Impact of Microbiota Depletion by Antibiotics on SARS-CoV-2 Infection of K18-hACE2 Mice.

Clinical and experimental data indicate that severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection is associated with significant changes in the composition and function of intestinal microbiota. However, the relevance of these effects for SARS-CoV-2 pathophysiology is unknown. In this study, we analyzed the impact of microbiota depletion after antibiotic treatment on the clinical and immunological responses of K18-hACE2 mice to SARS-CoV-2 infection. Mice were treated with a combination of antibiotics (kanamycin, gentamicin, metronidazole, vancomycin, and colistin, Abx) for 3 days, and 24 h later, they were infected with SARS-CoV-2 B lineage. Here, we show that more than 80% of mice succumbed to infection by day 11 post-infection. Treatment with Abx had no impact on mortality. However, Abx-treated mice presented better clinical symptoms, with similar weight loss between infected-treated and non-treated groups. We observed no differences in lung and colon histopathological scores or lung, colon, heart, brain and kidney viral load between groups on day 5 of infection. Despite some minor differences in the expression of antiviral and inflammatory markers in the lungs and colon, no robust change was observed in Abx-treated mice. Together, these findings indicate that microbiota depletion has no impact on SARS-CoV-2 infection in mice.

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

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.

PI3K Signaling in Mechanisms and Treatments of Pulmonary Fibrosis Following Sepsis and Acute Lung Injury.

Pulmonary fibrosis is a pathological fibrotic process affecting the lungs of five million people worldwide. The incidence rate will increase even more in the next years due to the long-COVID-19 syndrome, but a resolving treatment is not available yet and usually prognosis is poor. The emerging role of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling in fibrotic processes has inspired the testing of drugs targeting the PI3K/Akt pathway that are currently under clinical evaluation. This review highlights the progress in understanding the role of PI3K/Akt in the development of lung fibrosis and its causative pathological context, including sepsis as well as acute lung injury (ALI) and its consequent acute respiratory distress syndrome (ARDS). We further summarize current knowledge about PI3K inhibitors for pulmonary fibrosis treatment, including drugs under development as well as in clinical trials. We finally discuss how the design of inhaled compounds targeting the PI3K pathways might potentiate efficacy and improve tolerability.

PI3K Signaling in Mechanisms and Treatments of Pulmonary Fibrosis Following Sepsis and Acute Lung Injury

Pulmonary fibrosis is a pathological fibrotic process affecting the lungs of five million people worldwide. The incidence rate will increase even more in the next years due to the long-COVID-19 syndrome, but a resolving treatment is not available yet and usually prognosis is poor. The emerging role of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling in fibrotic processes has inspired the testing of drugs targeting the PI3K/Akt pathway that are currently under clinical evaluation. This review highlights the progress in understanding the role of PI3K/Akt in the development of lung fibrosis and its causative pathological context, including sepsis as well as acute lung injury (ALI) and its consequent acute respiratory distress syndrome (ARDS). We further summarize current knowledge about PI3K inhibitors for pulmonary fibrosis treatment, including drugs under development as well as in clinical trials. We finally discuss how the design of inhaled compounds targeting the PI3K pathways might potentiate efficacy and improve tolerability.

Gasdermin D inhibition prevents multiple organ dysfunction during sepsis by blocking NET formation.

Multiple organ dysfunction is the most severe outcome of sepsis progression and is highly correlated with a worse prognosis. Excessive neutrophil extracellular traps (NETs) are critical players in the development of organ failure during sepsis. Therefore, interventions targeting NET release would likely effectively prevent NET-based organ injury associated with this disease. Herein, we demonstrate that the pore-forming protein gasdermin D (GSDMD) is active in neutrophils from septic humans and mice and plays a crucial role in NET release. Inhibition of GSDMD with disulfiram or genic deletion abrogated NET formation, reducing multiple organ dysfunction and sepsis lethality. Mechanistically, we demonstrate that during sepsis, activation of the caspase-11/GSDMD pathway controls NET release by neutrophils during sepsis. In summary, our findings uncover a novel therapeutic use for disulfiram and suggest that GSDMD is a therapeutic target to improve sepsis treatment.

SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 pathology.

Severe COVID-19 patients develop acute respiratory distress syndrome that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that neutrophil extracellular traps (NETs) have been described as important mediators of tissue damage in inflammatory diseases, we investigated whether NETs would be involved in COVID-19 pathophysiology. A cohort of 32 hospitalized patients with a confirmed diagnosis of COVID-19 and healthy controls were enrolled. The concentration of NETs was augmented in plasma, tracheal aspirate, and lung autopsies tissues from COVID-19 patients, and their neutrophils released higher levels of NETs. Notably, we found that viable SARS-CoV-2 can directly induce the release of NETs by healthy neutrophils. Mechanistically, NETs triggered by SARS-CoV-2 depend on angiotensin-converting enzyme 2, serine protease, virus replication, and PAD-4. Finally, NETs released by SARS-CoV-2-activated neutrophils promote lung epithelial cell death in vitro. These results unravel a possible detrimental role of NETs in the pathophysiology of COVID-19. Therefore, the inhibition of NETs represents a potential therapeutic target for COVID-19.