Lipid body function in eicosanoid synthesis: an update

Eicosanoids (prostaglandins, leukotrienes and lipoxins) are signaling lipids derived from arachidonic acid metabolism that have important roles in physiological and pathological processes. Lately, intracellular compartmentalization of eicosanoid-synthetic machinery has emerged as a key component in the regulation of eicosanoid synthesis and functions. Over the past years substantial progresses have been made demonstrating that precursors and enzymes involved in eicosanoid synthesis localize at lipid bodies (also known as lipid droplets) and lipid bodies are distinct sites for eicosanoid generation. Here we will review the current knowledge on the functions of lipid bodies as specialized intracellular sites of compartmentalization of signaling with major roles in eicosanoid formation within cells engaged in inflammatory, infectious and neoplastic process.     

Leukocyte lipid bodies — Biogenesis and functions in inflammation

Lipid body accumulation within leukocytes is a common feature in both clinical and experimental infectious, neoplasic and other inflammatory conditions. Here, we will review the contemporary evidence related to the biogenesis and structure of leukocyte lipid bodies (also known as lipid droplets) as inflammatory organelles. Studies of leukocyte lipid bodies are providing functional, ultrastructural and protein compositional evidences that lipid bodies are not solely storage depots of neutral lipid. Over the past years substantial progresses have been made to demonstrate that lipid body biogenesis is a highly regulated process, that culminate in the compartmentalization of a specific set of proteins and lipids, that place leukocyte lipid bodies as inducible cytoplasmic organelles with roles in cell signaling and activation, regulation of lipid metabolism, membrane trafficking and control of the synthesis and secretion of inflammatory mediators. Pertinent to the roles of lipid bodies in inflammation and cell signaling, enzymes involved in eicosanoid synthesis are localized at lipid bodies and lipid bodies are sites for eicosanoid generation. Collectively, lipid bodies in leukocytes are emerging as critical regulators of different inflammatory diseases, key markers of leukocyte activation and attractive targets for novel anti-inflammatory therapies.     

Lipid droplets in inflammation and cancer

Accumulation of lipid droplets (also known as lipid bodies or adiposomes) within leukocytes, epithelial cells, hepatocytes and other non-adipocytic cells is a frequently observed phenotype in infectious, neoplastic and other inflammatory conditions. Lipid droplet biogenesis is a regulated cellular process that culminates in the compartmentalization of lipids and of an array of enzymes, protein kinases and other proteins, suggesting that lipid droplets are inducible organelles with roles in cell signaling, regulation of lipid metabolism, membrane trafficking and control of the synthesis and secretion of inflammatory mediators. Enzymes involved in eicosanoid synthesis are localized at lipid droplets and lipid droplets are sites for eicosanoid generation in cells during inflammation and cancer. In this review, we discuss the current evidence related to the biogenesis and function of lipid droplets in cell metabolism and signaling in inflammation and cancer. Moreover, the potential of lipid droplets as markers of disease and targets for novel anti-inflammatory and antineoplastic therapies will be discussed.     

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.     

Extranuclear lipid bodies, elicited by CCR3-mediated signaling pathways, are the sites of chemokine-enhanced leukotriene C4 production in eosinophils and basophils

Eosinophils and basophils, when activated, become major sources of cysteinyl leukotrienes, eicosanoid mediators pertinent to allergic inflammation. We show that the C-C chemokines, eotaxin and RANTES (regulated upon activation normal T cell expressed and secreted), activate eosinophils and basophils for enhanced leukotriene C(4) (LTC(4)) generation by distinct signaling and compartmentalization mechanisms involving the induced formation of new cytoplasmic lipid body organelles. Chemokine-induced lipid body formation and enhanced LTC(4) release were both mediated by CCR3 receptor G protein-linked downstream signaling involving activation of phosphoinositide 3-kinase, extracellular signal-regulated kinases 1 and 2, and p38 mitogen-activated protein kinases. Chemokine-elicited lipid body numbers correlated with increased calcium ionophore-stimulated LTC(4) production; and as demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid, lipid bodies were the predominant sites of LTC(4) synthesis in both chemokine-stimulated eosinophils and chemokine-primed and ionophore-activated eosinophils. Eotaxin and RANTES initiated signaling via phosphoinositide 3-kinase and mitogen-activated protein kinases both elicits the formation of lipid body domains and promotes LTC(4) formation at these specific extranuclear sites.     

