The immune response is crucial for coronavirus disease 19 (COVID-19) progression, with the participation of proinflammatory cells and cytokines, inducing lung injury and loss of respiratory function. CLEC5A expression on monocytes can be triggered by viral and bacterial infections, leading to poor outcomes. SARS-CoV-2 is able to induce neutrophil activation by CLEC5A and Toll-like receptor 2, leading to an aggressive inflammatory cascade, but little information is known about the molecular interactions between CLEC5A and SARS-CoV-2 proteins.Objectives: Here, we aimed to explore how CLEC5A expression could be affected by SARS-CoV-2 infection using immunological tools with in vitro, in vivo and in silico assays.Methodology: Molecular docking modeling was performed through the ClusPro 2.0 and Pymol 2.5 software. PBMCs were subjected to assays ex vivo immunophenotyping with commercial antibodies to characterize the monocyte subpopulations and Clec5a expression. The PBMC were isolated by Ficoll-Paque® and analyzed by flow cytometry. The samples were divided into three groups: unexposed (n=18), mild COVID-19 (n=17) and severe . Quantification of the cytokines IL-2, IFN-γ, IL-6, and IL-1β was performed using an in-house multiplex liquid microarray test. Detection of CLEC5A gene expressed in blood from hamsters was performed by RT-qPCR. Blood samples were obtained at days 3, 5, 10, and 15 through exsanguination by cardiac puncture from a 36 Syrian golden hamster (Mesocricetus auratus) at 1 year of age and 150 ± 1.4 g infected intranasally with SARS-CoV-2 strains Delta (1.0 × 106 PFU/ml) and Omicron (1.0 × 106 PFU/ml). Results:The findings revealed that high levels of CLEC5A expression were found in monocytes from severe COVID-19 patients in comparison with mild COVID-19 and unexposed subjects, but not in vaccinated subjects who developed mild COVID-19. In hamsters, we detected CLEC5A gene expression during 3-15 days of Omicron strain viral challenge. Our results also showed that CLEC5A can interact with SARS-CoV-2, promoting inflammatory cytokine production, probably through an interaction with the receptor binding domain in the N-acetylglucosamine binding site (NAG-601). The high expression of CLEC5A and high levels of proinflammatory cytokine production were reduced in vitro by a human CLEC5A monoclonal antibody. Conclusion:CLEC5A was triggered by spike glycoprotein, suggesting its involvement in COVID-19 progression; therapy with a monoclonal antibody could be a good strategy for COVID-19 treatment, but vaccines are still the best option to avoid hospitalization/deaths.
Introduction: Clec5a, is a lectin superfamily receptor expressed abundantly in myeloid lineage cells. Its has been know that macrophages infected with Dengue virus and anti-Clec5a have produced decrease pro-inflammatory cytokines. Clec5a also appears as a contributor increase in cytokine production by monocytes in cases of infection with Zika Virus and immunomodulation after Yellow Fever vaccination. Cytokines storms are related in severe COVID-19 cases and Clec5a could be an important signaling pathway for the worsening clinical evolution and thus be a strategic therapeutic target.Objective: Evaluate the expression of Clec5a in monocytes in Covid-19. Methodology:To evaluate the possibility of interaction between proteins and to investigate sites of protein binding, molecular docking modeling was performed through the Cluspro 2.0 and Pymol 2.5 software. These data were essential for predictive analysis of potential receptor-ligand interactions. PBMC were subjected to assays ex vivo immunophenotyping with commercial antibodies to characterize the monocyte subpopulations and Clec5a expression. PBMC were isolated by Ficoll-Paque®, PBMC samples were analyzed by flow cytometry and subsequently evaluated by the software FlowJo Tree Star. The samples were divided into three groups: unexposed, mild COVID19 and severe COVID-19.Results: Our finding showed that Clec5a is capable of interact with regions of the S1 subunit spike protein of SARS-CoV-2. Immunophenotyping shows that Clec5a is overexpressed on intermediate and non-classical monocytes. To establish a cut-off for Clec5A expression on non-classical monocytes related to severity, ROC curve was performed showing that 20,9% (p = 0.007) is the limit to define mild (20.9%). Our data also revealed an association of Clec5a expression with severe COVID-19 (OD=58.5;95%CI: 5.75-594.53, p = 0.0006). Conclusion:These results indicated that exists interaction between Clec5a and spike protein of SARS-CoV-2. The expression of Clec5a was elevated in severe COVID-19 cases, which may explain the inflammatory status in this syndrome. In addiction, findings shows that Clec5a may be a good therapeutic target for new treatments for viral infections, including cases of COVID-19.
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