Lipid Metabolism Modulation during SARS-CoV-2 Infection: A Spotlight on Extracellular Vesicles and Therapeutic Prospects.

Extracellular vesicles (EVs) have a significant impact on the pathophysiological processes associated with various diseases such as tumors, inflammation, and infection. They exhibit molecular, biochemical, and entry control characteristics similar to viral infections. Viruses, on the other hand, depend on host metabolic machineries to fulfill their biosynthetic requirements. Due to potential advantages such as biocompatibility, biodegradation, and efficient immune activation, EVs have emerged as potential therapeutic targets against the SARS-CoV-2 infection. Studies on COVID-19 patients have shown that they frequently have dysregulated lipid profiles, which are associated with an increased risk of severe repercussions. Lipid droplets (LDs) serve as organelles with significant roles in lipid metabolism and energy homeostasis as well as having a wide range of functions in infections. The down-modulation of lipids, such as sphingolipid ceramide and eicosanoids, or of the transcriptional factors involved in lipogenesis seem to inhibit the viral multiplication, suggesting their involvement in the virus replication and pathogenesis as well as highlighting their potential as targets for drug development. Hence, this review focuses on the role of modulation of lipid metabolism and EVs in the mechanism of immune system evasion during SARS-CoV-2 infection and explores the therapeutic potential of EVs as well as application for delivering therapeutic substances to mitigate viral infections.

Lipid droplets as multifunctional organelles related to the mechanism of evasion during mycobacterial infection.

Tuberculosis (TB) is an infectious disease caused by the bacteria of the Mycobaterium tuberculosis (Mtb) complex. The modulation of the lipid metabolism has been implicated in the immune response regulation, including the formation of lipid droplets (LD)s, LD-phagosome association and eicosanoid synthesis. Mtb, M. bovis BCG and other pathogenic mycobacteria, as well as wall components, such as LAM, can induce LDs formation in a mechanism involving surface receptors, for instance TLRs, CD36, CD14, CD11b/CD18 and others. In addition, the activation of the lipid-activated nuclear receptor PPARγ is involved in the mechanisms of LD biogenesis, as well as in the modulation of the synthesis of lipid mediators. In infected cells, LDs are sites of compartmentalized prostaglandin E2 synthesis involved in macrophage deactivation, bacterial replication and regulation of the host cytokine profile. LDs also have a function in vesicle traffic during infection. Rab7 and RILP, but not Rab5, are located on LDs of infected macrophages, suggesting that LDs and phagosomes could exchange essential proteins for phagosomal maturation, interfering in mycobacterial survival. The pharmacological inhibition of LDs biogenesis affects the bacterial replication and the synthesis of lipid mediators and cytokines, suggesting that LDs may be new targets for antimicrobial therapies. However, it is still controversial if the accumulation of LDs favors the mycobacterial survival acting as an escape mechanism, or promotes the host resistance to infection. Thus, in this mini-review we discuss recent advances in understanding the important role of LDs in the course of infections and the implications for the pathophysiology of mycobacteriosis.

Accessing BCG in infected macrophages by antibody-mediated drug delivery system and tracking by surface-enhanced Raman scattering spectroscopy.

Gold nanoparticles (AuNP) modified with antibody and rifampicin (RP) were tested against Mycobacterium bovis Bacillus Calmette-Guérin (BCG), which previously generated in vitro infection of macrophages from mice. Such a drug delivery system works as nanocarrier for RP and presented lower toxicity for macrophages cells than each separated component. Surface-enhanced Raman scattering (SERS) spectroscopy and fluorescence microscopy were used as analytical tools for the characterization of the internalization of gold nanocarriers into macrophage cells. The effective antibiotic action of RP, when combined with gold nanocarrier, was confirmed by dead-live assay of BCG bacteria lysed from macrophages after incubation. Such results indicate the delivery of RP to BCG bacteria, which were infecting macrophages, occurred with remarkable efficiency. It was rationalized based on the strategy used for the adsorption of antibody molecules on gold surface.

Leptin Mediates In Vivo Neutrophil Migration: Involvement of Tumor Necrosis Factor-Alpha and CXCL1.

