Orientia tsutsugamushi in human scrub typhus eschars shows tropism for dendritic cells and monocytes rather than endothelium

Scrub typhus is a common and underdiagnosed cause of febrile illness in Southeast Asia, caused by infection with Orientia tsutsugamushi. Inoculation of the organism at a cutaneous mite bite site commonly results in formation of a localized pathological skin reaction termed an eschar. The site of development of the obligate intracellular bacteria within the eschar and the mechanisms of dissemination to cause systemic infection are unclear. Previous postmortem and in vitro reports demonstrated infection of endothelial cells, but recent pathophysiological investigations of typhus patients using surrogate markers of endothelial cell and leucocyte activation indicated a more prevalent host leucocyte than endothelial cell response in vivo. We therefore examined eschar skin biopsies from patients with scrub typhus to determine and characterize the phenotypes of host cells in vivo with intracellular infection by O. tsutsugamushi, using histology, immunohistochemistry, double immunofluorescence confocal laser scanning microscopy and electron microscopy. Immunophenotyping of host leucocytes infected with O. tsutsugamushi showed a tropism for host monocytes and dendritic cells, which were spatially related to different histological zones of the eschar. Infected leucocyte subsets were characterized by expression of HLADR+, with an "inflammatory" monocyte phenotype of CD14/LSP-1/CD68 positive or dendritic cell phenotype of CD1a/DCSIGN/S100/FXIIIa and CD163 positive staining, or occasional CD3 positive T-cells. Endothelial cell infection was rare, and histology did not indicate a widespread inflammatory vasculitis as the cause of the eschar. Infection of dendritic cells and activated inflammatory monocytes offers a potential route for dissemination of O. tsutsugamushi from the initial eschar site. This newly described cellular tropism for O. tsutsugamushi may influence its interaction with local host immune responses.     

The MAP1-LC3 conjugation system is involved in lipid droplet formation

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.     

Lipid droplet changes in proliferating and quiescent 3T3 fibroblasts

Lipid droplets (LDs) are fat-storing organelles present in virtually all eukaryotic cells and involved in many aspects of cell biology related to lipid metabolism and cholesterol homeostasis. In this study, we investigated the presence of LDs in proliferating and quiescent (contact-inhibited) 3T3 fibroblasts to verify a correlation with cell growth. LDs were characterized by Nile red staining, positivity to adipophilin and negativity to perilipin. LDs were numerous in proliferating cells, but very few in quiescent cells. However, the fraction of quiescent cells, which resumed proliferation after scratch-wound assay, also resumed the formation of LDs. In proliferating cells, the number of LDs correlated with the DNA content, suggesting a continuous accumulation of LDs during cell growth. These findings were supported by biochemical data showing much higher rates of cholesterol esterification and triglyceride synthesis in proliferating cells. Both filipin staining and the fluorescent cholesterol analog dehydroergosterol revealed the presence of an intense traffic of free cholesterol, mediated by acidic vesicles, in proliferating cells. Nile red ratiometric measurements revealed a different lipid composition of LDs in proliferating and quiescent cells. Changes in the number and composition of LDs were also found in growing cells treated with inhibitors of cholesterol esterification (Sandoz 58-035), endosomal cholesterol efflux (U18666A) and V-ATPase (bafilomycin-A1).     

LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation

The cytoplasmic lipid droplet (LD) is one of organelles that has a neutral lipid core with a single phospholipid layer. LDs are believed to be generated between the two leaflets of the endoplasmic reticulum (ER) membrane and to play various roles, such as high effective energy storage. However, it remains largely unknown how LDs are generated and grow in the cytoplasm. We have previously shown that the Atg conjugation system that is essential for autophagosome formation is involved in LD formation in hepatocytes and cardiac myocytes. We show here that LC3 itself is involved in LD formation by using RNA interference (RNAi). All cultured cell lines examined, in which the expression of LC3 was suppressed by RNAi, showed reduced LD formation. Triacylglycerol, a major component of LDs, was synthesized and degraded in LC3 mRNA-knockdown cells as well as in control cells. Interestingly, potential of the bulk protein degradation in the knockdown-cells was also evident in the control cells. These findings indicate that LC3 is involved in the LD formation regardless of the bulk degradation, and that LC3 has two pivotal roles in cellular homeostasis mediated by autophagy and lipid metabolism.     

Mycobacterium tuberculosis induces delayed lipid droplet accumulation in dendritic cells depending on bacterial viability and virulence

Tuberculosis remains a global health threat with high morbidity. Dendritic cells (DCs) participate in the acute and chronic inflammatory responses to Mycobacterium tuberculosis (Mtb) by directing the adaptive immune response and are present in lung granulomas. In macrophages, the interaction of lipid droplets (LDs) with mycobacteria-containing phagosomes is central to host-pathogen interactions. However, the data available for DCs are still a matter of debate. Here, we reported that bone marrow-derived DCs (BMDCs) were susceptible to Mtb infection and replication at similar rate to macrophages. Unlike macrophages, the analysis of gene expression showed that Mtb infection induced a delayed increase in lipid droplet-related genes and proinflammatory response. Hence, LD accumulation has been observed by high-content imaging in late periods. Infection of BMDCs with killed H37Rv demonstrated that LD accumulation depends on Mtb viability. Moreover, infection with the attenuated strains H37Ra and Mycobacterium bovis-BCG induced only an early transient increase in LDs, whereas virulent Mtb also induced delayed LD accumulation. In addition, infection with the BCG strain with the reintroduced virulence RD1 locus induced higher LD accumulation and bacterial replication when compared to parental BCG. Collectively, our data suggest that delayed LD accumulation in DCs is dependent on mycobacterial viability and virulence.     

Genetic typing of the 56-kDa type-specific antigen gene of contemporary Orientia tsutsugamushi isolates causing human scrub typhus at two sites in north-eastern and western Thailand

Orientia tsutsugamushi is the causative agent of scrub typhus, a major cause of febrile illness in the rural areas of Southeast Asia. Twenty-three strains of O. tsutsugamushi were isolated from patients with scrub typhus in north-east (Udorn Thani province) and western Thailand (Tak province) between 2003 and 2005. The isolates were characterized by sequencing the entire ORF of the 56-kDa-type-specific antigen gene, followed by phylogenetic analysis. The majority (15/23) of isolates clustered with the Karp-type strain, six with a Gilliam-type strain and one each with the TA716- and TA763-type strains. Overall, there was considerable diversity in sequence, comparable to that seen in strains from across the rest of the scrub typhus-endemic world. There was no significant difference in the distributions of strains between the two provinces (P=0.08, Fisher's exact) nor a temporal change in distribution with year of isolation (P=0.80, Fisher's exact). Within this diversity there were also examples of isolates with identical 56-kDa genotypes that were cultured from patients from the same geographical areas.     

Possible participation of mitochondria in lipid yolk formation in oocytes of paddlefish and sturgeon

The ovary of paddlefish and sturgeons (Acipenseriformes) is composed of discrete units: the ovarian nests and ovarian follicles. The ovarian nests comprise oogonia and numerous early dictyotene oocytes surrounded by somatic prefollicular cells. Each ovarian follicle consists of a spherical oocyte and a layer of follicular cells situated on a thick basal lamina, encompassed by thecal cells. The cytoplasm of previtellogenic oocytes is differentiated into two distinct zones: the homogeneous and granular zones. The homogeneous cytoplasm is organelle-free, whereas the granular cytoplasm contains numerous organelles, including mitochondria and lipid droplets. We have analyzed the cytoplasm of early dictyotene and previtellogenic oocytes ultrastructurally and histologically. In the cytoplasm of early dictyotene oocytes, two morphologically different types of mitochondria can be distinguished: (1) with well-developed cristae and (2) with distorted and fused cristae. In previtellogenic oocytes, the mitochondria of the second type show various stages of cristae distortion; they contain and release material morphologically similar to that of lipid droplets and eventually degenerate. This process of mitochondrial transformation is accompanied by an accumulation of lipid droplets that form a single large accumulation (lipid body) located in the vicinity of the oocyte nucleus (germinal vesicle). The lipid body eventually disperses in the oocyte center. The possible participation of these mitochondria in the formation of oocyte lipid droplets is discussed. This work was supported by funds from the research grant BW/IZ/2005 to M.Ż. An erratum to this article can be found at http://dx.doi.org/. An erratum to this article can be found at     

Function and structure of lipid storage droplet protein 1 studied in lipoprotein complexes

Triglycerides (TG) stored in lipid droplets (LDs) are the main energy reserve in all animals. The mechanism by which animals mobilize TG is complex and not fully understood. Several proteins surrounding the LDs have been implicated in TG homeostasis such as mammalian perilipin A and insect lipid storage proteins (Lsd). Most of the knowledge on LD-associated proteins comes from studies using cells or LDs leaving biochemical properties of these proteins uncharacterized. Here we describe the purification of recombinant Lsd1 and its reconstitution with lipids to form lipoprotein complexes suitable for functional and structural studies. Lsd1 in the lipid bound state is a predominately α-helical protein. Using lipoprotein complexes containing triolein it is shown that PKA mediated phosphorylation of Lsd1 promoted a 1.7-fold activation of the main fat body lipase demonstrating the direct link between Lsd1 phosphorylation and activation of lipolysis. Serine 20 was identified as the Lsd1-phosphorylation site triggering this effect.     

