Pulmonary inflammation and viral replication define distinct clinical outcomes in fatal cases of COVID-19.

COVID-19 has affected more than half a billion people worldwide, with more than 6.3 million deaths, but the pathophysiological mechanisms involved in lethal cases and the host determinants that determine the different clinical outcomes are still unclear. In this study, we assessed lung autopsies of 47 COVID-19 patients and examined the inflammatory profiles, viral loads, and inflammasome activation. Additionally, we correlated these factors with the patient's clinical and histopathological conditions. Robust inflammasome activation was detected in the lungs of lethal cases of SARS-CoV-2. Experiments conducted on transgenic mice expressing hACE2 and infected with SARS-CoV-2 showed that Nlrp3-/- mice were protected from disease development and lethality compared to Nlrp3+/+ littermate mice, supporting the involvement of this inflammasome in disease exacerbation. An analysis of gene expression allowed for the classification of COVID-19 patients into two different clusters. Cluster 1 died with higher viral loads and exhibited a reduced inflammatory profile than Cluster 2. Illness time, mechanical ventilation time, pulmonary fibrosis, respiratory functions, histopathological status, thrombosis, viral loads, and inflammasome activation significantly differed between the two clusters. Our data demonstrated two distinct profiles in lethal cases of COVID-19, thus indicating that the balance of viral replication and inflammasome-mediated pulmonary inflammation led to different clinical outcomes. We provide important information to understand clinical variations in severe COVID-19, a process that is critical for decisions between immune-mediated or antiviral-mediated therapies for the treatment of critical cases of COVID-19.

CASP4/11 Contributes to NLRP3 Activation and COVID-19 Exacerbation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection triggers activation of the NLRP3 inflammasome, which promotes inflammation and aggravates severe COVID-19. Here, we report that SARS-CoV-2 induces upregulation and activation of human caspase-4/CASP4 (mouse caspase-11/CASP11), and this process contributes to NLRP3 activation. In vivo infections performed in transgenic hACE2 humanized mice, deficient or sufficient for Casp11, indicate that hACE2 Casp11-/- mice were protected from disease development, with the increased pulmonary parenchymal area, reduced clinical score of the disease, and reduced mortality. Assessing human samples from fatal cases of COVID-19, we found that CASP4 was expressed in patient lungs and correlated with the expression of inflammasome components and inflammatory mediators, including CASP1, IL1B, IL18, and IL6. Collectively, our data establish that CASP4/11 promotes NLRP3 activation and disease pathology, revealing a possible target for therapeutic interventions for COVID-19.

Identification of immunomodulatory drugs that inhibit multiple inflammasomes and impair SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces mild or asymptomatic COVID-19 in most cases, but some patients develop an excessive inflammatory process that can be fatal. As the NLRP3 inflammasome and additional inflammasomes are implicated in disease aggravation, drug repositioning to target inflammasomes emerges as a strategy to treat COVID-19. Here, we performed a high-throughput screening using a 2560 small-molecule compound library and identified FDA-approved drugs that function as pan-inflammasome inhibitors. Our best hit, niclosamide (NIC), effectively inhibits both inflammasome activation and SARS-CoV-2 replication. Mechanistically, induction of autophagy by NIC partially accounts for inhibition of NLRP3 and AIM2 inflammasomes, but NIC-mediated inhibition of NAIP/NLRC4 inflammasome are autophagy independent. NIC potently inhibited inflammasome activation in human monocytes infected in vitro, in PBMCs from patients with COVID-19, and in vivo in a mouse model of SARS-CoV-2 infection. This study provides relevant information regarding the immunomodulatory functions of this promising drug for COVID-19 treatment.

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.

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.

Targeting alpha-synuclein with a microRNA-embedded silencing vector in the rat substantia nigra: positive and negative effects.

BACKGROUND: Alpha-synuclein (SNCA) downregulation shows therapeutic potential for synucleinopathies, including Parkinson's disease (PD). Previously we showed that human (h)SNCA gene silencing using a short hairpin (sh)RNA in rat substantia nigra (SN) protects against a hSNCA-induced forelimb deficit, but not dopamine (DA) neuron loss. Furthermore, the shRNA increases cell death in vitro, but the same target sequence embedded in a microRNA30 transcript (mir30-hSNCA) does not. OBJECTIVE: Examine hSNCA gene silencing using mir30-hSNCA in vivo. METHODS: Rats were stereotaxically injected into one SN with adeno-associated virus serotype 2/8 (AAV)-hSNCA, AAV-hSNCA plus AAV-mir30-SNCA or AAV-hSNCA plus a control non-silencing mir30-embedded siRNA and DA neuron markers and associated behavior were examined. RESULTS: AAV2/8-mediated SN hSNCA expression induces a forelimb deficit and tyrosine hydroxylase-immunoreactive (TH-IR) neuron loss. hSNCA gene silencing using mir30-hSNCA protects against this forelimb deficit at 2 m and ameliorates TH-IR neuron loss. Striatal (ST) TH-IR fiber density and DA markers, assessed by western blot, are unaffected by AAV-hSNCA alone. Co-expression of either silencing vector reduces ST TH-IR fibers, panTH in SN and Ser40 phosphorylated TH in SN and ST, but does not affect vesicular monoamine transporter-2. However, hSNCA gene silencing promotes partial TH-IR fiber recovery by 2 m. Co-expression of either silencing vector also induces SN inflammation, although some recovery was observed by 2 m in hSNCA-silenced SN. CONCLUSION: hSNCA gene silencing with AAV-mir30-hSNCA has positive effects on forelimb behavior and SN DA neurons, which are compromised by inflammation and reduced TH expression, suggesting that AAV2/8-mir30-hSNCA-mediated gene silencing, although promising in vitro, is not a candidate for therapeutic translation for PD.