Collagen-Specific HSP47+ Myofibroblasts and CD163+ Macrophages Identify Profibrotic Phenotypes in Deceased Hearts With SARS-CoV-2 Infections.

Background Cardiac fibrosis complicates SARS-CoV-2 infections and has been linked to arrhythmic complications in survivors. Accordingly, we sought evidence of increased HSP47 (heat shock protein 47), a stress-inducible chaperone protein that regulates biosynthesis and secretion of procollagen in heart tissue, with the goal of elucidating molecular mechanisms underlying cardiac fibrosis in subjects with this viral infection. Methods and Results Using human autopsy tissue, immunofluorescence, and immunohistochemistry, we quantified Hsp47+ cells and collagen α 1(l) in hearts from people with SARS-CoV-2 infections. Because macrophages are also linked to inflammation, we measured CD163+ cells in the same tissues. We observed irregular groups of spindle-shaped HSP47+ and CD163+ cells as well as increased collagen α 1(I) deposition, each proximate to one another in "hot spots" of ≈40% of hearts after SARS-CoV-2 infection (HSP47+ P<0.05 versus nonfibrotics and P<0.001 versus controls). Because HSP47+ cells are consistent with myofibroblasts, subjects with hot spots are termed "profibrotic." The remaining 60% of subjects dying with COVID-19 without hot spots are referred to as "nonfibrotic." No control subject exhibited hot spots. Conclusions Colocalization of myofibroblasts, M2(CD163+) macrophages, and collagen α 1(l) may be the first evidence of a COVID-19-related "profibrotic phenotype" in human hearts in situ. The potential public health and diagnostic implications of these observations require follow-up to further define mechanisms of viral-mediated cardiac fibrosis.

Cytotoxic CD8-positive T-lymphocyte infiltration in the lungs as a histological pattern of SARS-CoV-2 pneumonitis.

Despite millions of PCR confirmed cases of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, the long-term pathophysiological changes induced by this infection in the lungs and their relationship with possible immune triggers remain incompletely understood. Acute respiratory distress syndrome and subsequent respiratory failure are the most common causes of mortality in hospitalised patients. Severe lung tissue destruction can be due to an overactive immune system that far exceeds the harm that would have been caused by direct virus replication. This study extends our previous investigation and presents detailed histopathological findings on cryotransbronchial biopsy in patients with persistent (range 31-182 days) pneumonitis and severe interstitial inflammatory infiltration in the lungs due to SARS-CoV-2 infection. We describe a novel lung injury pattern associated with SARS-CoV-2 pneumonitis, which manifests as a marked interstitial CD8-positive T-cell lymphocytic infiltration. These findings provide a better understanding of the changes in the lungs that ensue due to SARS-CoV-2 infection.

Cytotoxic CD8 positive T-lymphocyte infiltration in the lungs as a histological pattern of SARS-CoV-2 pneumonitis

Despite millions of PCR confirmed cases of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, the long-term pathophysiological changes induced by this infection in the lungs and their relationship with possible immune triggers remain incompletely understood. Acute respiratory distress syndrome and subsequent respiratory failure are the most common cause of mortality in hospitalised patients. Severe lung tissue destruction can be due to an overactivity immune system that far exceeds the harm that would have been caused by direct virus replication. This study extends our previous investigation and presents detailed histopathological findings on cryotransbronchial biopsy in patients with persistent (range 31–182 days) pneumonitis and severe interstitial inflammatory infiltration in the lungs due to SARS-CoV-2 infection. We describe a novel lung injury pattern associated with SARS-CoV-2 pneumonitis, which manifests as a marked interstitial CD8 positive T-cell lymphocytic infiltration. These findings provide a better understanding of the changes in the lungs that ensue due to SARS-CoV-2 infection.

Pneumocytes are distinguished by highly elevated expression of the ER stress biomarker GRP78, a co-receptor for SARS-CoV-2, in COVID-19 autopsies.

