Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators.

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent pathogen responsible for the coronavirus disease 2019 (COVID-19). Since its emergence, the novel coronavirus has rapidly achieved pandemic proportions causing remarkably increased morbidity and mortality around the world. A hypercoagulability state has been reported as a major pathologic event in COVID-19, and thromboembolic complications listed among life-threatening complications of the disease. Platelets are chief effector cells of hemostasis and pathological thrombosis. However, the participation of platelets in the pathogenesis of COVID-19 remains elusive. This report demonstrates that increased platelet activation and platelet-monocyte aggregate formation are observed in severe COVID-19 patients, but not in patients presenting mild COVID-19 syndrome. In addition, exposure to plasma from severe COVID-19 patients increased the activation of control platelets ex vivo. In our cohort of COVID-19 patients admitted to the intensive care unit, platelet-monocyte interaction was strongly associated with tissue factor (TF) expression by the monocytes. Platelet activation and monocyte TF expression were associated with markers of coagulation exacerbation as fibrinogen and D-dimers, and were increased in patients requiring invasive mechanical ventilation or patients who evolved with in-hospital mortality. Finally, platelets from severe COVID-19 patients were able to induce TF expression ex vivo in monocytes from healthy volunteers, a phenomenon that was inhibited by platelet P-selectin neutralization or integrin αIIb/ß3 blocking with the aggregation inhibitor abciximab. Altogether, these data shed light on new pathological mechanisms involving platelet activation and platelet-dependent monocyte TF expression, which were associated with COVID-19 severity and mortality.

In vitro antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19.

BACKGROUND: Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. METHODS: SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19. RESULTS: Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 µM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. CONCLUSIONS: Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.

Eosinophils increase macrophage ability to control intracellular Leishmania amazonensis infection via PGD2 paracrine activity in vitro.

Clinical and experimental studies have described eosinophil infiltration in Leishmania amazonensis infection sites, positioning eosinophils strategically adjacent to the protozoan-infected macrophages in cutaneous leishmaniasis. Here, by co-culturing mouse eosinophils with L. amazonensis-infected macrophages, we studied the impact of eosinophils on macrophage ability to regulate intracellular L. amazonensis infection. Eosinophils prevented the increase in amastigote numbers within macrophages by a mechanism dependent on a paracrine activity mediated by eosinophil-derived prostaglandin (PG) D2 acting on DP2 receptors. Exogenous PGD2 mimicked eosinophil-mediated effect on managing L. amazonensis intracellular infection by macrophages and therefore may function as a complementary tool for therapeutic intervention in L. amazonensis-driven cutaneous leishmaniasis.

Repeated Allergen Exposure in A/J Mice Causes Steroid-Insensitive Asthma via a Defect in Glucocorticoid Receptor Bioavailability.

The importance of developing new animal models to assess the pathogenesis of glucocorticoid (GC)-insensitive asthma has been stressed. Because of the asthma-prone background of A/J mice, we hypothesized that asthma changes in these animals would be or become resistant to GCs under repeated exposures to an allergen. A/J mice were challenged with OVA for 2 or 4 consecutive d, starting on day 19 postsensitization. Oral dexamethasone or inhaled budesonide were given 1 h before challenge, and analyses were done 24 h after the last challenge. Airway hyperreactivity, leukocyte infiltration, tissue remodeling, and cytokine levels as well as phosphorylated GC receptor (p-GCR), p-GATA-3, p-p38, MAPK phosphatase-1 (MKP-1), and GC-induced leucine zipper (GILZ) levels were assessed. A/J mice subjected to two daily consecutive challenges reacted with airway hyperreactivity, subepithelial fibrosis, and marked accumulation of eosinophils in both bronchoalveolar lavage fluid and peribronchial space, all of which were clearly sensitive to dexamethasone and budesonide. Conversely, under four provocations, most of these changes were steroid resistant. A significant reduction in p-GCR/GCR ratio following 4- but not 2-d treatment was observed, as compared with untreated positive control. Accordingly, steroid efficacy to transactivate MKP-1 and GILZ and to downregulate p-p38, p-GATA-3 as well as proinflammatory cytokine levels was also seen after two but not four provocations. In conclusion, we report that repeated allergen exposure causes GC-insensitive asthma in A/J mice in a mechanism associated with decrease in GCR availability and subsequent loss of steroid capacity to modulate pivotal regulatory proteins, such as GATA-3, p-p38, MKP-1, and GILZ.