Lipopolysaccharide-induced leukocyte lipid body formation in vivo: innate immunity elicited intracellular Loci involved in eicosanoid metabolism

Lipid bodies are rapidly inducible, specialized cytoplasmic domains for eicosanoid-forming enzyme localization, which we hypothesize to have specific roles in enhanced inflammatory mediator production during pathological conditions, including sepsis. However, little is known about the origins, composition, or functions of lipid bodies in vivo. We show that lipid body numbers were increased in leukocytes from septic patients in comparison with healthy subjects. Analogously, the intrathoracic administration of LPS into mice induced a dose- and time-dependent increase in lipid body numbers. Pretreatment with anti-CD14 or anti-CD11b/CD18 mAb drastically inhibited LPS-induced lipid body formation. Moreover, LPS failed to form lipid bodies in C3H/HeJ (TLR4 mutated) mice, demonstrating a requisite role for LPS receptors in lipid body formation. LPS-induced lipid body formation was also inhibited by the platelet-activating factor-receptor antagonists, suggesting a role for endogenous platelet-activating factor. The eicosanoid-forming enzymes, 5-lipoxygenase and cyclooxygenase-2, were immunolocalized within experimentally induced (LPS in mice) or naturally occurring (septic patients) lipid bodies. The proinflammatory cytokine involved in the pathogenesis of sepsis, TNF-alpha, was also shown to colocalize within lipid bodies. Prior stimulation of leukocytes to form lipid bodies enhanced the capacity of leukocytes to produce leukotriene B(4) and PGE(2). In conclusion, our studies indicate that lipid bodies formed after LPS stimulation and sepsis are sites for eicosanoid-forming enzymes and cytokine localization and may develop and function as structurally distinct, intracellular sites for paracrine eicosanoid synthesis during inflammatory conditions.     

Monocyte chemoattractant protein-1/CC chemokine ligand 2 controls microtubule-driven biogenesis and leukotriene B4-synthesizing function of macrophage lipid bodies elicited by innate immune response

Lipid bodies (also known as lipid droplets) are emerging as inflammatory organelles with roles in the innate immune response to infections and inflammatory processes. In this study, we identified MCP-1 as a key endogenous mediator of lipid body biogenesis in infection-driven inflammatory disorders and we described the cellular mechanisms and signaling pathways involved in the ability of MCP-1 to regulate the biogenesis and leukotriene B4 (LTB4) synthetic function of lipid bodies. In vivo assays in MCP-1-/- mice revealed that endogenous MCP-1 produced during polymicrobial infection or LPS-driven inflammatory responses has a critical role on the activation of lipid body-assembling machinery, as well as on empowering enzymatically these newly formed lipid bodies with LTB4 synthetic function within macrophages. MCP-1 triggered directly the rapid biogenesis of distinctive LTB4-synthesizing lipid bodies via CCR2-driven ERK- and PI3K-dependent intracellular signaling in in vitro-stimulated macrophages. Disturbance of microtubule organization by microtubule-active drugs demonstrated that MCP-1-induced lipid body biogenesis also signals through a pathway dependent on microtubular dynamics. Besides biogenic process, microtubules control LTB4-synthesizing function of MCP-1-elicited lipid bodies, in part by regulating the compartmentalization of key proteins, as adipose differentiation-related protein and 5-lipoxygenase. Therefore, infection-elicited MCP-1, besides its known CCR2-driven chemotactic function, appears as a key activator of lipid body biogenic and functional machineries, signaling through a microtubule-dependent manner.     

