Mitochondrial DNA and TLR9 activation contribute to SARS-CoV-2-induced endothelial cell damage.

BACKGROUND AND PURPOSE: Mitochondria play a central role in the host response to viral infection and immunity, being key to antiviral signaling and exacerbating inflammatory processes. Mitochondria and Toll-like receptor (TLR) have been suggested as potential targets in SARS-CoV-2 infection. However, the involvement of TLR9 in SARS-Cov-2-induced endothelial dysfunction and potential contribution to cardiovascular complications in COVID-19 have not been demonstrated. This study determined whether infection of endothelial cells by SARS-CoV-2 affects mitochondrial function and induces mitochondrial DNA (mtDNA) release. We also questioned whether TLR9 signaling mediates the inflammatory responses induced by SARS-CoV-2 in endothelial cells. EXPERIMENTAL APPROACH: Human umbilical vein endothelial cells (HUVECs) were infected by SARS-CoV-2 and immunofluorescence was used to confirm the infection. Mitochondrial function was analyzed by specific probes and mtDNA levels by real-time polymerase chain reaction (RT-PCR). Inflammatory markers were measured by ELISA, protein expression by western blot, intracellular calcium (Ca2+) by FLUOR-4, and vascular reactivity with a myography. KEY RESULTS: SARS-CoV-2 infected HUVECs, which express ACE2 and TMPRSS2 proteins, and promoted mitochondrial dysfunction, i.e. it increased mitochondria-derived superoxide anion, mitochondrial membrane potential, and mtDNA release, leading to activation of TLR9 and NF-kB, and release of cytokines. SARS-CoV-2 also decreased nitric oxide synthase (eNOS) expression and inhibited Ca2+ responses in endothelial cells. TLR9 blockade reduced SARS-CoV-2-induced IL-6 release and prevented decreased eNOS expression. mtDNA increased vascular reactivity to endothelin-1 (ET-1) in arteries from wild type, but not TLR9 knockout mice. These events were recapitulated in serum samples from COVID-19 patients, that exhibited increased levels of mtDNA compared to sex- and age-matched healthy subjects and patients with comorbidities. CONCLUSION AND APPLICATIONS: SARS-CoV-2 infection impairs mitochondrial function and activates TLR9 signaling in endothelial cells. TLR9 triggers inflammatory responses that lead to endothelial cell dysfunction, potentially contributing to the severity of symptoms in COVID-19. Targeting mitochondrial metabolic pathways may help to define novel therapeutic strategies for COVID-19.

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.

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

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.

Heparin prevents in vitro glycocalyx shedding induced by plasma from COVID-19 patients.

The severe forms and worsened outcomes of COVID-19 (coronavirus disease 19) are closely associated with hypertension and cardiovascular disease. Endothelial cells express Angiotensin-Converting Enzyme 2 (ACE2), which is the entrance door for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The hallmarks of severe illness caused by SARS-CoV-2 infection are increased levels of IL-6, C-reactive protein, D-dimer, ferritin, neutrophilia and lymphopenia, pulmonary intravascular coagulopathy and microthrombi of alveolar capillaries. The endothelial glycocalyx, a proteoglycan- and glycoprotein-rich layer covering the luminal side of endothelial cells, contributes to vascular homeostasis. It regulates vascular tonus and permeability, prevents thrombosis, and modulates leukocyte adhesion and inflammatory response. We hypothesized that cytokine production and reactive oxygen species (ROS) generation associated with COVID-19 leads to glycocalyx degradation. A cohort of 20 hospitalized patients with a confirmed COVID-19 diagnosis and healthy subjects were enrolled in this study. Mechanisms associated with glycocalyx degradation in COVID-19 were investigated. Increased plasma concentrations of IL-6 and IL1-ß, as well as increased lipid peroxidation and glycocalyx components were detected in plasma from COVID-19 patients compared to plasma from healthy subjects. Plasma from COVID-19 patients induced glycocalyx shedding in cultured human umbilical vein endothelial cells (HUVECs) and disrupted redox balance. Treatment of HUVECs with low molecular weight heparin inhibited the glycocalyx perturbation. In conclusion, plasma from COVID-19 patients promotes glycocalyx shedding and redox imbalance in endothelial cells, and heparin treatment potentially inhibits glycocalyx disruption.

Beneficial effects of colchicine for moderate to severe COVID-19: a randomised, double-blinded, placebo-controlled clinical trial.

OBJECTIVE: To evaluate whether the addition of colchicine to standard treatment for COVID-19 results in better outcomes. DESIGN: We present the results of a randomised, double-blinded, placebo-controlled clinical trial of colchicine for the treatment of moderate to severe COVID-19, with 75 patients allocated 1:1 from 11 April to 30 August 2020. Colchicine regimen was 0.5 mg thrice daily for 5 days, then 0.5 mg twice daily for 5 days. The primary endpoints were the need for supplemental oxygen, time of hospitalisation, need for admission and length of stay in intensive care unit and death rate. RESULTS: Seventy-two patients (36 for placebo and 36 for colchicine) completed the study. Median (and IQR) time of need for supplemental oxygen was 4.0 (2.0-6.0) days for the colchicine group and 6.5 (4.0-9.0) days for the placebo group (p<0.001). Median (IQR) time of hospitalisation was 7.0 (5.0-9.0) days for the colchicine group and 9.0 (7.0-12.0) days for the placebo group (p=0.003). At day 2, 67% versus 86% of patients maintained the need for supplemental oxygen, while at day 7, the values were 9% versus 42%, in the colchicine and the placebo groups, respectively (log rank; p=0.001). Two patients died, both in placebo group. Diarrhoea was more frequent in the colchicine group (p=0.26). CONCLUSION: Colchicine reduced the length of both, supplemental oxygen therapy and hospitalisation. The drug was safe and well tolerated. Once death was an uncommon event, it is not possible to ensure that colchicine reduced mortality of COVID-19. TRIAL REGISTRATION NUMBER: RBR-8jyhxh.

Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in 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-1ß, and IL-18. Although participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease are unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and is active in COVID-19 patients. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of postmortem 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 inflammasomes participate in the pathophysiology of the disease, indicating that these platforms might be a marker of disease severity and a potential therapeutic target for COVID-19.