE | ID: covidwho-1388446
7.
Neurology ; 97(8): e849-e858, 2021 08 24.
Article in English | MEDLINE | ID: covidwho-1261289

ABSTRACT

OBJECTIVE: To explore the spectrum of skeletal muscle and nerve pathology of patients who died after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and to assess for direct viral invasion of these tissues. METHODS: Psoas muscle and femoral nerve sampled from 35 consecutive autopsies of patients who died after SARS-CoV-2 infection and 10 SARS-CoV-2-negative controls were examined under light microscopy. Clinical and laboratory data were obtained by chart review. RESULTS: In SARS-CoV-2-positive patients, mean age at death was 67.8 years (range 43-96 years), and the duration of symptom onset to death ranged from 1 to 49 days. Four patients had neuromuscular symptoms. Peak creatine kinase was elevated in 74% (mean 959 U/L, range 29-8,413 U/L). Muscle showed type 2 atrophy in 32 patients, necrotizing myopathy in 9, and myositis in 7. Neuritis was seen in 9. Major histocompatibility complex-1 (MHC-1) expression was observed in all cases of necrotizing myopathy and myositis and in 8 additional patients. Abnormal expression of myxovirus resistance protein A (MxA) was present on capillaries in muscle in 9 patients and in nerve in 7 patients. SARS-CoV-2 immunohistochemistry was negative in muscle and nerve in all patients. In the 10 controls, muscle showed type 2 atrophy in all patients, necrotic muscle fibers in 1, MHC-1 expression in nonnecrotic/nonregenerating fibers in 3, MxA expression on capillaries in 2, and inflammatory cells in none, and nerves showed no inflammatory cells or MxA expression. CONCLUSIONS: Muscle and nerve tissue demonstrated inflammatory/immune-mediated damage likely related to release of cytokines. There was no evidence of direct SARS-CoV-2 invasion of these tissues. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that muscle and nerve biopsies document a variety of pathologic changes in patients dying of coronavirus disease 2019 (COVID-19).


Subject(s)
COVID-19/pathology , Muscle, Skeletal/pathology , Peripheral Nerves/pathology , Adult , Aged , Aged, 80 and over , Autopsy , COVID-19/immunology , COVID-19/virology , Female , Humans , Male , Middle Aged , Muscle, Skeletal/immunology , Muscle, Skeletal/virology , Peripheral Nerves/immunology , Peripheral Nerves/virology
8.
Arch Pathol Lab Med ; 145(5): 529-535, 2021 05 01.
Article in English | MEDLINE | ID: covidwho-1207909

ABSTRACT

CONTEXT.­: This study represents the largest compilation to date of clinical and postmortem data from decedents with coronavirus disease 2019 (COVID-19). It will augment previously published small series of autopsy case reports, refine clinicopathologic considerations, and improve the accuracy of future vital statistical reporting. OBJECTIVE.­: To accurately reflect the preexisting diseases and pathologic conditions of decedents with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection through autopsy. DESIGN.­: Comprehensive data from 135 autopsy evaluations of COVID-19-positive decedents is presented, including histologic assessment. Postmortem examinations were performed by 36 pathologists at 19 medical centers or forensic institutions in the United States and Brazil. Data from each autopsy were collected through the online submission of multiple-choice and open-ended survey responses. RESULTS.­: Patients dying of or with COVID-19 had an average of 8.89 pathologic conditions documented at autopsy, spanning a combination of prior chronic disease and acute conditions acquired during hospitalization. Virtually all decedents were cited as having more than 1 preexisting condition, encompassing an average of 2.88 such diseases each. Clinical conditions during terminal hospitalization were cited 395 times for the 135 autopsied decedents and predominantly encompassed acute failure of multiple organ systems and/or impaired coagulation. Myocarditis was rarely cited. CONCLUSIONS.­: Cause-of-death statements in both autopsy reports and death certificates may not encompass the severity or spectrum of comorbid conditions in those dying of or with COVID-19. If supported by additional research, this finding may have implications for public health decisions and reporting moving forward through the pandemic.


