Lung cancer-derived extracellular vesicles induced myotube atrophy and adipocyte lipolysis via the extracellular IL-6-mediated STAT3 pathway

Cancer-associated cachexia (CAC) constitutes a metabolic dysfunction characterized by systemic inflammation and body weight loss. Muscle atrophy and adipose tissue lipolysis might explain weight loss in CAC. Specific functions of numerous hormones and cytokines derived from tumours can provoke cachexia. Extracellular vesicles (EVs) can be involved in intercellular communication. However, whether EVs participate in this process has not been investigated thoroughly. Using Lewis lung carcinoma (LLC) cell cultures, we tested whether LLC-derived EVs induced C2C12 myotube atrophy and 3T3-L1 adipocyte lipolysis. EVs derived from LLC cells and serum from patients with lung cancer, non-lung cancer controls, tumour-bearing mice, and non-tumour-bearing control mice were isolated and characterized biochemically and biophysically. LLC cell-derived EVs induced dose-dependent effects of atrophy in C2C12 myotubes and lipolysis in 3T3-L1 adipocytes. Mechanistically, EVs directly fused with target C2C12 myotubes and 3T3-L1 adipocytes, and transferred interleukin-6 (IL-6) activates the STAT3 signalling pathway in C2C12 myotubes and 3T3-L1 adipocytes. Neutralization of extracellular IL-6 prevented the atrophy and lipolysis effects of EVs. Inhibiting the STAT3 signalling pathway also prevented the atrophy and lipolysis effects of EVs. PKH67-labelled (PKH 67 is a lipid dye that can be used to label extracellular vesicles) LLC-EVs were readily internalized into myotubes and adipocytes. Our data showed that LLC cell-derived EVs induced myotube atrophy and adipocyte lipolysis via the extracellular IL-6-mediated STAT3 pathway in target cells. These findings represent a potentially novel basis for further research in this field towards identifying targets and developing strategies for maintaining weight in CAC.     

The density of extracellular matrix proteins regulates inflammation and insulin signaling in adipocytes

Cells can not only sense the type of extracellular matrix (ECM) protein that is present in the microenvironment, but they can also sense its density. Here, we investigated the effects of ECM protein density on adipokine secretion and insulin signaling in adipocytes. To this end, 3T3-L1 adipocytes were cultured on the surface of polyacrylamide gels that were coated with gradient densities of a collagen type I and fibronectin mixture. We found that high density ECM causes a decrease in insulin signaling and adiponectin secretion, whereas the secretion of monocyte chemoattractant protein-1 (MCP-1) was increased via the activation of nuclear factor-κB (NF-κB). These results indicate that the density of the ECM directly regulates the inflammatory response and insulin sensitivity of adipocytes.

Structured summary

MINT-7992217: Irs1 (uniprotkb:P35569) physically interacts (MI:0915) with phosphatidylinositol 3-kinase 85 kDa regulatory subunit alpha (uniprotkb:P26450) by anti bait coimmunoprecipitation (MI:0006)     

An emerging interplay between extracellular vesicles and cytokines

Extracellular vesicles (EVs) are small membrane-bound particles that are naturally released from cells. They are recognized as potent vehicles of intercellular communication both in prokaryotes and eukaryotes. Because of their capacity to carry biological macromolecules such as proteins, lipids and nucleic acids, EVs influence different physiological and pathological functions of both parental and recipient cells. Although multiple pathways have been proposed for cytokine secretion beyond the classical ER/Golgi route, EVs have recently recognized as an alternative secretory mechanism. Interestingly, cytokines/chemokines exploit these vesicles to be released into the extracellular milieu, and also appear to modulate their release, trafficking and/or content. In this review, we provide an overview of the cytokines/chemokines that are known to be associated with EVs or their regulation with a focus on TNFα, IL-1β and IFNs.     

Human primary adipocytes exhibit immune cell function: adipocytes prime inflammation independent of macrophages

Background

Obesity promotes inflammation in adipose tissue (AT) and this is implicated in pathophysiological complications such as insulin resistance, type 2 diabetes and cardiovascular disease. Although based on the classical hypothesis, necrotic AT adipocytes (ATA) in obese state activate AT macrophages (ATM) that then lead to a sustained chronic inflammation in AT, the link between human adipocytes and the source of inflammation in AT has not been in-depth and systematically studied. So we decided as a new hypothesis to investigate human primary adipocytes alone to see whether they are able to prime inflammation in AT.

