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Influenza A virus (IAV) causes a major public health concern, because it is one of the leading causes of respiratory tract infections and hospitalization. Severe influenza has been associated with the cytokine storm, and IAV productive infection cell death in airway epithelial cells may contribute to the exacerbation of this proinflammatory event. On the other hand, IAV replication in macrophages is non-permissive and whether this immune cell may contribute to severe influenza physiopathology requires more details. Here, we investigated IAV-induced macrophage death, along with potential therapeutic intervention. We found that IAV or simply its surface glycoprotein hemagglutinin (HA) triggers necroptosis in human and murine macrophages in a Toll-like receptor-4 (TLR4) and TNF-dependent manner. Anti-TNF treatment, with the clinically approved drug etanercept, prevented the engagement of the necroptotic loop and mice mortality. impaired IAV-induced pro-inflammatory cytokine storm and lung injury. Our results implicate macrophage necroptosis with severe influenza in experimental models and potentially repurpose a clinically available therapy.Author SummaryVarious fates of cell death have an integral role in the influenza A virus (IAV) pathogenesis and lung/respiratory dysfunction. IAV physiopathology is not restricted to airway epithelial cells, where this virus actively replicated. Macrophages should support both viral clearance and priming of adaptative immune response in patients that adequately control influenza. However, during severe IAV infection, macrophages – which are unable to support a permissive viral replication - undergo disruptive cell death and contribute to the exacerbated production of proinflammatory cytokines/chemokines. We characterized this process by showing that IAV or just its surface glycoprotein hemagglutinin (HA) trigger necroptosis, a disruptive and TNF-dependent cells death. Since TNF is a hallmark of pro-inflammatory cell death, we blocked this mediator with a repurposed biomedicine etanercept, which prevented the severe IAV infection in the experimental model. The present work improves the knowledge of influenza pathophysiology by highlighting the importance of macrophage cell death during severe infection.

Salmonellosis is a public health problem caused by Salmonella sp., a highly adapted facultative intracellular pathogen. After internalization, Salmonella sp. manipulates several host processes, mainly through the activation of the type III secretion system (T3SS), including modification of host lipid metabolism and lipid droplet (LD) accumulation. LDs are dynamic and complex lipid-rich organelles involved in several cellular processes. The present study investigated the mechanism involved in LD biogenesis in Salmonella-infected macrophages and its role in bacterial pathogenicity. Here, we reported that S. Typhimurium induced a rapid time-dependent increase of LD formation in macrophages. The LD biogenesis was demonstrated to depend on Salmonella's viability and SPI1-related T3SS activity, with the participation of Toll-Like Receptor (TLR) signaling. We also observed that LD accumulation occurs through TLR2 dependent signaling and is counter-regulated by TLR4. Lastly, the pharmacological modulation of LD formation by inhibiting diacylglycerol O-acyltransferase 1 (DGAT1) and cytosolic phospholipase A2 (cPLA2) significantly reduced the intracellular bacterial proliferation and impaired the prostaglandin E2 (PGE2 ) synthesis. Collectively, our data suggest the role of LDs on S. Typhimurium intracellular survival and replication in macrophages. This data set provides new perspectives for future investigations about LDs in host-pathogen interaction.

Introduction Lipid droplets (LDs) are dynamic and evolutionary conserved lipid-enriched organelles composed of a core of neutral lipids surrounded by a monolayer of phospholipids associated with a diverse array of proteins that are cell- and stimulus-regulated. Far beyond being simply a deposit of neutral lipids, accumulating evidence demonstrate that LDs act as spatial and temporal local for lipid and protein compartmentalization and signaling organization. Areas covered This review focuses on the progress in our understanding of LD protein diversity and LD functions in the context of cell signaling and immune responses, highlighting the relationship between LD composition with the multiple roles of this organelle in immunometabolism, inflammation and host-response to infection. Expert opinion LDs are essential platforms for various cellular processes, including metabolic regulation, cell signaling, and immune responses. The functions of LD in infection and inflammatory disease are associated with the dynamic and complexity of their proteome. Our contemporary view place LDs as critical regulators of different inflammatory and infectious diseases and key markers of leukocyte activation.

Anticoagulants are associated with clinical benefit against the 2019 coronavirus disease (COVID-19), preventing COVID-19 associated coagulopathy. Blood coagulation factor Xa (FXa) and SARS-CoV-2 major protease (Mpro) share over 80% homology at the three-dimensional protein level. Thus, it is worth interrogating whether there is crosstalk between inhibitors and substrates between these enzymes. Here, we found that the clinically-approved FXa inhibitor apixaban targets SARS-CoV-2 Mpro with a 21-fold higher potency than boceprevir (GC376). Apixaban displayed a non-competitive mechanism of inhibition towards Mpro, since it targets the enzyme/substrate complex and the allosteric site onto the viral protease. Enzymatic assays were further validated in infected Calu-3 cells, which reveal that apixaban decreases the production of infectious viral particles in a dose-dependent manner, with an inhibitory potency in the micromolar range. Our results are in line with the proposed early use of anticoagulants, including FXa inhibitors, to improve clinical outcome of COVID-19 patients. In this context, apixaban may display a dual mechanism of action by targeting FXa to prevent coagulopathy and, at some level, SARS-CoV-2 Mpro.