Vaccinations are widely credited with reducing death rates from COVID-19, but the underlying host-viral mechanisms/interactions for morbidity and mortality of SARS-CoV-2 infection remain poorly understood. Acute respiratory distress syndrome (ARDS) describes the severe lung injury, which is pathologically associated with alveolar damage, inflammation, non-cardiogenic edema, and hyaline membrane formation. Because proteostatic pathways play central roles in cellular protection, immune modulation, protein degradation, and tissue repair, we examined the pathological features for the unfolded protein response (UPR) using the surrogate biomarker glucose-regulated protein 78 (GRP78) and co-receptor for SARS-CoV-2. At autopsy, immunostaining of COVID-19 lungs showed highly elevated expression of GRP78 in both pneumocytes and macrophages compared with that of non-COVID control lungs. GRP78 expression was detected in both SARS-CoV-2-infected and un-infected pneumocytes as determined by multiplexed immunostaining for nucleocapsid protein. In macrophages, immunohistochemical staining for GRP78 from deceased COVID-19 patients was increased but overlapped with GRP78 expression taken from surgical resections of non-COVID-19 controls. In contrast, the robust in situ GRP78 immunostaining of pneumocytes from COVID-19 autopsies exhibited no overlap and was independent of age, race/ethnicity, and gender compared with that from non-COVID-19 controls. Our findings bring new insights for stress-response pathways involving the proteostatic network implicated for host resilience and suggest that targeting of GRP78 expression with existing therapeutics might afford an alternative therapeutic strategy to modulate host-viral interactions during SARS-CoV-2 infections.

Sensitive and Specific Immunohistochemistry Protocol for Nucleocapsid Protein from All Common SARS-CoV-2 Virus Strains in Formalin-Fixed, Paraffin Embedded Tissues.

Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 viral proteins in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identified commercially available affinity-purified polyclonal antibodies raised against nucleocapsid and spike proteins of SARS-CoV-2 that provide sensitive and specific detection of the virus by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Two immunohistochemistry protocols are presented that are mutually validated by the matched detection patterns of the two distinct viral antigens in virus-infected cells within autopsy lung tissue of COVID-19 deceased patients. Levels of nucleocapsid protein in the lungs of COVID-19 decedents, as measured by quantitative histo-cytometry of immunohistochemistry images, showed an excellent log-linear relationship with levels of viral nucleocapsid RNA levels, as measured by qRT-PCR. Importantly, since the nucleocapsid protein sequence is conserved across all known viral strains, the nucleocapsid immunohistochemistry protocol is expected to recognize all common variants of SARS-CoV-2. Negative controls include autopsy lung tissues from patients who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. Sensitive detection of SARS-CoV-2 in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection, as well as provide insight into the clearance rate of virus in later COVID-19 disease stages. The protocols are also expected to be readily transferable to detect SARS-CoV-2 proteins in tissues of experimental animal models or animals suspected to serve as viral reservoirs.

Diffuse interstitial pneumonia-like/macrophage activation syndrome-like changes in patients with COVID-19 correlate with length of illness.

OBJECTIVES: Assess the pathologic changes in the lungs of COVID-19 decedents and correlate these changes with demographic data, clinical course, therapies, and duration of illness. METHODS: Lungs of 12 consecutive COVID-19 decedents consented for autopsy were evaluated for gross and histopathologic abnormalities. A complete Ghon "en block" dissection was performed on all cases; lung weights and gross characteristics recorded. Immunohistochemical studies were performed to characterize lymphocytic infiltrates and to assess SARS-CoV-2 capsid protein. RESULTS: Two distinct patterns of pulmonary involvement were identified. Three of 12 cases demonstrated a predominance of acute alveolar damage (DAD) while 9 of 12 cases demonstrated a marked increase in intra-alveolar macrophages in a fashion resembling desquamative interstitial pneumonia or macrophage activation syndrome (DIP/MAS). Two patterns were correlated solely with a statistically significant difference in the duration of illness. The group exhibiting DAD had duration of illness of 5.7 days while the group with DIP/MAS had duration of illness of 21.5 days (t-test p = 0.014). CONCLUSIONS: The pulmonary pathology of COVID-19 patients demonstrates a biphasic pattern, an acute phase demonstrating DAD changes while the patients with a more prolonged course exhibit a different pattern that resembles DIP/MAS-like pattern. The potential mechanisms and clinical significance are discussed.

Acute SARS-CoV-2 pneumonitis with cytotoxic CD8 positive T-lymphocytes: Case report and review of the literature.

As of October 2020, there are over 40 million confirmed cases, and more than 1 million confirmed deaths of Covid-19 worldwide. The main cause of death in hospitalized patients is a respiratory failure due to acute respiratory distress syndrome. It has been suggested that the very intense immune response induces diffuse alveolar damage that far exceeds the harm that would have been caused by virus replication per se, resulting in lethal tissue destruction. We present a detailed report of the histopathological findings on cryo transbronchial biopsy in the patient with persistent (3 months) interstitial pneumonitis and severe CD8 positive cell infiltration in the lungs due to SARS-CoV-2 infection. CD8 positive T-lymphocytes have a great potential to damage tissue either through direct cytotoxicity or through cytokines release.