Ultrastructural immunogold localization of prostaglandin endoperoxide synthase (cyclooxygenase) to non-membrane-bound cytoplasmic lipid bodies in human lung mast cells, alveolar macrophages, type II pneumocytes, and neutrophils.

Lipid bodies are non-membrane-bound, lipid-rich cytoplasmic inclusions that occur in many mammalian cell types. Because lipid bodies are more prominent in cells associated with inflammation and are repositories of arachidonyl-phospholipids, a role for lipid bodies in the oxidative metabolism of arachidonic acid to form eicosanoids has been suggested. To evaluate further whether lipid bodies, in addition to serving as non-membranous sources of substrate arachidonate, are involved in eicosanoid formation, we used cells isolated from human lung to investigate the intracellular localization of prostaglandin endoperoxide (PGH) synthase (cyclooxygenase), the key initial, rate-limiting enzyme in the formation of prostaglandins and thromboxanes. Isolated lung cells containing a mixture of mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils from short-term cultures were fixed in suspension in a dilute aldehyde mixture, post-fixed in osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in a graded series of alcohols, and embedded in Epon. A post-embedding immunogold procedure was used with a primary PGH synthase monoclonal antibody and 20-nm gold-conjugated secondary antibody to demonstrate enzyme locations. Specificity controls were also done. We found PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils. Specific secretory and lysosomal granules and plasma membranes did not express PGH synthase. Specificity controls, including omission of the primary antibody or substitution with an irrelevant antibody, were negative. Absorption of the specific PGH synthase antibody with purified solid-phase PGH synthase resulted in a marked reduction of label in lipid bodies of all four cell types. These findings establish the presence of PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils and, in concert with previous studies, suggest that these cytoplasmic lipid-rich organelles may be non-membrane sites of eicosanoid formation.     

Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism

Leptin is an adipocyte-derived hormone/cytokine that links nutritional status with neuroendocrine and immune functions. Lipid bodies (lipid droplets) are emerging as dynamic organelles with roles in lipid metabolism and inflammation. Here we investigated the roles of leptin in signaling pathways involved in cytoplasmic lipid body biogenesis and leukotriene B(4) synthesis in macrophages. Our results demonstrated that leptin directly activated macrophages and induced the formation of adipose differentiation-related protein-enriched lipid bodies. Newly formed lipid bodies were sites of 5-lipoxygenase localization and correlated with an enhanced capacity of leukotriene B(4) production. We demonstrated that leptin-induced macrophage activation was dependent on phosphatidylinositol 3-kinase (PI3K) activity, since the lipid body formation was inhibited by LY294002 and was absent in the PI3K knock-out mice. Leptin induces phosphorylation of p70(S6K) and 4EBP1 key downstream signaling intermediates of the mammalian target of rapamycin (mTOR) pathway in a rapamycin-sensitive mechanism. The mTOR inhibitor, rapamycin, inhibited leptin-induced lipid body formation, both in vivo and in vitro. In addition, rapamycin inhibited leptin-induced adipose differentiation-related protein accumulation in macrophages and lipid body-dependent leukotriene synthesis, demonstrating a key role for mTOR in lipid body biogenesis and function. Our results establish PI3K/mTOR as an important signaling pathway for leptin-induced cytoplasmic lipid body biogenesis and adipose differentiation-related protein accumulation. Furthermore, we demonstrate a previously unrecognized link between intracellular (mTOR) and systemic (leptin) nutrient sensors in macrophage lipid metabolism. Leptin-induced increased formation of cytoplasmic lipid bodies and enhanced inflammatory mediator production in macrophages may have implications for obesity-related cardiovascular diseases.     

