Rossana C. N. Melo

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.

Roles and origins of leukocyte lipid bodies: proteomic and ultrastructural studies

Lipid bodies (LBs), multifunctional organelles present in most eukaryotic cells, are sites of eicosanoid formation in leukocytes; but little is known about the composition of leukocyte LBs or the biogenesis and internal structures of LBs from mammalian cells. Proteomic analyses of LBs purified from human monocytic U937 cells detected, common to LBs in other cells, proteins involved in cholesterol and triglyceride metabolism, Rab GTPases, and many membrane and endoplasmic reticulum (ER)‐associated proteins. Newly lipid body (LB)‐associated proteins included MRP‐14, potentially involved in arachidonate transport, and ribosomal subunit proteins and translation regulatory proteins. Ultrastructurally, in U937 cells as well as human neutrophils and eosinophils, ribosomes are attached to and distributed within LBs, and LBs contain extensive ER‐like membranes. The presence of ribosomes, ER‐like membranes and many membrane‐associated and ER luminal proteins within LBs, supports a new model by which enveloped ER‐membranes and domains form LBs and indicates that LBs may be sites of protein synthesis. Wan, H‐C., Melo, R. C. N., Jin, Z., Dvorak, A. M., Weller, P. F. Roles and origins of leukocyte lipid bodies: proteomic and ultrastructural studies. FASEB J. 21, 167–178 (2007)

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.

Mechanisms of eosinophil secretion: large vesiculotubular carriers mediate transport and release of granule-derived cytokines and other proteins

Eosinophils generate and store a battery of proteins, including classical cationic proteins, cytokines, chemokines, and growth factors. Rapid secretion of these active mediators by eosinophils is central to a range of inflammatory and immunoregulatory responses. Eosinophil products are packaged within a dominant population of cytoplasmic specific granules and generally secreted by piecemeal degranulation, a process mediated by transport vesicles. Large, pleiomorphic vesiculotubular carriers were identified recently as key players for moving eosinophil proteins from granules to the plasma membrane for extracellular release. During secretion, these specialized, morphologically distinct carriers, termed eosinophil sombrero vesicles, are actively formed and direct differential and rapid release of eosinophil proteins. This review highlights recent discoveries concerning the organization of the human eosinophil secretory pathway. These discoveries are defining a broader role for large vesiculotubular carriers in the intracellular trafficking and secretion of proteins, including selective receptor‐mediated mobilization and transport of cytokines.

Modulation of lipid droplets by Mycobacterium leprae in Schwann cells: a putative mechanism for host lipid acquisition and bacterial survival in phagosomes

The predilection of Mycobacterium leprae (ML) for Schwann cells (SCs) leads to peripheral neuropathy, a major concern in leprosy. Highly infected SCs in lepromatous leprosy nerves show a foamy, lipid‐laden appearance; but the origin and nature of these lipids, as well as their role in leprosy, have remained unclear. The data presented show that ML has a pronounced effect on host‐cell lipid homeostasis through regulation of lipid droplet (lipid bodies, LD) biogenesis and intracellular distribution. Electron microscopy and immunohistochemical analysis of lepromatous leprosy nerves for adipose differentiation‐related protein expression, a classical LD marker, revealed accumulating LDs in close association to ML in infected SCs. The capacity of ML to induce LD formation was confirmed in in vitro studies with human SCs. Moreover, via confocal and live‐cell analysis, it was found that LDs are promptly recruited to bacterial phagosomes and that this process depends on cytoskeletal reorganization and PI3K signalling. ML‐induced LD biogenesis and recruitment were found to be independent of TLR2 bacterial sensing. Notably, LD recruitment impairment by cytoskeleton drugs decreased intracellular bacterial survival. Altogether, our data revealed SC lipid accumulation in ML‐containing phagosomes, which may represent a fundamental aspect of bacterial pathogenesis in the nerve.

Macrophage lipid body induction by Chagas disease in vivo: putative intracellular domains for eicosanoid formation during infection

Lipid bodies (LB), lipid-rich inclusions abundantly present in cells engaged in inflammation, are specialized intracellular domains involved in generating inflammatory mediators, the eicosanoids. Since the acute Trypanosoma cruzi infection triggers a potent inflammatory reaction characterized by a great increase of peripheral blood monocyte (PBM) and macrophage numbers, we investigated the LB occurrence in these cells. The experimental rat infection by T. cruzi (Y strain) induced significant increase of the LB numbers in peritoneal macrophages at day 6 and 12, accompanied by significant enhancement of Prostaglandin E(2) (PGE(2)) production, as measured by EIA. At day 12, ultrastructural analysis of the heart, a target organ of the disease, showed numerous macrophages with LB prominently increased in number (mean of 8.3 per section view, range of 1-25) compared to controls (mean of 2.6 per section view, range of 0-3) and size. PBM from all groups rarely showed LB. Our results demonstrate, for the first time, that T. cruzi infection in rats elicits important LB formation in inflammatory macrophages but not in PBM. The increase in LB numbers during infection positively correlates with increased generation of PGE(2), suggesting that LB may have a role in the heightened eicosanoid production observed during T. cruzi infection.

Eosinophil granules function extracellularly as receptor-mediated secretory organelles

Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-γ, and G protein–coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.

Human Eosinophils Secrete Preformed, Granule-Stored Interleukin-4 Through Distinct Vesicular Compartments

Secretion of interleukin‐4 (IL‐4) by leukocytes is important for varied immune responses including allergic inflammation. Within eosinophils, unlike lymphocytes, IL‐4 is stored in granules (termed specific granules) and can be rapidly released by brefeldin A (BFA)‐inhibitable mechanisms upon stimulation with eotaxin, a chemokine that activates eosinophils. In studying eotaxin‐elicited IL‐4 secretion, we identified at the ultrastructural level distinct vesicular IL‐4 transport mechanisms. Interleukin‐4 traffics from granules via two vesicular compartments, large vesiculotubular carriers, which we term eosinophil sombrero vesicles (EoSV), and small classical spherical vesicles. These two vesicles may represent alternative pathways for transport to the plasma membrane. Loci of both secreted IL‐4 and IL‐4‐loaded vesicles were imaged at the plasma membranes by a novel EliCell assay using a fluoronanogold probe. Three dimensional electron tomographic reconstructions revealed EoSVs to be folded, flattened and elongated tubules with substantial membrane surfaces. As documented with quantitative electron microscopy, eotaxin‐induced significant formation of EoSVs while BFA pretreatment suppressed eotaxin‐elicited EoSVs. Electron tomography showed that both EoSVs and small vesicles interact with and arise from granules in response to stimulation. Thus, this intracellular vesicular system mediates the rapid mobilization and secretion of preformed IL‐4 by activated eosinophils. These findings, highlighting the participation of large tubular carriers, provide new insights into vesicular trafficking of cytokines.

Neutrophils recruited to the site of Mycobacterium bovis BCG infection undergo apoptosis and modulate lipid body biogenesis and prostaglandin E2 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 E2 (PGE2) and TGF‐β1 synthesis, occurred in parallel to macrophage uptake of apoptotic cells. Lipid body and PGE2 formation was observed after macrophage exposure to apoptotic, but not necrotic or live neutrophils. Blockage of BCG‐induced lipid body formation significantly inhibited PGE2 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‐β1 generation and PGE2‐derived lipid body formation, and may have modulator roles in mycobacterial pathogenesis.

Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans

Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane-enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets.