Targeting Neutrophils Extracellular Traps (NETs) reduces multiple organ injury in a COVID-19 mouse model

COVID-19 is characterized by severe acute lung injury, which is associated with neutrophils infiltration and release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment. Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potential deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I. Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.

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

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. Using a single-cell transcriptome analysis we observed that the expression of GSDMD and inflammasome-related genes were increased in neutrophils from COVID-19 patients. Furthermore, high expression of GSDMD was found associated with NETs structures in the lung tissue of COVID-19 patients. The activation of GSDMD in neutrophils requires live SARS-CoV-2 and occurs after neutrophil infection via ACE2 receptors and serine protease TMPRSS2. In a mouse model of SARS-CoV-2 infection, the treatment with GSDMD inhibitor (disulfiram) reduced NETs release and organ damage. These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology, and suggests that GSDMD inhibitors, can be useful to COVID-19 treatment. In BriefHere, we showed that the activation of the Gasdermin-D (GSDMD) pathway in neutrophils controls NET release during COVID-19. The inhibition of GSDMD with disulfiram, abrogated NET formation reducing lung inflammation and tissue damage. These findings suggest GSDMD as a target for improving the COVID-19 therapy.

Vandetanib Reduces Inflammatory Cytokines and Ameliorates COVID-19 in Infected Mice

The portfolio of SARS-CoV-2 small molecule drugs is currently limited to a handful that are either approved (remdesivir), emergency approved (dexamethasone, baricitinib) or in advanced clinical trials. We have tested 45 FDA-approved kinase inhibitors in vitro against murine hepatitis virus (MHV) as a model of SARS-CoV-2 replication and identified 12 showing inhibition in the delayed brain tumor (DBT) cell line. Vandetanib, which targets the vascular endothelial growth factor receptor (VEGFR), the epidermal growth factor receptor (EGFR), and the RET-tyrosine kinase showed the most promising results on inhibition versus toxic effect on SARS-CoV-2-infected Caco-2 and A549-hACE2 cells (IC50 0.79 M) while also showing a reduction of > 3 log TCID50/mL for HCoV-229E. The in vivo efficacy of vandetanib was assessed in a mouse model of SARS-CoV-2 infection and statistically significantly reduced the levels of IL-6, IL-10, TNF-, and mitigated inflammatory cell infiltrates in the lungs of infected animals but did not reduce viral load. Vandetanib rescued the decreased IFN-1{beta} caused by SARS-CoV-2 infection in mice to levels similar to that in uninfected animals. Our results indicate that the FDA-approved vandetanib is a potential therapeutic candidate for COVID-19 positioned for follow up in clinical trials either alone or in combination with other drugs to address the cytokine storm associated with this viral infection.

Pyronaridine Protects Against SARS-CoV-2 in Mouse

There are currently relatively few small-molecule antiviral drugs that are either approved or emergency approved for use against SARS-CoV-2. One of these is remdesivir, which was originally repurposed from its use against Ebola and functions by causing early RNA chain termination. We used this as justification to evaluate three molecules we had previously identified computationally with antiviral activity against Ebola and Marburg. Out of these we previously identified pyronaridine, which inhibited the SARS-CoV-2 replication in A549-ACE2 cells. Herein, the in vivo efficacy of pyronaridine has now been assessed in a K18-hACE transgenic mouse model of COVID-19. Pyronaridine treatment demonstrated a statistically significant reduction of viral load in the lungs of SARS CoV-2 infected mice. Furthermore, the pyronaridine treated group reduced lung pathology, which was also associated with significant reduction in the levels of pro-inflammatory cytokines/chemokine and cell infiltration. Notably, pyronaridine inhibited the viral PLpro activity in vitro (IC50 of 1.8 {micro}M) without any effect on Mpro, indicating a possible molecular mechanism involved in its ability to inhibit SARS-CoV-2 replication. Interestingly, pyronaridine also selectively inhibits the host kinase CAMK1 (IC50 of 2.4 {micro}M). We have also generated several pyronaridine analogs to assist in understanding the structure activity relationship for PLpro inhibition. Our results indicate that pyronaridine is a potential therapeutic candidate for COVID-19. One sentence summaryThere is currently intense interest in discovering small molecules with direct antiviral activity against the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Pyronaridine, an antiviral drug with in vitro activity against Ebola, Marburg and SARS-CoV-2 has now statistically significantly reduced the viral load in mice along with IL-6, TNF-, and IFN-{beta} ultimately demonstrating a protective effect against lung damage by infection to provide a new potential treatment for testing clinically.

Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory programming and continual 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-CoV2-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 suppress macrophage anti-inflammation and efficient tissue repair programs and provide mechanistic insights for the excessive production of pro-inflammatory cytokines and accumulation of tissue damage associated with COVID-19 immunopathogenesis.

Inflammasome activation in COVID-19 patients

Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1{beta} and IL-18. Although the participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease is unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and it is active in COVID-19, influencing the clinical outcome of the disease. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of post-mortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that the inflammasome is key in the pathophysiology of the disease, indicating this platform as a marker of disease severity and a potential therapeutic target for COVID-19.

Infection of human lymphomononuclear cells by SARS-CoV-2

Although SARS-CoV-2 severe infection is associated with a hyperinflammatory state, lymphopenia is an immunological hallmark, and correlates with poor prognosis in COVID-19. However, it remains unknown if circulating human lymphocytes and monocytes are susceptible to SARS-CoV-2 infection. In this study, SARS-CoV-2 infection of human peripheral blood mononuclear cells (PBMCs) was investigated both in vitro and in vivo. We found that in vitro infection of whole PBMCs from healthy donors was productive of virus progeny. Results revealed that monocytes, as well as B and T lymphocytes, are susceptible to SARS-CoV-2 active infection and viral replication was indicated by detection of double-stranded RNA. Moreover, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from COVID-19 patients, and less frequently in CD4+T lymphocytes. The rates of SARS-CoV-2-infected monocytes in PBMCs from COVID-19 patients increased over time from symptom onset. Additionally, SARS-CoV-2-positive monocytes and B and CD4+T lymphocytes were detected by immunohistochemistry in post mortem lung tissue. SARS-CoV-2 infection of blood circulating leukocytes in COVID-19 patients may have important implications for disease pathogenesis, immune dysfunction, and virus spread within the host.