S1P metabolism in cancer and other pathological conditions

Nearly two decades ago, the sphingolipid metabolite sphingosine 1-phosphate was discovered to function as a lipid mediator and regulator of cell proliferation. Since that time, sphingosine 1-phosphate has been shown to mediate a diverse array of fundamental biological processes including cell proliferation, migration, invasion, angiogenesis, vascular maturation and lymphocyte trafficking. Sphingosine 1-phosphate acts primarily via signaling through five ubiquitously expressed G protein-coupled receptors. Intracellular sphingosine 1-phosphate molecules are transported extracellularly and gain access to cognate receptors for autocrine and paracrine signaling and for signaling at distant sites reached through blood and lymphatic circulation systems. Intracellular pools of sphingosine 1-phosphate available for signaling are tightly regulated primarily by three enzymes: sphinosine kinase, S1P lyase and S1P phosphatase. Alterations in sphingosine 1-phosphate as well as the enzymes involved in its synthesis and catabolism have been observed in many types of malignancy. These enzymes are being evaluated for their role in mediating cancer formation and progression, as well as their potential to serve as targets of anti-cancer therapeutics. In this review, the impact of sphingosine 1-phosphate, its cognate receptors, and the enzymes of sphingosine 1-phosphate metabolism on cell survival, apoptosis, autophagy, cellular transformation, invasion, angiogenesis and hypoxia in relation to cancer biology and treatment are discussed.     

The Internal Architecture of Leukocyte Lipid Body Organelles Captured by Three-Dimensional Electron Microscopy Tomography

Lipid bodies (LBs), also known as lipid droplets, are complex organelles of all eukaryotic cells linked to a variety of biological functions as well as to the development of human diseases. In cells from the immune system, such as eosinophils, neutrophils and macrophages, LBs are rapidly formed in the cytoplasm in response to inflammatory and infectious diseases and are sites of synthesis of eicosanoid lipid mediators. However, little is known about the structural organization of these organelles. It is unclear whether leukocyte LBs contain a hydrophobic core of neutral lipids as found in lipid droplets from adipocytes and how diverse proteins, including enzymes involved in eicosanoid formation, incorporate into LBs. Here, leukocyte LB ultrastructure was studied in detail by conventional transmission electron microscopy (TEM), immunogold EM and electron tomography. By careful analysis of the two-dimensional ultrastructure of LBs from human blood eosinophils under different conditions, we identified membranous structures within LBs in both resting and activated cells. Cyclooxygenase, a membrane inserted protein that catalyzes the first step in prostaglandin synthesis, was localized throughout the internum of LBs. We used fully automated dual-axis electron tomography to study the three-dimensional architecture of LBs in high resolution. By tracking 4 nm-thick serial digital sections we found that leukocyte LBs enclose an intricate system of membranes within their “cores”. After computational reconstruction, we showed that these membranes are organized as a network of tubules which resemble the endoplasmic reticulum (ER). Our findings explain how membrane-bound proteins interact and are spatially arranged within LB “cores” and support a model for LB formation by incorporating cytoplasmic membranes of the ER, instead of the conventional view that LBs emerge from the ER leaflets. This is important to understand the functional capabilities of leukocyte LBs in health and during diverse diseases in which these organelles are functionally involved.     

Lipid bodies in oxidized LDL-induced foam cells are leukotriene-synthesizing organelles: a MCP-1/CCL2 regulated phenomenon