Importance of lipid metabolism for intracellular and mitochondrial membrane fusion/fission processes

Mitochondria move along cytoskeletal tracks, fuse and divide. These dynamic features have been shown to be critical for several mitochondrial functions in cell viability and cell death. After a rapid recall of the proteic machineries that are known to be involved, the review will focus on lipids, other key molecular actors of membrane dynamics. A summary of the current knowledge on lipids and their implication in various cellular membrane fusion/fission processes will be first presented. The review will then report what has been discovered or can be expected on the role of the different families of lipids in mitochondrial membrane fusion and fission processes.     

Adipose differentiation related protein induces lipid accumulation and lipid droplet formation in hepatic stellate cells

Summary The function of adipose differentiation-related protein (ADRP) is known to be the uptake of long-chain fatty acids and formation of lipid droplets in lipid-accumulating cells. We hypothesized that ADRP might stimulate activated hepatic stellate cells (HSCs) to accumulate lipids, resulting in their transition to the quiescent state. In this study, cultured HSCs in fifth passages isolated from rat were infected by adenovirus vector expressing ADRP (Ad.GFP-ADRP), and morphologic and functional changes were evaluated in comparison with control HSCs infected by recombinant adenovirus-expressing β-galactosidase (Ad. LacZ). In Ad. GFP-ADRP-infected cells only, many tiny lipid droplets were apparent in the cytoplasm, while the outline of the cells was not changed. The ADRP was detected around the lipid droplets. In HSCs with intracellular actin filaments, the staining pattern of the filaments before and after infection with Ad.GFP-ADRP or Ad.LacZ did not differ. The cell proliferation rate was not influenced by infection with Ad.LacZ or Ad.GFP-ADRP. Type I collagen secretion from cells overexpressing ADRP was not significantly different from that of Ad.LacZ-infected cells. In our in vitro study, ADRP overexpression induced the formation of cytoplasmic lipid droplets in activated HSCs but could not convert other characteristics of the activated form into those of the quiescent form.     

Leptin activation of mTOR pathway in intestinal epithelial cell triggers lipid droplet formation,cytokine production and increased cell proliferation

Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma.     

HDAC6 regulates lipid droplet turnover in response to nutrient deprivation via p62-mediated selective autophagy

Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively degraded through autophagy. Recently, the lipid storage organelle, lipid droplet(LD), has been reported to be the cargo of starvation-induced autophagy. However, it remains largely unknown how the autophagy machinery recognizes the LDs and whether it can selectively degrade LDs. In this study, we show that Drosophila histone deacetylase 6(dHDAC6), a key regulator of selective autophagy, is required for the LD turnover in the hepatocyte-like oenocytes in response to starvation. HDAC6 regulates LD turnover via p62/SQSTM1(sequestosome 1)-mediated aggresome formation, suggesting that the selective autophagy machinery is required for LD recognition and degradation. Furthermore, our results show that the loss of dHDAC6 causes steatosis in response to starvation. Our findings suggest that there is a potential link between selective autophagy and susceptible predisposition to lipid metabolism associated diseases in stress conditions.     

Lipid droplets as metabolic determinants for stemness and chemoresistance in cancer

Previously regarded as simple fat storage particles, new evidence suggests that lipid droplets (LDs) are dynamic and functional organelles involved in key cellular processes such as membrane biosynthesis, lipid metabolism, cell signalling and inflammation. Indeed, an increased LD content is one of the most apparent features resulting from lipid metabolism reprogramming necessary to support the basic functions of cancer cells. LDs have been associated to different cellular processes involved in cancer progression and aggressiveness, such as tumorigenicity, invasion and metastasis, as well as chemoresistance. Interestingly, all of these processes are controlled by a subpopulation of highly aggressive tumoral cells named cancer stem cells (CSCs), suggesting that LDs may be fundamental elements for stemness in cancer. Considering the key role of CSCs on chemoresistance and disease relapse, main factors of therapy failure, the design of novel therapeutic approaches targeting these cells may be the only chance for long-term survival in cancer patients. In this sense, their biology and functional properties render LDs excellent candidates for target discovery and design of combined therapeutic strategies. In this review, we summarise the current knowledge identifying LDs and CSCs as main contributors to cancer aggressiveness, metastasis and chemoresistance.     