Leptin directly activates macrophages and lymphocytes, but the role of leptin in neutrophil activation and migration is still controversial. Here, we investigate the in vivo mechanisms of neutrophil migration induced by leptin. The intraperitoneal injection of leptin (1 mg/kg) induces a time- and concentration-dependent neutrophil influx. We did not observe the enhancement of lipid bodies/droplets in neutrophils, after leptin treatment, as we had observed previously in peritoneal macrophages. The participation of leukotriene B4 (LTB4) in neutrophil recruitment triggered by leptin was investigated using different strategies. Leptin-induced neutrophil recruitment occurs both in the absence of 5-lipoxygenase activity in 5-lipoxygenase (5-LO)-/- mice and after the administration of either 5-LO inhibitor (Zileuton) or the LTB4 receptor antagonist (U-75302). Moreover, no direct induction of LTB4 by leptin could be observed. Neutrophil influx could not be prevented by the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, contrasting with the leptin-induced signaling for lipid body formation in macrophage that is mTOR-dependent. Leptin administration led to tumor necrosis factor-alpha (TNFα) production by the peritoneal cells both in vivo and in vitro. In addition, neutrophil recruitment was inhibited in tumor necrosis factor receptor 1 (TNFR1-/-) mice, indicating a role for TNF in leptin-induced neutrophil recruitment to the peritoneal cavity. Leptin-induced neutrophil influx was PI3Kγ-dependent, as it was absent in PI3Kγ-/- mice. Accordingly, leptin induced the peritoneal cells to produce CXCL1, both in vivo and in vitro, and the neutrophil influx was ablated after using an antibody against CXCL1. Our results establish TNFα/TNFR1- and CXCL1-dependent signaling as important pathways for leptin-induced neutrophil migration in vivo.

Oleic acid induces lung injury in mice through activation of the ERK pathway.

Oleic acid (OA) can induce acute lung injury in experimental models. In the present work, we used intratracheal OA injection to show augmented oedema formation, cell migration and activation, lipid mediator, and cytokine productions in the bronchoalveolar fluids of Swiss Webster mice. We also demonstrated that OA-induced pulmonary injury is dependent on ERK1/2 activation, since U0126, an inhibitor of ERK1/2 phosphorylation, blocked neutrophil migration, oedema, and lipid body formation as well as IL-6, but not IL-1ß production. Using a mice strain carrying a null mutation for the TLR4 receptor, we proved that increased inflammatory parameters after OA challenges were not due to the activation of the TLR4 receptor. With OA being a Na/K-ATPase inhibitor, we suggest the possible involvement of this enzyme as an OA target triggering lung inflammation.

Lipid bodies: inflammatory organelles implicated in host-Trypanosoma cruzi interplay during innate immune responses.

The flagellated protozoa Trypanosoma cruzi is the causal agent of Chagas' disease, a significant public health issue and still a major cause of morbidity and mortality in Latin America. Acute Chagas' disease elicits a strong inflammatory response. In order to control the parasite multiplication, cells of the monocytic lineage are highly mobilized. Monocyte differentiation leads to the formation of phagocytosing macrophages, which are strongly activated and direct host defense. A distinguishing feature of Chagas' disease-triggered macrophages is the presence of increased numbers of distinct cytoplasmic organelles termed lipid bodies or lipid droplets. These organelles are actively formed in response to the parasite and are sites for synthesis and storage of inflammatory mediators. This review covers current knowledge on lipid bodies elicited by the acute Chagas' disease within inflammatory macrophages and discusses the role of these organelles in inflammation. The increased knowledge of lipid bodies in pathogenic mechanisms of infections may not only contribute to the understanding of pathogen-host interactions but may also identify new targets for intervention.

Host cell lipid bodies triggered by Trypanosoma cruzi infection and enhanced by the uptake of apoptotic cells are associated with prostaglandin E2 generation and increased parasite growth.