Subject(s)
COVID-19/pathology , Adult , Aged , Aged, 80 and over , Autopsy , Brazil/epidemiology , COVID-19/diagnosis , COVID-19/epidemiology , Cause of Death , Chronic Disease , Comorbidity , Female , Humans , Male , Middle Aged , Pandemics , Surveys and Questionnaires , United States/epidemiology
9.
Elife ; 102021 04 23.
Article in English | MEDLINE | ID: covidwho-1200330

ABSTRACT

Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.


Subject(s)
COVID-19/pathology , Giant Cells/pathology , Host-Pathogen Interactions , SARS-CoV-2/physiology , Virus Internalization , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Cholesterol , Coculture Techniques , Humans , Lung/pathology , Membrane Fusion , Membrane Lipids/metabolism
10.
medRxiv ; 2022 Jan 11.
Article in English | MEDLINE | ID: covidwho-808244

ABSTRACT

SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited by anti-KIM-1 antibodies and TW-37, a newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 binds to the SARS-CoV-2 Spike protein in vitro . KIM-1 expressing cells, not expressing angiotensin-converting enzyme 2 (ACE2), are permissive to SARS-CoV-2 infection. Thus, KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.

12.
Cell ; 183(1): 143-157.e13, 2020 10 01.
Article in English | MEDLINE | ID: covidwho-720447

ABSTRACT

Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.


Subject(s)
Coronavirus Infections/immunology , Germinal Center/immunology , Pneumonia, Viral/immunology , T-Lymphocytes, Helper-Inducer/immunology , Aged , Aged, 80 and over , B-Lymphocytes/immunology , COVID-19 , Female , Germinal Center/pathology , Humans , Male , Middle Aged , Pandemics , Proto-Oncogene Proteins c-bcl-6/genetics , Proto-Oncogene Proteins c-bcl-6/metabolism , Spleen/immunology , Spleen/pathology , Tumor Necrosis Factor-alpha/metabolism
13.
Mod Pathol ; 33(11): 2104-2114, 2020 11.
Article in English | MEDLINE | ID: covidwho-606994

ABSTRACT

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has led to a global public health crisis. In elderly individuals and those with comorbidities, COVID-19 is associated with high mortality, frequently caused by acute respiratory distress syndrome. We examine in situ expression of SARS-CoV-2 in airways and lung obtained at autopsy of individuals with confirmed COVID-19 infection. Seven autopsy cases (male, N = 5; female, N = 2) with reverse transcriptase-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infection and a median age of 66 years (range, 50-77 years) were evaluated using a rabbit polyclonal antibody against SARS Nucleocapsid protein in correlation with clinical parameters. The median time from symptom onset to death was 9 days (range, 6-31 days), from hospitalization 7 days (range, 1-21 days), from positive RT-PCR 7 days (range, 0-18 days), and from intensive care unit admission defining onset of respiratory failure 3 days (range, 1-18 days). Chest imaging identified diffuse airspace disease in all patients corresponding to acute and (N = 5) or organizing (N = 2) diffuse alveolar damage (DAD) on histologic examination. Among five patients with acute-phase DAD (≤7 days from onset of respiratory failure), SARS-CoV-2 was detected in pulmonary pneumocytes and ciliated airway cells (N = 5), and in upper airway epithelium (N = 2). In two patients with organizing DAD (>14 days from onset of respiratory failure), no virus was detected in lungs or airways. No endothelial cell infection was observed. The findings suggest that SARS-CoV-2 infection of epithelial cells in lungs and airways of patients with COVID-19 who developed respiratory failure can be detected during the acute phase of lung injury and is absent in the organizing phase.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/pathology , Coronavirus Infections/virology , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Severe Acute Respiratory Syndrome/pathology , Severe Acute Respiratory Syndrome/virology , Aged , Autopsy , COVID-19 , Female , Humans , Male , Middle Aged , Pandemics , Respiratory System/pathology , Respiratory System/virology , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2
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