JM25-1, a Lidocaine Analog Combining Airway Relaxant and Antiinflammatory Properties: Implications for New Bronchospasm Therapy.

BACKGROUND: Inhaled lidocaine antagonized bronchospasm in animal models and patients, but adverse effects limited its efficacy. This study evaluated the antibronchospasm potential of the analog JM25-1, exploring in vitro mechanisms and translation to an animal model. METHODS: The effectiveness of JM25-1 was assessed in GH3 cells, rat tracheal rings, mouse lymphocytes, and human eosinophil systems in vitro, assessing changes in Na current, contraction, proliferation, and survival, respectively. Lung function and inflammatory changes were studied in ovalbumin-sensitized mice. RESULTS: The efficacy of JM25-1 was higher than lidocaine in inhibiting carbachol-induced and calcium-induced tracheal contractions (maximum effect inhibition at 1 mM [%]: 67 ± 10 [JM25-1] vs. 41 ± 11 [lidocaine] [P < 0.001] for carbachol; 100 ± 3 [JM25-1] vs. 36 ± 26 [lidocaine] [P < 0.001] for Ca; mean ± SD; n = 9 each) but lower in Na current (50% inhibitory concentration = 151.5, n = 8 vs. 0.2 mM; n = 5; P < 0.001). JM25-1 also inhibited eosinophil survival (dead cells [%]: 65 ± 6; n = 4; P < 0.001 at 1 mM) and lymphocyte proliferation (cells in phase S + G2 [%]: 94 ± 10; n = 6; P < 0.001) at 0.6 mM. Aerosolized JM25-1 (1%) decreased lung eosinophil numbers from 13.2 ± 2.4 to 1.7 ± 0.7 × 10/µm (n = 6; P < 0.001) and neutrophils from 1.9 ± 0.4 to 0.2 ± 0.1 × 10/µm (n = 7; P < 0.001). Other parameters, including airway hyperreactivity, cytokines, mucus, and extracellular matrix deposition, were also sensitive to aerosolized JM25-1. CONCLUSION: These findings highlight the potential of JM25-1, emphasizing its putative value in drug development for clinical conditions where there is bronchospasm.

Nebulized lidocaine prevents airway inflammation, peribronchial fibrosis, and mucus production in a murine model of asthma.

BACKGROUND: Evidence suggests that nebulized lidocaine is beneficial in asthma therapy, but to what extent and the mechanisms underlying this effect remain poorly understood. The aim of this study was to assess the impact of lidocaine treatment using a murine model of allergic asthma characterized by expression of pivotal features of the disease: inflammation, mucus production, and lung remodeling. METHODS: A/J mice sensitized with ovalbumin were treated with inhaled lidocaine or vehicle immediately after ovalbumin intranasal challenges. Lung function, total and differential leukocytes in bronchoalveolar lavage fluid, peribronchial eosinophil density, interleukin (IL)-4, IL-5 and eotaxin-1 levels, epithelial mucus, collagen, extracellular-matrix deposition, matrix metalloproteinase-9 activity, and GATA-3 expression were evaluated. Between five and eight animals per group were used. RESULTS: Inhaled lidocaine inhibited ovalbumin-induced airway hyperreactivity to methacholine, and accumulation of lymphocytes, neutrophils, and eosinophils in bronchoalveolar lavage fluid 24 h after the last allergen provocation. Lidocaine administration also prevented other pathophysiological changes triggered by ovalbumin in lung tissue, including peribronchial eosinophil and neutrophil infiltration, subepithelial fibrosis, increased content of collagen and mucus, matrix metalloproteinase-9 activity, and increased levels of IL-4, IL-5, IL-13, and eotaxin-1. Furthermore, inhaled lidocaine inhibited lung tissue GATA-3 expression in ovalbumin-challenged mice. We also demonstrated that lidocaine inhibited the expression of GATA-3 in ovalbumin-stimulated T cells in vitro. CONCLUSIONS: Inhaled lidocaine prevents eosinophilic inflammation, overproduction of mucus, and peribronchial fibrosis in a murine model of asthma, and impaired airway hyperreactivity, possibly by inhibiting allergen-evoked GATA-3 expression and the subsequent up-regulation of proinflammatory cytokines and chemokines.