Lipid bodies (lipid droplets) are lipid-rich organelles with functions in cell metabolism and signaling. Here, we investigate the mechanisms of Trypanosoma cruzi-induced lipid body formation and their contributions to host-parasite interplay. We demonstrate that T. cruzi-induced lipid body formation in macrophages occurs in a Toll-like receptor 2-dependent mechanism and is potentiated by apoptotic cell uptake. Lipid body biogenesis and prostaglandin E2 (PGE2) production triggered by apoptotic cell uptake was largely dependent of α(v)ß3 and transforming growth factor-ß signaling. T. cruzi-induced lipid bodies act as sites of increased PGE synthesis. Inhibition of lipid body biogenesis by the fatty acid synthase inhibitor C75 reversed the effects of apoptotic cells on lipid body formation, eicosanoid synthesis, and parasite replication. Our findings indicate that lipid bodies are highly regulated organelles during T. cruzi infection with roles in lipid mediator generation by macrophages and are potentially involved in T. cruzi-triggered escape mechanisms.

Lipid bodies in inflammatory cells: structure, function, and current imaging techniques.

Lipid bodies (LBs), also known as lipid droplets, have increasingly been recognized as functionally active organelles linked to diverse biological functions and human diseases. These organelles are actively formed in vivo within cells from the immune system, such as macrophages, neutrophils, and eosinophils, in response to different inflammatory conditions and are sites for synthesis and storage of inflammatory mediators. In this review, the authors discuss structural and functional aspects of LBs and current imaging techniques to visualize these organelles in cells engaged in inflammatory processes, including infectious diseases. The dynamic morphological aspects of LBs in leukocytes as inducible, newly formable organelles, elicitable in response to stimuli that lead to cellular activation, contribute to the evolving understanding of LBs as organelles that are critical regulators of different inflammatory diseases, key markers of leukocyte activation, and attractive targets for novel anti-inflammatory therapies.

Lipid droplet formation in leprosy: Toll-like receptor-regulated organelles involved in eicosanoid formation and Mycobacterium leprae pathogenesis.

A hallmark of LL is the accumulation of Virchow's foamy macrophages. However, the origin and nature of these lipids, as well as their function and contribution to leprosy disease, remain unclear. We herein show that macrophages present in LL dermal lesions are highly positive for ADRP, suggesting that their foamy aspect is at least in part derived from LD (also known as lipid bodies) accumulation induced during ML infection. Indeed, the capacity of ML to induce LD formation was confirmed in vivo via an experimental model of mouse pleurisy and in in vitro studies with human peripheral monocytes and murine peritoneal macrophages. Furthermore, infected cells were shown to propagate LD induction to uninfected, neighboring cells by generating a paracrine signal, for which TLR2 and TLR6 were demonstrated to be essential. However, TLR2 and TLR6 deletions affected LD formation in bacterium-bearing cells only partially, suggesting the involvement of alternative receptors of the innate immune response besides TLR2/6 for ML recognition by macrophages. Finally, a direct correlation between LD formation and PGE(2) production was observed, indicating that ML-induced LDs constitute intracellular sites for eicosanoid synthesis and that foamy cells may be critical regulators in subverting the immune response in leprosy.

Cytosolic phospholipase A2-driven PGE2 synthesis within unsaturated fatty acids-induced lipid bodies of epithelial cells.

Cytoplasmic lipid bodies (also known as lipid droplets) are intracellular deposits of arachidonic acid (AA), which can be metabolized for eicosanoid generation. PGE2 is a major AA metabolite produced by epithelial cells and can modulate restoration of epithelium homeostasis after injury. We studied lipid body biogenesis and their role in AA metabolic pathway in an epithelial cell line derived from normal rat intestinal epithelium, IEC-6 cells. Lipid bodies were virtually absent in confluent IEC-6 cells. Stimulation of confluent IEC-6 cells with unsaturated fatty acids, including AA or oleic acid (OA), induced rapid lipid body assembly that was independent on its metabolism to PGE(2), but dependent on G-coupled receptor-driven signaling through p38, PKC, and PI3 K. Newly formed lipid bodies compartmentalized cytosolic phospholipase (cPL)A(2)-alpha, while facilitated AA mobilization and synthesis of PGE(2) within epithelial cells. Thus, both lipid body-related events, including highly regulated biogenesis and functional assembly of cPLA (2)-alpha-driven enhanced AA mobilization and PGE(2)production, may have key roles in epithelial cell-driven inflammatory functions, and may represent relevant therapeutic targets of epithelial pathologies.