The NTPase activity of the double FYVE domain–containing protein 1 regulates lipid droplet metabolism

Lipid droplets (LDs) are transient lipid storage organelles that can be readily tapped to resupply cells with energy or lipid building blocks and therefore play a central role in cellular metabolism. However, the molecular factors and underlying mechanisms that regulate the growth and degradation of LDs are poorly understood. It has emerged that proteins that establish contacts between LDs and the endoplasmic reticulum play a critical role in regulating LD metabolism. Recently, the autophagy-related protein, double FYVE domain–containing protein 1 (DFCP1/ZFYVE1) was shown to reside at the interface of the endoplasmic reticulum and LDs, however, little is known about the involvement of DFCP1 in autophagy and LD metabolism. Here, we show that DFCP1 is a novel NTPase that regulates free fatty acid metabolism. Specifically, we show that DFPC1-knockdown, particularly during starvation, increases cellular free fatty acids and decreases the levels of cellular TAGs, resulting in accumulated small LDs. Using selective truncations, we demonstrate that DFCP1 accumulation on LDs in cells and in vitro is regulated by a previously unknown NTPase domain. Using spectroscopic approaches, we show that this NTPase domain can dimerize and can hydrolyze both ATP and GTP. Furthermore, mutations in DFCP1 that either impact nucleotide hydrolysis or dimerization result in changes in the accumulation of DFCP1 on LDs, changes in LD density and size, and colocalization of LDs to autophagosomes. Collectively, our findings suggest that DFCP1 is an NTPase that modulates the metabolism of LDs in cells.     

The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide induces large lipid droplet accumulation and highlights the effect of LAMP-2 deficiency on lipid droplet degradation

Excess accumulation of intracellular lipids leads to various diseases. Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage. LDs are hydrolyzed via cytosolic lipases (lipolysis) and also degraded in lysosomes through autophagy; namely, lipophagy. A recent study has shown the size-dependent selection of LDs by the two major catabolic pathways (lipolysis and lipophagy), and thus experimental systems that can manipulate the size of LDs are now needed. The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide (PDMP) affects the structures and functions of lysosomes/late endosomes and the endoplasmic reticulum (ER), and alters cholesterol homeostasis. We previously reported that PDMP induces autophagy via the inhibition of mTORC1. In the present study, we found that PDMP induced the accumulation of LDs, especially that of large LDs, in mouse fibroblast (L cells). Surprisingly, the LD accumulation was relieved by PDMP in L cells deficient in lysosome-associated membrane protein-2 (LAMP-2), which is reportedly important for lipophagy. An electron microscopy analysis demonstrated that the LAMP-2 deficiency caused enlarged autophagosomes/autolysosomes in L cells, which may promote the sequestration and degradation of the PDMP-dependent large LDs. Accordingly, PDMP will be useful to explore the mechanism of LD degradation, by inducing large LDs.     

The stoichiometry of protein phosphorylation in adipocyte lipid droplets: Analysis by N‐terminal isotope tagging and enzymatic dephosphorylation

Most phosphoproteomic studies to date have been limited to the identification of phosphoproteins and their phosphorylation sites, and have not assessed the stoichiometry of protein phosphorylation, a critical parameter reflecting the dynamic equilibrium between phosphorylated and non‐phosphorylated pools of proteins. Here, we used a method for measuring phosphorylation stoichiometry through isotope tagging and enzymatic dephosphorylation of tryptic peptides. Using this method, protein digests are divided into two equal aliquots that are modified with either light or heavy isotope tags. One aliquot is dephosphorylated by alkaline phosphatase. Finally, the peptide mixtures are recombined and LC‐MS/MS analysis is performed. With this method, we studied adipocytes of mice stimulated with CL316,243, a β‐3 adrenergic agonist known to induce lipolysis and marked phosphorylation changes in proteins of the lipid droplet surface. In lipid droplet preparations, CL316,243 administration increased phosphorylation of proteins related to regulation of signaling, metabolism and intracellular trafficking in white adipose tissue, including hormone‐sensitive lipase which was 80% phosphorylated at the previously reported site, Ser‐559, and the lipid surface protein perilipin, which was phosphorylated by ～60 and ～40% at previously unreported sites, Ser‐410 and Ser‐460.     

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.