Deregulated miRNA expression is associated with endothelial dysfunction in post-mortem lung biopsies of COVID-19 patients.

MicroRNAs (miRNAs) are critical modulators of endothelial homeostasis, which highlights their involvement in vascular diseases, including the ones caused by virus infections. Our main objective was to identify miRNAs involved in the endothelial function and determine their expression in post-mortem lung biopsies of COVID-19 patients with severe respiratory injuries and thrombotic events. Based on functional enrichment analysis, miR-26a-5p, miR-29b-3p, and miR-34a-5p were identified as regulators of mRNA targets involved in endothelial, and inflammatory signaling pathways as well as in viral diseases. A miRNA/mRNA network, constructed based on protein-protein interactions of the miRNA targets and the inflammatory biomarkers characterized in the patients, revealed a close interconnection of these miRNAs with relevance to the endothelial activation/dysfunction. Reduced expression levels of selected miRNAs were observed in the lung biopsies of COVID-19 patients (n=9) compared to the Controls (n=10)(P<0.01-0.0001). MiR-26a-5p and miR-29b-3p presented the best power to discriminate these groups (AUC=0.8286, and AUC=0.8125, respectively). The correlation analysis of the miRNAs with inflammatory biomarkers in the COVID-19 patients was significant for miR-26a-5p [IL-6 (r2=0.5414), and ICAM-1(r2=0.5624)], and miR-29b-3p [IL-4 (r2=0.8332), and IL-8 (r2=0.2654)]. Altogether, these findings demonstrate the relevance and the non-random involvement of miR-26a-5p, miR-29b-3p, and miR-34a-5p in endothelial dysfunction and inflammatory response in patients with SARS-CoV-2 infection and the occurrence of severe lung injury and immunothrombosis.

Lung Inflammasome Activation in SARS-CoV-2 Post-Mortem Biopsies.

The inflammasome complex is a key part of chronic diseases and acute infections, being responsible for cytokine release and cell death mechanism regulation. The SARS-CoV-2 infection is characterized by a dysregulated cytokine release. In this context, the inflammasome complex analysis within SARS-CoV-2 infection may prove beneficial to understand the disease's mechanisms. Post-mortem minimally invasive autopsies were performed in patients who died from COVID-19 (n = 24), and lung samples were compared to a patient control group (n = 11) and an Influenza A virus H1N1 subtype group from the 2009 pandemics (n = 10). Histological analysis was performed using hematoxylin-eosin staining. Immunohistochemical (IHC) staining was performed using monoclonal antibodies against targets: ACE2, TLR4, NF-κB, NLRP-3 (or NALP), IL-1ß, IL-18, ASC, CASP1, CASP9, GSDMD, NOX4, TNF-α. Data obtained from digital analysis underwent appropriate statistical tests. IHC analysis showed biomarkers that indicate inflammasome activation (ACE2; NF-κB; NOX4; ASC) were significantly increased in the COVID-19 group (p < 0.05 for all) and biomarkers that indicate cell pyroptosis and inflammasome derived cytokines such as IL-18 (p < 0.005) and CASP1 were greatly increased (p < 0.0001) even when compared to the H1N1 group. We propose that the SARS-CoV-2 pathogenesis is connected to the inflammasome complex activation. Further studies are still warranted to elucidate the pathophysiology of the disease.

Immune Response Gaps Linked to SARS-CoV-2 Infection: Cellular Exhaustion, Senescence, or Both?

The COVID-19 pandemic, promoted by the SARS-CoV-2 respiratory virus, has resulted in widespread global morbidity and mortality. The immune response against this pathogen has shown a thin line between protective effects and pathological reactions resulting from the massive release of cytokines and poor viral clearance. The latter is possibly caused by exhaustion, senescence, or both of TCD8+ cells and reduced activity of natural killer (NK) cells. The imbalance between innate and adaptive responses during the early stages of infection caused by SARS-CoV-2 contributes to the ineffective control of viral spread. The present study evaluated the tissue immunoexpression of the tissue biomarkers (Arginase-1, CCR4, CD3, CD4, CD8, CD20, CD57, CD68, CD138, IL-4, INF-α, INF-γ, iNOS, PD-1, Perforin and Sphingosine-1) to understand the cellular immune response triggered in patients who died of COVID-19. We evaluated twenty-four paraffin-embedded lung tissue samples from patients who died of COVID-19 (COVID-19 group) and compared them with ten lung tissue samples from patients who died of H1N1pdm09 (H1N1 group) with the immunohistochemical markers mentioned above. In addition, polymorphisms in the Perforin gene were genotyped through Real-Time PCR. Significantly increased tissue immunoexpression of Arginase, CD4, CD68, CD138, Perforin, Sphingosine-1, and IL-4 markers were observed in the COVID-19 group. A significantly lower immunoexpression of CD8 and CD57 was also found in this group. It is suggested that patients who died from COVID-19 had a poor cellular response concerning viral clearance and adaptive response going through tissue repair.