Induction of autophagy correlates with increased parasite load of Leishmania amazonensis in BALB/c but not C57BL/6 macrophages.

We investigated the role of autophagy in infection of macrophages by Leishmania amazonensis. Induction of autophagy by IFN-gamma or starvation increased intracellular parasite load and the percentages of infected macrophages from BALB/c but not from C57BL/6 mice. In contrast, starvation did not affect the replication of either Leishmania major or Trypanosoma cruzi in BALB/c macrophages. In BALB/c macrophages, starvation resulted in increased monodansylcadaverine staining and in the appearance of double-membrane and myelin-like vesicles characteristic of autophagosomes. Increased parasite load was associated with a reduction in NO levels and was attenuated by wortmannin, an inhibitor of autophagy. In infected macrophages from BALB/c, but not from C57BL/6 mice, starvation increased the number of lipid bodies and the amounts of PGE(2) produced. Exogenous PGE(2) increased parasite load in macrophages from BALB/c, but not C57BL/6 mice. The cyclooxygenase inhibitor indomethacin prevented the increase of parasite load in starved BALB/c macrophages, and actually induced parasite killing. These results suggest that autophagy regulates the outcome of L. amazonensis infection in macrophages in a host strain specific manner.

Neutrophils recruited to the site of Mycobacterium bovis BCG infection undergo apoptosis and modulate lipid body biogenesis and prostaglandin E production by macrophages.

Neutrophil influx to sites of mycobacterial infections is one of the first events of tuberculosis pathogenesis. However, the role of early neutrophil recruitment in mycobacterial infection is not completely understood. We investigated the rate of neutrophil apoptosis and the role of macrophage uptake of apoptotic neutrophils in a pleural tuberculosis model induced by BCG. Recruited neutrophils were shown to phagocyte BCG and a large number of neutrophils undergo apoptosis within 24 h. Notably, the great majority of apoptotic neutrophils were infected by BCG. Increased lipid body (lipid droplets) formation, accompanied by prostaglandin E(2) (PGE(2)) and TGF-beta1 synthesis, occurred in parallel to macrophage uptake of apoptotic cells. Lipid body and PGE(2) formation was observed after macrophage exposure to apoptotic, but not necrotic or live neutrophils. Blockage of BCG-induced lipid body formation significantly inhibited PGE(2) synthesis. Pre-treatment with the pan-caspase inhibitor zVAD inhibited BCG-induced neutrophil apoptosis and lipid body formation, indicating a role for apoptotic neutrophils in macrophage lipid body biogenesis in infected mice. In conclusion, BCG infection induced activation and apoptosis of infected neutrophils at the inflammatory site. The uptake of apoptotic neutrophils by macrophages leads to TGF-beta1 generation and PGE(2)-derived lipid body formation, and may have modulator roles in mycobacterial pathogenesis.

Mycobacterium bovis bacillus Calmette-Guérin induces TLR2-mediated formation of lipid bodies: intracellular domains for eicosanoid synthesis in vivo.

Differentiation of macrophages into foamy (lipid-laden) macrophages is a common pathological observation in tuberculous granulomas both in experimental settings as well as in clinical conditions; however, the mechanisms that regulate intracellular lipid accumulation in the course of mycobacterial infection and their significance to pathophysiology of tuberculosis are not well understood. In this study, we investigated the mechanisms of formation and function of lipid-laden macrophages in a murine model of tuberculosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG), but not Mycobacterium smegmatis, induced a dose- and time-dependent increase in lipid body-inducible nonmembrane-bound cytoplasmic lipid domain size and numbers. Lipid body formation was drastically inhibited in TLR2-, but not in TLR4-deficient mice, indicating a role for TLR2 in BCG recognition and signaling to form lipid bodies. Increase in lipid bodies during infection correlated with increased generation of PGE2 and localization of cyclooxygenase-2 within lipid bodies. Moreover, we demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid that lipid bodies were the predominant sites of PGE2 synthesis in activated macrophages. Our findings demonstrated that BCG-induced lipid body formation is TLR2 mediated and these structures function as signaling platforms in inflammatory mediator production, because compartmentalization of substrate and key enzymes within lipid bodies has impact on the capacity of activated leukocytes to generate increased amounts of eicosanoids during experimental infection by BCG.