VIP plasma levels associate with survival in severe COVID-19 patients, correlating with protective effects in SARS-CoV-2-infected cells.

Infection by SARS-CoV-2 may elicit uncontrolled and damaging inflammatory responses. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the inflammatory reaction. Here, we show that the plasma levels of the immunoregulatory neuropeptide VIP are elevated in patients with severe COVID-19, correlating with reduced inflammatory mediators and with survival on those patients. In vitro, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), highly similar neuropeptides, decreased the SARS-CoV-2 RNA content in human monocytes and viral production in lung epithelial cells, also reducing cell death. Both neuropeptides inhibited the production of proinflammatory mediators in lung epithelial cells and in monocytes. VIP and PACAP prevented in monocytes the SARS-CoV-2-induced activation of NF-kB and SREBP1 and SREBP2, transcriptions factors involved in proinflammatory reactions and lipid metabolism, respectively. They also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF-kB. Specific inhibition of NF-kB and SREBP1/2 reproduced the anti-inflammatory, antiviral, and cell death protection effects of VIP and PACAP. Our results support further clinical investigations of these neuropeptides against COVID-19.

Simvastatin Downregulates the SARS-CoV-2-Induced Inflammatory Response and Impairs Viral Infection Through Disruption of Lipid Rafts.

Coronavirus disease 2019 (COVID-19) is currently a worldwide emergency caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In observational clinical studies, statins have been identified as beneficial to hospitalized patients with COVID-19. However, experimental evidence of underlying statins protection against SARS-CoV-2 remains elusive. Here we reported for the first-time experimental evidence of the protective effects of simvastatin treatment both in vitro and in vivo. We found that treatment with simvastatin significantly reduced the viral replication and lung damage in vivo, delaying SARS-CoV-2-associated physiopathology and mortality in the K18-hACE2-transgenic mice model. Moreover, simvastatin also downregulated the inflammation triggered by SARS-CoV-2 infection in pulmonary tissue and in human neutrophils, peripheral blood monocytes, and lung epithelial Calu-3 cells in vitro, showing its potential to modulate the inflammatory response both at the site of infection and systemically. Additionally, we also observed that simvastatin affected the course of SARS-CoV-2 infection through displacing ACE2 on cell membrane lipid rafts. In conclusion, our results show that simvastatin exhibits early protective effects on SARS-CoV-2 infection by inhibiting virus cell entry and inflammatory cytokine production, through mechanisms at least in part dependent on lipid rafts disruption.

SARS-CoV-2 engages inflammasome and pyroptosis in human primary monocytes.

Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with leukopenia and uncontrolled inflammatory response in critically ill patients. A better comprehension of SARS-CoV-2-induced monocyte death is essential for the identification of therapies capable to control the hyper-inflammation and reduce viral replication in patients with 2019 coronavirus disease (COVID-19). Here, we show that SARS-CoV-2 engages inflammasome and triggers pyroptosis in human monocytes, experimentally infected, and from patients under intensive care. Pyroptosis associated with caspase-1 activation, IL-1ß production, gasdermin D cleavage, and enhanced pro-inflammatory cytokine levels in human primary monocytes. At least in part, our results originally describe mechanisms by which monocytes, a central cellular component recruited from peripheral blood to respiratory tract, succumb to control severe COVID-19.