Lipid-laden foam macrophages are emerging as key players in early atherogenesis. Even though cytoplasmic lipid bodies (lipid droplets) are now recognized as organelles with cell functions beyond lipid storage, the mechanisms controlling lipid body biogenesis within macrophages and their additional functions in atherosclerosis are not completely elucidated. Here we studied oxLDL-elicited macrophage machinery involved in lipid body biogenesis as well as lipid body roles in leukotriene (LT) synthesis. Both in vivo and in vitro, oxLDL (but not native LDL) induced rapid assembly of cytoplasmic lipid bodies-bearing ADRP within mice macrophages. Such oxLDL-elicited foamy-like phenotype was a pertussis toxin-sensitive process that depended on a paracrine activity of endogenous MCP-1/CCL2 and activation of ERK. Pretreatment with neutralizing anti-MCP-1/CCL2 inhibited macrophage ADRP protein expression induced by oxLDL. By directly immuno-localizing leukotrienes at their sites of synthesis, we showed that oxLDL-induced newly formed lipid bodies function as active sites of LTB₄ and LTC₄ synthesis, since oxLDL-induced lipid bodies within foam macrophages compartmentalized the enzyme 5-lipoxygenase and five lipoxygenase-activating protein (FLAP) as well as newly formed LTB₄ and LTC₄. Consistent with MCP-1/CCL-2 role in ox-LDL-induced lipid body biogenesis, in CCR2 deficient mice both ox-LDL-induced lipid body assembly and LT release were reduced as compared to wild type mice. In conclusion, oxLDL-driven foam cells are enriched with leukotriene-synthesizing lipid bodies – specialized organelles whose biogenic process is mediated by MCP-1/CCL2-triggered CCR2 activation and ERK-dependent downstream signaling – that may amplify inflammatory mediator production in atherosclerosis.     

Phospholipase A(2) regulation of arachidonic acid mobilization

Phospholipase A(2) (PLA(2)) constitutes a growing superfamily of lipolytic enzymes, and to date, at least 19 distinct enzymes have been found in mammals. This class of enzymes has attracted considerable interest as a pharmacological target in view of its role in lipid signaling and its involvement in a variety of inflammatory conditions. PLA(2)s hydrolyze the sn-2 ester bond of cellular phospholipids, producing a free fatty acid and a lysophospholipid, both of which are lipid signaling molecules. The free fatty acid produced is frequently arachidonic acid (AA, 5,8,11,14-eicosatetraenoic acid), the precursor of the eicosanoid family of potent inflammatory mediators that includes prostaglandins, thromboxanes, leukotrienes and lipoxins. Multiple PLA(2) enzymes are active within and surrounding the cell and these enzymes have distinct, but interconnected roles in AA release.     

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 PGE₂, but dependent on G-coupled receptor-driven signaling through p38, PKC, and PI3K. Newly formed lipid bodies compartmentalized cytosolic phospholipase (cPL)A₂-α, while facilitated AA mobilization and synthesis of PGE₂ within epithelial cells. Thus, both lipid body-related events, including highly regulated biogenesis and functional assembly of cPLA₂-α-driven enhanced AA mobilization and PGE₂ production, may have key roles in epithelial cell-driven inflammatory functions, and may represent relevant therapeutic targets of epithelial pathologies.     

Ras signaling from plasma membrane and endomembrane microdomains

Ras proteins are compartmentalized by dynamic interactions with both plasma membrane microdomains and intracellular membranes. The mechanisms underlying Ras compartmentalization involve a series of protein/lipid, lipid/lipid and cytoskeleton interactions, resulting in the generation of discrete microdomains from which Ras operates. Segregation of Ras proteins to these different platforms regulates the formation of Ras signaling complexes and the generation of discrete signal outputs. This temporal and spatial modulation of Ras signal transduction provides a mechanism for the generation of different biological outcomes from different Ras isoforms, as well as flexibility in the signal output from a single activated isoform.     

Adhesion promotes transcellular leukotriene biosynthesis during neutrophil-glomerular endothelial cell interactions: inhibition by antibodies against CD18 and L-selectin.

Eicosanoid formation by transcellular routes can amplify the levels and types of lipid mediators within a local milieu. To evaluate the role of adhesion in this process, we assessed the influence of mAb against adhesion molecules on LTC4 generation by PMN-endothelial cell interaction. Transcellular LTC4 generation was initiated by addition of fMLP to coincubations of GM-CSF-primed PMN and TNF-activated endothelial cells cultured from kidney glomeruli. Both PMN-endothelial cell adhesion and transcellular LTC4 generation were inhibited by mAb against leukocyte L-selectin and CD18. These results indicate that cytokine-treated PMN and endothelial cells generate LTC4 via transcellular routes by receptor-triggered mechanisms. They suggest that adhesion promotes transcellular eicosanoid biosynthesis and that adhesion molecules may also be targets for blockade of transcellular biosynthesis of lipid mediators.     

