Identification of Lipid Biomarkers for Chronic Joint Pain Associated with Different Joint Diseases.

Lipids, especially lysophosphatidylcholine LPC16:0, have been shown to be involved in chronic joint pain through the activation of acid-sensing ion channels (ASIC3). The aim of the present study was to investigate the lipid contents of the synovial fluids from controls and patients suffering from chronic joint pain in order to identify characteristic lipid signatures associated with specific joint diseases. For this purpose, lipids were extracted from the synovial fluids and analyzed by mass spectrometry. Lipidomic analyses identified certain choline-containing lipid classes and molecular species as biomarkers of chronic joint pain, regardless of the pathology, with significantly higher levels detected in the patient samples. Moreover, correlations were observed between certain lipid levels and the type of joint pathologies. Interestingly, LPC16:0 levels appeared to correlate with the metabolic status of patients while other choline-containing lipids were more specifically associated with the inflammatory state. Overall, these data point at selective lipid species in synovial fluid as being strong predictors of specific joint pathologies which could help in the selection of the most adapted treatment.

Lipidomic Profile Analysis of Lung Tissues Revealed Lipointoxication in Pulmonary Veno-Occlusive Disease.

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary arterial hypertension (PAH) occurring in a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2, general control nonderepressible 2) or in a sporadic form in older age (sPVOD), following exposure to chemotherapy or organic solvents. In contrast to PAH, PVOD is characterized by a particular remodeling of the pulmonary venous system and the obliteration of small pulmonary veins by fibrous intimal thickening and patchy capillary proliferation. The pathobiological knowledge of PVOD is poor, explaining the absence of medical therapy for PVOD. Lung transplantation remains the only therapy for eligible PVOD patients. As we recently demonstrated, respiratory diseases, chronic obstructive pulmonary disease, or cystic fibrosis exhibit lipointoxication signatures characterized by excessive levels of saturated phospholipids contributing to the pathological features of these diseases, including endoplasmic reticulum stress, pro-inflammatory cytokines production, and bronchoconstriction. In this study, we investigated and compared the clinical data and lung lipid signature of control (10 patients), idiopathic PAH (7 patients), heritable PAH (9 BMPR2 mutations carriers), hPVOD (10 EIF2AK4 mutation carriers), and sPVOD (6 non-carriers) subjects. Mass spectrometry analyses demonstrated lung lipointoxication only in hPVOD patients, characterized by an increased abundance of saturated phosphatidylcholine (PC) at the expense of the polyunsaturated species in the lungs of hPVOD patients. The present data suggest that lipointoxication could be a potential player in the etiology of PVOD.

Lysophosphatidylcholine 16:0 mediates chronic joint pain associated to rheumatic diseases through acid-sensing ion channel 3.

ABSTRACT: Rheumatic diseases are often associated to debilitating chronic pain, which remains difficult to treat and requires new therapeutic strategies. We had previously identified lysophosphatidylcholine (LPC) in the synovial fluids from few patients and shown its effect as a positive modulator of acid-sensing ion channel 3 (ASIC3) able to induce acute cutaneous pain in rodents. However, the possible involvement of LPC in chronic joint pain remained completely unknown. Here, we show, from 2 independent cohorts of patients with painful rheumatic diseases, that the synovial fluid levels of LPC are significantly elevated, especially the LPC16:0 species, compared with postmortem control subjects. Moreover, LPC16:0 levels correlated with pain outcomes in a cohort of osteoarthritis patients. However, LPC16:0 do not appear to be the hallmark of a particular joint disease because similar levels are found in the synovial fluids of a second cohort of patients with various rheumatic diseases. The mechanism of action was next explored by developing a pathology-derived rodent model. Intra-articular injections of LPC16:0 is a triggering factor of chronic joint pain in both male and female mice, ultimately leading to persistent pain and anxiety-like behaviors. All these effects are dependent on ASIC3 channels, which drive sufficient peripheral inputs to generate spinal sensitization processes. This study brings evidences from mouse and human supporting a role for LPC16:0 via ASIC3 channels in chronic pain arising from joints, with potential implications for pain management in osteoarthritis and possibly across other rheumatic diseases.