Role of Genetic Polymorphism Present in Macrophage Activation Syndrome Pathway in Post Mortem Biopsies of Patients with COVID-19.

COVID-19 is a viral disease associated with an intense inflammatory response. Macrophage Activation Syndrome (MAS), the complication present in secondary hemophagocytic lymphohistiocytosis (sHLH), shares many clinical aspects observed in COVID-19 patients, and investigating the cytolytic function of the responsible cells for the first line of the immune response is important. Formalin-fixed paraffin-embedded lung tissue samples obtained by post mortem necropsy were accessed for three groups (COVID-19, H1N1, and CONTROL). Polymorphisms in MAS cytolytic pathway (PRF1; STX11; STXBP2; UNC13D and GZMB) were selected and genotyping by TaqMan® assays (Thermo Fisher Scientific, MA, USA) using Real-Time PCR (Applied Biosystems, MA USA). Moreover, immunohistochemistry staining was performed with a monoclonal antibody against perforin, CD8+ and CD57+ proteins. Histopathological analysis showed high perforin tissue expression in the COVID-19 group; CD8+ was high in the H1N1 group and CD57+ in the CONTROL group. An association could be observed in two genes related to the cytolytic pathway (PRF1 rs885822 G/A and STXBP2 rs2303115 G/A). Furthermore, PRF1 rs350947132 was associated with increased immune tissue expression for perforin in the COVID-19 group. The genotype approach could help identify patients that are more susceptible, and for this reason, our results showed that perforin and SNPs in the PRF1 gene can be involved in this critical pathway in the context of COVID-19.

Association Between COVID-19 Pregnant Women Symptoms Severity and Placental Morphologic Features.

Since the beginning of the pandemic, few papers describe the placenta's morphological and morphometrical features in SARS-CoV-2-positive pregnant women. Alterations, such as low placental weight, accelerated villous maturation, decidual vasculopathy, infarcts, thrombosis of fetal placental vessels, and chronic histiocytic intervillositis (CHI), have been described. Objective: To analyze clinical data and the placental morphological and morphometric changes of pregnant women infected with SARS-CoV-2 (COVID-19 group) in comparison with the placentas of non-infected pregnant women, matched for maternal age and comorbidities, besides gestational age of delivery (Control group). Method: The patients in the COVID-19 and the Control group were matched for maternal age, gestational age, and comorbidities. The morphological analysis of placentas was performed using Amsterdam Placental Workshop Group Consensus Statement. The quantitative morphometric evaluation included perimeter diameter and number of tertiary villi, number of sprouts and knots, evaluation of deposition of villous fibrin, and deposition of intra-villous collagen I and III by Sirius Red. Additionally, Hofbauer cells (HC) were counted within villi by immunohistochemistry with CD68 marker. Results: Compared to controls, symptomatic women in the COVID-19 group were more likely to have at least one comorbidity, to evolve to preterm labor and infant death, and to have positive SARS-CoV-2 RNA testing in their concepts. Compared to controls, placentas in the COVID-19 group were more likely to show features of maternal and fetal vascular malperfusion. In the COVID-19 group, placentas of symptomatic women were more likely to show CHI. No significant results were found after morphometric analysis. Conclusion: Pregnant women with symptomatic SARS-CoV-2 infection, particularly with the severe course, are more likely to exhibit an adverse fetal outcome, with slightly more frequent histopathologic findings of maternal and fetal vascular malperfusion, and CHI. The morphometric changes found in the placentas of the COVID-19 group do not seem to be different from those observed in the Control group, as far as maternal age, gestational age, and comorbidities are paired. Only the deposition of villous fibrin could be more accentuated in the COVID-19 group (p = 0.08 borderline). The number of HC/villous evaluated with CD68 immunohistochemistry did not show a difference between both groups.

Lung Neutrophilic Recruitment and IL-8/IL-17A Tissue Expression in COVID-19.

