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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.

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.

Mast cells (MCs) have relevant participation in inflammatory and vascular hyperpermeability events, responsible for the action of the kallikrein–kinin system (KKS), that affect patients inflicted by the severe form of COVID-19. Given a higher number of activated MCs present in COVID-19 patients and their association with vascular hyperpermeability events, we investigated the factors that lead to the activation and degranulation of these cells and their harmful effects on the alveolar septum environment provided by the action of its mediators. Therefore, the pyroptotic processes throughout caspase-1 (CASP-1) and alarmin interleukin-33 (IL-33) secretion were investigated, along with the immunoexpression of angiotensin-converting enzyme 2 (ACE2), bradykinin receptor B1 (B1R) and bradykinin receptor B2 (B2R) on post-mortem lung samples from 24 patients affected by COVID-19. The results were compared to 10 patients affected by H1N1pdm09 and 11 control patients. As a result of the inflammatory processes induced by SARS-CoV-2, the activation by immunoglobulin E (IgE) and degranulation of tryptase, as well as Toluidine Blue metachromatic (TB)-stained MCs of the interstitial and perivascular regions of the same groups were also counted. An increased immunoexpression of the tissue biomarkers CASP-1, IL-33, ACE2, B1R and B2R was observed in the alveolar septum of the COVID-19 patients, associated with a higher density of IgE+ MCs, tryptase+ MCs and TB-stained MCs, in addition to the presence of intra-alveolar edema. These findings suggest the direct correlation of MCs with vascular hyperpermeability, edema and diffuse alveolar damage (DAD) events that affect patients with a severe form of this disease. The role of KKS activation in events involving the exacerbated increase in vascular permeability and its direct link with the conditions that precede intra-alveolar edema, and the consequent DAD, is evidenced. Therapy with drugs that inhibit the activation/degranulation of MCs can prevent the worsening of the prognosis and provide a better outcome for the patient.

MicroRNAs (miRNAs) are critical modulators of endothelial homeostasis, which highlights their involvement in vascular diseases, including those 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 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 in association 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 (area under the curve (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.

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.

Supplemental Digital Content is available in the text., 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.