Methods and Results

Using mRNA expression, human preadipocytes and adipocytes express the cytokines/chemokines and their receptors, MHC II molecule genes and 14 acute phase reactants including C-reactive protein. Using multiplex ELISA revealed the expression of 50 cytokine/chemokine proteins by human adipocytes. Upon lipopolysaccharide stimulation, most of these adipocyte-associated cytokines/chemokines and immune cell modulating receptors were up-regulated and a few down-regulated such as (ICAM-1, VCAM-1, MCP-1, IP-10, IL-6, IL-8, TNF-α and TNF-β highly up-regulated and IL-2, IL-7, IL-10, IL-13 and VEGF down-regulated. In migration assay, human adipocyte-derived chemokines attracted significantly more CD4+ T cells than controls and the number of migrated CD4+ cells was doubled after treating the adipocytes with LPS. Neutralizing MCP-1 effect produced by adipocytes reduced CD4+ migration by approximately 30%.

Conclusion

Human adipocytes express many cytokines/chemokines that are biologically functional. They are able to induce inflammation and activate CD4+ cells independent of macrophages. This suggests that the primary event in the sequence leading to chronic inflammation in AT is metabolic dysfunction in adipocytes, followed by production of immunological mediators by these adipocytes, which is then exacerbated by activated ATM, activation and recruitment of immune cells. This study provides novel knowledge about the prime of inflammation in human obese adipose tissue, opening a new avenue of investigations towards obesity-associated type 2 diabetes.     

Sofalcone,an anti-ulcer chalcone derivative,suppresses inflammatory crosstalk between macrophages and adipocytes and adipocyte differentiation: Implication of heme-oxygenase-1 induction

Sofalcone, 2′-carboxymethoxy-4,4′-bis(3-methyl-2-butenyloxy)chalcone, has been used as an anti-ulcer agent, although its precise molecular mechanism has not been completely understood. In the current study, we tested the effects of sofalcone on the inflammatory crosstalk between macrophages and adipocytes and on the differentiation of pre-adipocytes. We found that sofalcone has a strong suppressive effect on the production of nitric oxide (NO), tumor necrosis factor (TNF)α, and monocyte chemoattractant protein (MCP)-1 in the culture medium of a coculture system containing RAW264.7 macrophages and 3T3-F442A adipocytes stimulated with lipopolysaccharide (LPS). The suppressive effect of sofalcone on NO production was attenuated by treatment with tin-protoporphyrin (SnPP), a heme-oxygenase (HO)-1 inhibitor. Western blotting analysis showed that sofalcone increased HO-1 expression in both 3T3-F442A mature adipocytes and undifferentiated fibroblasts. Sofalcone also inhibited the differentiation of 3T3-F442A pre-adipocytes into adipocytes, which was restored by SnPP treatment. These results suggest that sofalcone has preferable properties for obesity or metabolic syndrome.     

Cancer cells copy migratory behavior and exchange signaling networks via extracellular vesicles

Recent data showed that cancer cells from different tumor subtypes with distinct metastatic potential influence each other's metastatic behavior by exchanging biomolecules through extracellular vesicles (EVs). However, it is debated how small amounts of cargo can mediate this effect, especially in tumors where all cells are from one subtype, and only subtle molecular differences drive metastatic heterogeneity. To study this, we have characterized the content of EVs shed in vivo by two clones of melanoma (B16) tumors with distinct metastatic potential. Using the Cre‐LoxP system and intravital microscopy, we show that cells from these distinct clones phenocopy their migratory behavior through EV exchange. By tandem mass spectrometry and RNA sequencing, we show that EVs shed by these clones into the tumor microenvironment contain thousands of different proteins and RNAs, and many of these biomolecules are from interconnected signaling networks involved in cellular processes such as migration. Thus, EVs contain numerous proteins and RNAs and act on recipient cells by invoking a multi‐faceted biological response including cell migration.     

Fractionation of endocytic vesicles and glucose-transporter-containing vesicles in rat adipocytes.

We subfractionated intracellular vesicles from rat adipocytes in order to examine the subcellular distribution of endocytic vesicles or endosomes with respect to insulin-regulatable glucose-transporter (GT)-containing vesicles [James, Lederman & Pilch (1987) J. Biol. Chem. 262, 11817-11824]. Vesicles mediating fluid-phase endocytosis sedimented as a single major peak of greater density than the single distinct peak of GT-containing vesicles. This difference was also apparent during cellular insulin exposure and after insulin removal. Endocytosis of insulin and IGF (insulin-like growth factor) II was also examined. In sucrose gradients, IGF II-containing vesicles were less dense than those containing internalized insulin. Receptor-mediated endocytic vesicles were distinct from fluid-phase endocytic vesicles, but overlapped with the GT-containing vesicles. Vesicles containing internalized ligand were further fractionated by agarose-gel electrophoresis after various times of internalization. At least three different vesicle subpopulations containing the iodinated ligands were resolved after 5 min of internalization. Endocytic vesicles containing rapidly internalized insulin (1.5 min at 37 degrees C) consistently co-migrated with GT-containing vesicles. These data indicate that fluid-phase and receptor-mediated endocytosis occur via different pathways in adipocytes. Furthermore, whereas the intracellular GT-containing vesicles are distinct from fluid-phase vesicles, a rapidly labelled pool of insulin-containing vesicles consistently co-fractionated with GT-containing vesicles when separation techniques based on size, density and charge were used. This suggests that the insulin receptor may directly interact with the intracellular GT-containing vesicles after insulin-induced endocytosis.     

MicroRNAs and extracellular vesicles in cholangiopathies

Cholangiopathies encompass a heterogeneous group of disorders affecting biliary epithelial cells (i.e. cholangiocytes). Early diagnosis, prognosis and treatment still remain clinically challenging for most of these diseases and are critical for adequate patient care. In the past decade, extensive research has emphasized microRNAs (miRs) as potential non-invasive biomarkers and tools to accurately identify, predict and treat cholangiopathies. MiRs can be released extracellularly conjugated with lipoproteins or encapsulated in extracellular vesicles (EVs). Research on EVs is also gaining attention since they are present in multiple biological fluids and may represent a relevant source of novel non-invasive biomarkers and be vehicles for new therapeutic approaches. This review highlights the most promising candidate miRs and EV-related biomarkers in cholangiopathies, as well as their relevant roles in biliary pathophysiology. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Human thermogenic adipocytes: a reflection on types of adipocyte,developmental origin,and potential application

Obesity is a leading health problem facing the modern world; however, no effective therapy for this health issue has yet been developed. A promising research direction to identify novel therapies to prevent obesity has emerged from discoveries on development and function of brown/brite adipocytes in mammals. Importantly, there is evidence for the presence and function of active thermogenic brown adipocytes in both infants and adult humans. Several new investigations have shown that thermogenic adipocytes are beneficial to maintain glucose homeostasis, insulin sensitivity, and a healthy body fat content. Such thermogenic adipocytes have been considered as targets to develop a therapy for preventing obesity. This short review seeks to highlight recent findings on the development and function of brown/brite adipocytes in humans and to discuss potential treatments based on these adipocytes to reduce obesity and its related disorders.     

CD40 signaling induces reciprocal outcomes in Leishmania-infected macrophages; roles of host genotype and cytokine milieu

We investigated the influence of CD40-CD40 ligand-mediated signaling on induction of microbicidal activity against Leishmania major in macrophages from resistant (B6) and susceptible (BALB) mouse strains. CD40 engagement induced leishmanicidal activity in resistant macrophages, but increased parasite replication in susceptible macrophages. CD40 engagement induced comparable TNF-alpha production in macrophages from both strains. However, increased IL-10 production was restricted to susceptible macrophages. Increased parasite replication in susceptible macrophages was prevented by a neutralizing anti-IL-10 antibody. In the presence of IFN-gamma, CD40 engagement induced Leishmania killing by macrophages from both strains. Therefore, the outcome of CD40 signaling on effector responses against L. major depends on host genotype and the cytokine milieu, and a source of IFN-gamma is required for a protective response.     

Efferocytes release extracellular vesicles to resolve inflammation and tissue injury via prosaposin-GPR37 signaling

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Human hepatocyte-derived extracellular vesicles attenuate the carbon tetrachloride-induced acute liver injury in mice

Acute liver injury (ALI) induced by chemicals or viruses can progress rapidly to acute liver failure (ALF), often resulting in death of patients without liver transplantation. Since liver transplantation is limited due to a paucity of donors, expensive surgical costs, and severe immune rejection, novel therapies are required to treat liver injury. Extracellular vesicles (EVs) are used for cellular communication, carrying RNAs, proteins, and lipids and delivering them intercellularly after being endocytosed by target cells. Recently, it was reported that EVs secreted from human hepatocytes have an ability to modulate the immune responses; however, these roles of EVs secreted from human hepatocytes were studied only with in vitro experiments. In the present study, we evidenced that EVs secreted from human hepatocytes attenuated the CCL₄-induced ALI by inhibiting the recruitment of monocytes through downregulation of chemokine receptor in the bone marrow and recruitment of neutrophils through the reduction of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2 expression levels in the liver.Subject terms: Acute inflammation, Liver diseases     

Human Papillomavirus and carcinogenesis: Novel mechanisms of cell communication involving extracellular vesicles

A small group of mucosal Human Papillomaviruses are the causative agents of cervical cancer and are also associated with other types of cancers. Certain cutaneous Human Papillomaviruses seem to have a role as co-factors in the UV-induced carcinogenesis of the skin. The main mechanism of the tumorigenesis induced by Human Papillomaviruses is linked to the transforming activity of the viral E6 and E7 oncoproteins. However, other mechanisms, such as the gene expression control by specific microRNAs expression and deregulation of immune inflammatory mediators, may be important in the process of transformation. In this context, the release of Extracellular Vesicles with a specific cargo (microRNAs involved in tumorigenesis, mRNAs of viral oncoproteins, cytokines, chemokines) appears to play a key role.     

Human pluripotent stem cell-derived extracellular vesicles: From now to the future

Extracellular vesicles (EVs) are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools. Accordingly, in various biological contexts, EVs are reported to engage in immune modulation, senescence, and cell proliferation and differentiation. Therefore, EVs could be key elements for potential off-the-shelf cell-free therapy. Little has been studied regarding EVs derived from human pluripotent stem cells (hPSC-EVs), even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability. In this review article, we provide an overview of studies using hPSC-EVs, focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs, how they are characterized, and applications already demonstrated. The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs’ prospective applications as PSC-derived cell-free therapy products.     

Caspase-1-mediated extracellular vesicles derived from pyroptotic alveolar macrophages promote inflammation in acute lung injury

The occurrence and development of acute lung injury (ALI) involve a variety of pathological factors and complex mechanisms. How pulmonary cells communicate with each other and subsequently trigger an inflammatory cascade remains elusive. Extracellular vesicles (EVs) are a critical class of membrane-bound structures that have been widely investigated for their roles in pathophysiological processes, especially in immune responses and tumor progression. Most of the current knowledge of the functions of EVs is related to functions derived from viable cells (e.g., microvesicles and exosomes) or apoptotic cells (e.g., apoptotic bodies); however, there is limited understanding of the rapidly progressing inflammatory response in ALI. Herein, a comprehensive analysis of micron-sized EVs revealed a mass production of 1-5 μm pyroptotic bodies (PyrBDs) release in the early phase of ALI induced by lipopolysaccharide (LPS). Alveolar macrophages were the main source of PyrBDs in the early phase of ALI, and the formation and release of PyrBDs were dependent on caspase-1. Furthermore, PyrBDs promoted the activation of epithelial cells, induced vascular leakage and recruited neutrophils through delivery of damage-associated molecular patterns (DAMPs). Collectively, these findings suggest that PyrBDs are mainly released by macrophages in a caspase-1-dependent manner and serve as mediators of LPS-induced ALI.