The new SARS-CoV-2 virus differs from the pandemic Influenza A virus H1N1 subtype (H1N1pmd09) how it induces a pro-inflammatory response in infected patients. This study aims to evaluate the involvement of SNPs and tissue expression of IL-17A and the neutrophils recruitment in post-mortem lung samples from patients who died of severe forms of COVID-19 comparing to those who died by H1N1pdm09. Twenty lung samples from patients SARS-CoV-2 infected (COVID-19 group) and 10 lung samples from adults who died from a severe respiratory H1N1pdm09 infection (H1N1 group) were tested. The tissue expression of IL-8/IL-17A was identified by immunohistochemistry, and hematoxylin and eosin (H&E) stain slides were used for neutrophil scoring. DNA was extracted from paraffin blocks, and genotyping was done in real time-PCR for two IL17A target polymorphisms. Tissue expression increasing of IL-8/IL-17A and a higher number of neutrophils were identified in samples from the H1N1 group compared to the COVID-19 group. The distribution of genotype frequencies in the IL17A gene was not statistically significant between groups. However, the G allele (GG and GA) of rs3819025 was correlated with higher tissue expression of IL-17A in the COVID-19 group. SARS-CoV-2 virus evokes an exacerbated response of the host's immune system but differs from that observed in the H1N1pdm09 infection since the IL-8/IL-17A tissue expression, and lung neutrophilic recruitment may be decreased. In SNP rs3819025 (G/A), the G allele may be considered a risk allele in the patients who died for COVID-19.

Endothelial Dysfunction and Thrombosis in Patients With COVID-19-Brief Report.

OBJECTIVE: Alveolar-capillary endothelial cells can be activated by severe acute respiratory syndrome coronavirus 2 infection leading to cytokine release. This could trigger endothelial dysfunction, pyroptosis, and thrombosis, which are the vascular changes, commonly referred to as coronavirus disease 2019 (COVID-19) endotheliopathy. Thus, this study aimed to identify tissue biomarkers associated with endothelial activation/dysfunction and the pyroptosis pathway in the lung samples of patients with COVID-19 and to compare them to pandemic influenza A virus H1N1 subtype 2009 and control cases. Approach and Results: Postmortem lung samples (COVID-19 group =6 cases; H1N1 group =10 cases, and control group =11 cases) were analyzed using immunohistochemistry and the following monoclonal primary antibodies: anti-IL (interleukin)-6, anti-TNF (tumor necrosis factor)-α, anti-ICAM-1 (intercellular adhesion molecule 1), and anticaspase-1. From the result, IL-6, TNF-α, ICAM-1, and caspase-1 showed higher tissue expression in the COVID-19 group than in the H1N1 and control groups. CONCLUSIONS: Our results demonstrated endothelial dysfunction and suggested the participation of the pyroptosis pathway in the pulmonary samples. These conditions might lead to systemic thrombotic events that could impair the clinical staff's efforts to avoid fatal outcomes. One of the health professionals' goals should be to identify the high risk of thrombosis patients early to block endotheliopathy and its consequences.

The role of the lectin pathway of the complement system in SARS-CoV-2 lung injury.

Although some evidence showed the activation of complement systems in COVID-19 patients, proinflammatory status and lectin pathway remain unclear. Thus, the present study aimed to demonstrate the role of MBL and ficolin-3 in the complement system activation and compared to pandemic Influenza A virus H1N1 subtype infection (H1N1pdm09) and control patients. A total of 27 lungs formalin-fixed paraffin-embedded samples (10 from H1N1 group, 6 from the COVID-19 group, and 11 from the control group) were analyzed by immunohistochemistry using anti-IL-6, TNF-alfa, CD163, MBL e FCN3 antibodies. Genotyping of target polymorphisms in the MBL2 gene was performed by real-time PCR. Proinflammatory cytokines such as IL-6 and TNF-alpha presented higher tissue expression in the COVID-19 group compared to H1N1 and control groups. The same results were observed for ICAM-1 tissue expression. Increased expression of the FCN3 was observed in the COVID-19 group and H1N1 group compared to the control group. The MBL tissue expression was higher in the COVID-19 group compared to H1N1 and control groups. The genotypes AA for rs180040 (G/A), GG for rs1800451 (G/A) and CC for rs5030737 (T/C) showed a higher prevalence in the COVID-19 group. The intense activation of the lectin pathway, with particular emphasis on the MBL pathway, together with endothelial dysfunction and a massive proinflammatory cytokines production, possibly lead to a worse outcome in patients infected with SARS-Cov-2. Moreover, 3 SNPs of our study presented genotypes that might be correlated with high MBL tissue expression in the COVID-19 pulmonary samples.