Repositioning of clinical approved drugs could represent the fastest way to identify therapeutic options during public health emergencies, the majority of drugs explored for repurposing as antivirals for 2019 coronavirus disease (COVID-19) have failed to demonstrate clinical benefit. Without specific antivirals, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to cause major global mortality. Antimalarial drugs, such as chloroquine (CQ)/hydroxychloroquine (HCQ) and mefloquine have emerged as potential anti-SARS-CoV-2 antivirals. CQ/HCQ entered the Solidarity and RECOVERY clinical trials against COVID-19 and showed lack of efficacy. Importantly, mefloquine is not a 4-aminoquinoline like CQ and HCQ and has been previously repurposed for other respiratory diseases. Unlike the 4-aminoquinolines that accumulate in the high pH of intracellular lysosomes of the lung, the high respiratory tract penetration of mefloquine is driven by its high lipophilicity. While CQ and HCQ exhibit activity in Vero E6 cells, their activity is obviated in TMPRSS2-expressing cells, such as Calu-3 cells, which more accurately recapitulate in vivo entry mechanisms for SARS-CoV-2. Accordingly, here we report the anti-SARS-CoV-2 activity of mefloquine in Calu-3 type II pneumocytes and primary human monocytes. Mefloquine inhibited SARS-CoV-2 replication in Calu-3 cells with low cytotoxicity and EC50and EC90values of 1.2 and 5.3 µM, respectively. In addition, mefloquine reduced up to 68% the SARS-CoV-2 RNA levels in infected monocytes, reducing viral-induced inflammation. Mefloquine blocked early steps of the SARS-CoV-2 replicative cycle and was less prone than CQ to induce drug-associated viral mutations and synergized with RNA polymerase inhibitor. The pharmacological parameters of mefloquine are consistent with its plasma exposure in humans and its tissue-to-plasma predicted coefficient points that this drug may accumulate in the lungs. These data indicate that mefloquine could represent an orally available clinically approved drug option against COVID-19 and should not be neglected on the basis of the failure of CQ and HCQ.

This review aims to describe and discuss the different functions of the endolysosomal system, from homeostasis to its vital role during viral infections. We will initially describe endolysosomal system’s main functions, presenting recent data on how its compartments are essential for host defense to explore later how SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) and other coronaviruses subvert these organelles for their benefit. It is clear that to succeed, pathogens’ evolution favored the establishment of ways to avoid, escape, or manipulate lysosomal function. The unavoidable coexistence with such an unfriendly milieu imposed on viruses the establishment of a vast array of strategies to make the most out of the invaded cell’s machinery to produce new viruses and maneuvers to escape the host’s defense system.

The immune system is a key component of tumorigenesis, with the latter promoting the development of cancer, its progression and metastasis. In fact, abundant infiltration of tumor-associated macrophages (TAM), which are M2-like macrophages, has been associated with a poor outcome in most types of cancers. Here, we show that lactate produced by murine melanoma B16F10 cells induces an M2-like profile in cultured macrophages. Further, we demonstrate that clotrimazole (CTZ), an off-target anti-tumor drug, abolishes lactate effects on the activation of macrophages and induces the expression of M1-like markers. We show that clotrimazole has cytotoxic effects on tumor cells by negatively modulating PI3K, which inhibits glycolytic metabolism and leads to a diminishing lactate production by these cells. These effects are more pronounced in cancer cells exposed to conditioned media of M2-polarized macrophages. Moreover, clotrimazole inhibits tumor growth in a murine model of implanted melanoma, reduces lactate content in a tumor microenvironment and decreases vascular endothelial growth factor expression. Finally, clotrimazole drastically diminishes TAM infiltration in the tumors, thereby inducing M1 polarization. Collectively, these findings identify a new antitumor mechanism of clotrimazole by modulating the tumor microenvironment (TME), particularly the activation and viability of TAM.

Sepsis is a complex life-threatening syndrome caused by dysregulated inflammatory and metabolic host response to infection. Alterations in lipid metabolism and an increased number of lipid droplets (LDs) are observed during sepsis; however LD involvement in maladaptive tissue tolerance that culminates in organ dysfunction during sepsis is poorly understood. Here we show that increased accumulation of LDs is associated with peroxidized lipid production, liver dysfunction and sepsis severity. Through the use of antioxidants or CRISPR/Cas9 deletion of the NADPH oxidase subunit p22phox we demonstrate that ROS induced LD biogenesis and oxidatively altered LD content. Moreover, the LDs associated with mitochondria and peridroplet mitochondria in cells from the septic mice showed increased ultrastructural damage. Strikingly, dampening LD accumulation by DGAT1 inhibition decreased the production of inflammatory mediators, reduced lipid peroxidation while improving tissue function. Altogether, we demonstrate that LDs contribute to sepsis-associated organ dysfunction by disrupting tissue tolerance through the amplification of lipid peroxidation, suggesting that LD and changes in lipid metabolism in sepsis may be a target for therapy.

Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40–150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes.