Role of amphiregulin (AREG) in the pathophysiology of COVID-19

Os casos graves de COVID-19 são frequentemente caracterizados por um grande processo inflamatório que pode levar à falência de órgãos e à morte do paciente. Neste contexto, a anfiregulina (AREG), um fator de crescimento epidérmico semelhante ao EGF, tem sido descrita como importante no processo de reparo por exercer um papel fundamental na mediação dos mecanismos de tolerância. Considerando que a AREG é um fator importante no processo de homeostasia durante e após o processo inflamatório, decidimos investigar seu papel na fisiopatologia da COVID-19. Para isso, utilizamos amostras de PBMCs, de autópsias pulmonares e lavado traqueal de pacientes com COVID-19 (leve, moderada e severa) e comparamos a presença de AREG por RT-PCR, imunofluorescência e ELISA com base em grupos controle. Além disso, usamos um modelo experimental de infecção usando como base camundongos K18-hACE2 para entender o papel do AREG na fisiopatologia da COVID-19, também bloqueamos a sinalização do EGFR com intuito entender como os sinais emitidos por esse receptor se relacionam com a doença. Observamos que os níveis de AREG em PBMCs de pacientes com COVID-19 estavam elevados em comparação aos doadores saudáveis e que, este aumento, estava correlacionado com manifestações clínicas mais graves, com um provável desfecho de morte, também observamos um aumento das concentrações de AREG no lavado traqueal de pacientes hospitalizados e no homogenato do pulmão, bem como uma coloração intensa na imunofluorescência de autópsias pulmonares de pacientes que morreram de COVID-19. Vimos também que no modelo experimental de infecção, o AREG estava em altos níveis, assim como outros mediadores inflamatórios. Além disso, o bloqueio da sinalização do receptor AREG melhorou os sinais clínicos da doença no modelo de experimental. Em resumo, nossos dados indicam que o AREG está envolvido no processo fisiopatológico desencadeado pelo SARS-CoV-2 e está relacionado com a gravidade da doença.Severe cases of COVID-19 are often characterized by a large inflammatory process that can lead to organ failure and patient death. In this context, amphiregulin (AREG), an epidermal growth factor EGF-like, has been described as important in the repair process by playing a key role in mediating tolerance mechanisms. Considering that AREG is an important factor in the homeostasis process during and after the inflammatory process, we decided to investigate its role in the pathophysiology of COVID-19. To this end, we used PBMCs, lung autopsy and tracheal lavage samples from patients with COVID-19 (mild, moderate and severe) and compared the presence of AREG by RT-PCR, immunofluorescence and ELISA based on control groups. In addition, we used an experimental infection model using K18-hACE2 mice to understand the role of AREG in the pathophysiology of COVID-19, we also blocked EGFR signaling in order to understand how the signals emitted by this receptor relate to the disease. We observed that AREG levels in PBMCs from patients with COVID-19 were elevated compared to healthy donors, and that this increase correlated with more severe clinical manifestations with a likely outcome of death, we also observed increased AREG concentrations in tracheal lavage from hospitalized patients and in lung homogenate, as well as intense staining in immunofluorescence from lung autopsies of patients who died of COVID-19. We also saw that in the experimental infection model, AREG was at high levels, as were other inflammatory mediators. Furthermore, blocking AREG receptor signaling improved the clinical signs of the disease in the experimental model. In summary, our data indicate that AREG is involved in the pathophysiological process triggered by SARS-CoV-2 and is related to disease severity

Sepsis expands a CD39+ plasmablast population that promotes immunosuppression via adenosine-mediated inhibition of macrophage antimicrobial activity

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease

SARS-CoV-2 productively infects primary human immune system cells in vitro and in COVID-19 patients

Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a hyperinflammatory state and lymphocytopenia, a hallmark that appears as both signature and prognosis of disease severity outcome. Although cytokine storm and a sustained inflammatory state are commonly associated with immune cell depletion, it is still unclear whether direct SARS-CoV-2 infection of immune cells could also play a role in this scenario by harboring viral replication. We found that monocytes, as well as both B and T lymphocytes, were susceptible to SARS-CoV-2 infection in vitro, accumulating double-stranded RNA consistent with viral RNA replication and ultimately leading to expressive T cell apoptosis. In addition, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from coronavirus disease 2019 (COVID-19) patients. The rates of SARS-CoV-2-infected monocytes in peripheral blood mononuclear cells from COVID-19 patients increased over time from symptom onset, with SARS-CoV-2-positive monocytes, B cells, and CD4+ T lymphocytes also detected in postmortem lung tissue. These results indicated that SARS-CoV-2 infection of blood-circulating leukocytes in COVID-19 patients might have important implications for disease pathogenesis and progression, immune dysfunction, and virus spread within the host

Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory functions and clearance of apoptotic cells

COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV-2-infected apoptotic cells exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence supporting that lung monocytes and macrophages from severe COVID-19 patients have compromised efferocytic capacity. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppresses macrophage anti-inflammation and efficient tissue repair programs and provides mechanistic insights for the excessive production of pro-inflammatory cytokines and accumulation of tissue damage associated with COVID-19 immunopathogenesis

Gasdermin-D activation by SARS-CoV-2 triggers NET and mediate COVID-19 immunopathology

Abstract: Background: The release of neutrophil extracellular traps (NETs) is associated with inflammation, coagulopathy, and organ damage found in severe cases of COVID-19. However, the molecular mechanisms underlying the release of NETs in COVID-19 remain unclear. Objectives: We aim to investigate the role of the Gasdermin-D (GSDMD) pathway on NETs release and the development of organ damage during COVID-19. Methods: We performed a single-cell transcriptome analysis in public data of bronchoalveolar lavage. Then, we enrolled 63 hospitalized patients with moderate and severe COVID-19. We analyze in blood and lung tissue samples the expression of GSDMD, presence of NETs, and signaling pathways upstreaming. Furthermore, we analyzed the treatment with disulfiram in a mouse model of SARS-CoV-2 infection. Results: We found that the SARS-CoV-2 virus directly activates the pore-forming protein GSDMD that triggers NET production and organ damage in COVID-19. Single-cell transcriptome analysis revealed that the expression of GSDMD and inflammasome-related genes were increased in COVID-19 patients. High expression of active GSDMD associated with NETs structures was found in the lung tissue of COVID-19 patients. Furthermore, we showed that activation of GSDMD in neutrophils requires active caspase1/4 and live SARS-CoV-2, which infects neutrophils. In a mouse model of SARS-CoV-2 infection, the treatment with disulfiram inhibited NETs release and reduced organ damage. Conclusion: These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology and suggests GSDMD as a novel potential target for improving the COVID-19 therapeutic strategy