Phospholipase A(2)

Considerable progress has been made in characterizing the individual participant enzymes and their relative contributions in the generation of eicosanoids, lipid mediators derived from arachidonic acid, such as prostaglandins and leukotrienes. However, the role of individual phospholipase (PL) A(2) enzymes in providing arachidonic acid to the downstream enzymes for eicosanoid generation in biologic processes has not been fully elucidated. In this review, we will provide an overview of the classification of the families of PLA(2) enzymes, their putative mechanisms of action, and their role(s) in eicosanoid generation and inflammation.     

Modulation of lipid and protein mediators of inflammation by cytosolic phospholipase A2alpha during experimental sepsis

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is one of the key enzymes in lipid mediator generation. It preferentially hydrolyzes arachidonoyl-phospholipid in response to cellular stimuli, liberating arachidonic acid, the shared precursor of PGs and leukotrienes. Mice with disruption of the cPLA(2)alpha gene exhibit a more than 80% decrease in the generation of these lipid mediators, leading to dramatic phenotypes in various models of inflammatory and allergic disease. In this study, we use the cecal ligation and puncture model of sepsis along with multiplex quantitation systems to explore interactions between eicosanoids and protein mediators. cPLA(2)alpha-deficient mice exhibited significantly less weight loss accompanied by decreased generation of PGs, leukotriene B(4), IL-6, and CCL2. Despite these differences, genetic ablation of cPLA(2)alpha did not provide any survival advantage. Unexpectedly, abundant production of 12-hydroxy-eicosatetraenoic acid, another arachidonic acid-derived lipid mediator, was found to be unaffected by disruption of the cPLA(2)alpha gene. Eicosanoid production preceded the production of cytokines. Eicosanoid modulation of IL-6 and CCL2 expression was suggested by scattergram analyses. These results provide in vivo evidence for the rapid generation of eicosanoids, regulatory role(s) for cPLA(2)alpha-derived lipid mediators on protein mediator production, and the existence of a robust cPLA(2)alpha-independent pathway(s) of eicosanoid generation.     

Rab7 controls lipid droplet-phagosome association during mycobacterial infection

Lipid droplets (LDs) are organelles that have multiple roles in inflammatory and infectious diseases. LD act as essential platforms for immunometabolic regulation, including as sites for lipid storage and metabolism, inflammatory lipid mediator production, and signaling pathway compartmentalization. Accumulating evidence indicates that intracellular pathogens may exploit host LDs as source of nutrients and as part of their strategy to promote immune evasion. Notably, numerous studies have demonstrated the interaction between LDs and pathogen-containing phagosomes. However, the mechanism involved in this phenomenon remains elusive. Here, we observed LDs and PLIN2 surrounding M. bovis BCG-containing phagosomes, which included observations of a bacillus cell surrounded by lipid content inside a phagosome and LAM from mycobacteria co-localizing with LDs; these results were suggestive of exchange of contents between these compartments. By using beads coated with M.tb lipids, we demonstrated that LD-phagosome associations are regulated through the mycobacterial cell wall components LAM and PIM. In addition, we demonstrated that Rab7 and RILP, but not Rab5, localizes to LDs of infected macrophages and observed the presence of Rab7 at the site of interaction with an infected phagosome. Moreover, treatment of macrophages with the Rab7 inhibitor CID1067700 significantly inhibited the association between LDs and LAM-coated beads. Altogether, our data demonstrate that LD-phagosome interactions are controlled by mycobacterial cell wall components and Rab7, which enables the exchange of contents between LDs and phagosomes and may represent a fundamental aspect of bacterial pathogenesis and immune evasion.