Turnover of neutrophils mediated by Fas ligand drives Leishmania major infection.

Apoptosis mediated by Fas ligand (FasL) initiates inflammation characterized by neutrophilic infiltration. Neutrophils undergo apoptosis and are ingested by macrophages. Clearance of dead neutrophils leads to prostaglandin- and transforming growth factor-beta-dependent replication of Leishmania major in macrophages from susceptible mice. How L. major induces neutrophil turnover in a physiological setting is unknown. We show that BALB/c FasL-sufficient mice are more susceptible to L. major infection than are FasL-deficient mice. FasL promotes the apoptosis of infected resident macrophages and attracts neutrophils. Furthermore, FasL-sufficient neutrophils exacerbate L. major replication in macrophages, whereas FasL-deficient neutrophils induce parasite killing. These contrasting effects are due to delaying apoptosis and the clearance of FasL-deficient neutrophils. The transfer of neutrophils exacerbates infection in FasL-sufficient mice but reduces infection in FasL-deficient mice. Depletion of neutrophils abolishes the susceptibility of FasL-sufficient mice. These data illustrate a deleterious role of the FasL-mediated turnover of neutrophils on L. major infection.

Morphological and biochemical characterization of macrophages activated by carrageenan and lipopolysaccharide in vivo.

Macrophages are able to recognize, internalize and destroy a large number of pathogens, thus restricting the infection until adaptive immunity is initiated. In this work our aim was to analyze the surface charge of cells activated by carrageenan (CAR) and lipopolysaccharide (LPS) through light and electron microscopy approaches as well as the release of inflammatory mediators in vitro. The ultrastuctural analysis and the light microscopy data showed that in vivo administration of CAR represents a potent inflammatory stimulation for macrophages leading to a high degree of spreading, an increase in their size, in the number of the intracellular vacuoles and membrane projections as compared to the macrophages collected from untreated animals as well as mice submitted to LPS. Our data demonstrated that CAR stimulated-macrophages displayed a remarkable increase in nitric oxide production and PGE2 release as compared to the cells collected from non-stimulated and stimulated mice with LPS in vivo. On the other hand, non-stimulated macrophages as well as macrophages stimulated by LPS produce almost the same quantities of TNF-alpha, while in vivo stimulation by CAR leads to a 30-40% increase of cytokine release in vitro compared to the other groups. In conclusion, our morphological and biochemical data clearly showed that in vivo stimulation with CAR induces a potent inflammatory response in macrophages representing an interesting model to analyze inflammatory responses.

Production of hydrogen peroxide by peripheral blood monocytes and specific macrophages during experimental infection with Trypanosoma cruzi in vivo.

Acute Chagas' disease triggers potent inflammatory reaction characterized by great increase of peripheral blood monocyte (PBM) and macrophage numbers. We studied the respiratory burst responses of PBM and peritoneal and splenic macrophages to in vivo infection (rats). The ultrastructure of heart inflammatory macrophages was also investigated. The infection increased the hydrogen peroxide (H2O2) production by PBM and splenic macrophages but not by peritoneal macrophages. Accordingly, the PBM and spleen cell numbers increased but the total number of peritoneal cells was similar to controls. Heart macrophages of infected rats exhibited increase (number and size) and activated morphology in parallel to high cardiomyocyte parasitism. Our data highlight the importance of innate immunity and H2O2production to host resistance during acute phase of T. cruzi infection. A novel finding is that H2O2production seems related to specific types of monocytes/macrophages that are able to release this agent when in presence of high parasite load.