Antibody-induced pain-like behavior and bone erosion: links to subclinical inflammation, osteoclast activity, and acid-sensing ion channel 3-dependent sensitization.

ABSTRACT: Several bone conditions, eg, bone cancer, osteoporosis, and rheumatoid arthritis (RA), are associated with a risk of developing persistent pain. Increased osteoclast activity is often the hallmark of these bony pathologies and not only leads to bone remodeling but is also a source of pronociceptive factors that sensitize the bone-innervating nociceptors. Although historically bone loss in RA has been believed to be a consequence of inflammation, both bone erosion and pain can occur years before the symptom onset. Here, we have addressed the disconnection between inflammation, pain, and bone erosion by using a combination of 2 monoclonal antibodies isolated from B cells of patients with RA. We have found that mice injected with B02/B09 monoclonal antibodies (mAbs) developed a long-lasting mechanical hypersensitivity that was accompanied by bone erosion in the absence of joint edema or synovitis. Intriguingly, we have noted a lack of analgesic effect of naproxen and a moderate elevation of few inflammatory factors in the ankle joints suggesting that B02/B09-induced pain-like behavior does not depend on inflammatory processes. By contrast, we found that inhibiting osteoclast activity and acid-sensing ion channel 3 signaling prevented the development of B02/B09-mediated mechanical hypersensitivity. Moreover, we have identified secretory phospholipase A2 and lysophosphatidylcholine 16:0 as critical components of B02/B09-induced pain-like behavior and shown that treatment with a secretory phospholipase A2 inhibitor reversed B02/B09-induced mechanical hypersensitivity and bone erosion. Taken together, our study suggests a potential link between bone erosion and pain in a state of subclinical inflammation and offers a step forward in understanding the mechanisms of bone pain in diseases such as RA.

Polyunsaturated Phospholipids Increase Cell Resilience to Mechanical Constraints.

If polyunsaturated fatty acids (PUFAs) are generally accepted to be good for health, the mechanisms of their bona fide benefits still remain elusive. Membrane phospholipids (PLs) of the cardiovascular system and skeletal muscles are particularly enriched in PUFAs. The fatty acid composition of PLs is known to regulate crucial membrane properties, including elasticity and plasticity. Since muscle cells undergo repeated cycles of elongation and relaxation, we postulated in the present study that PUFA-containing PLs could be central players for muscle cell adaptation to mechanical constraints. By a combination of in cellulo and in silico approaches, we show that PUFAs, and particularly the ω-3 docosahexaenoic acid (DHA), regulate important properties of the plasma membrane that improve muscle cell resilience to mechanical constraints. Thanks to their unique property to contortionate within the bilayer plane, they facilitate the formation of vacuole-like dilation (VLD), which, in turn, avoid cell breakage under mechanical constraints.

Glued in lipids: Lipointoxication in cystic fibrosis.

Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane regulator (CFTR) gene, which encodes a chloride channel located at the apical surface of epithelial cells. Unsaturated Fatty Acid (UFA) deficiency has been a persistent observation in tissues from patients with CF. However, the impacts of such deficiencies on the etiology of the disease have been the object of intense debates. The aim of the present review is first to highlight the general consensus on fatty acid dysregulations that emerges from, sometimes apparently contradictory, studies. In a second step, a unifying mechanism for the potential impacts of these fatty acid dysregulations in CF cells, based on alterations of membrane biophysical properties (known as lipointoxication), is proposed. Finally, the contribution of lipointoxication to the progression of the CF disease and how it could affect the efficacy of current treatments is also discussed.

A comprehensive study of phospholipid fatty acid rearrangements in metabolic syndrome: correlations with organ dysfunction.

The balance within phospholipids (PLs) between saturated fatty acids and monounsaturated or polyunsaturated fatty acids is known to regulate the biophysical properties of cellular membranes. As a consequence, in many cell types, perturbing this balance alters crucial cellular processes, such as vesicular budding and the trafficking/function of membrane-anchored proteins. The worldwide spread of the Western diet, which is highly enriched in saturated fats, has been clearly correlated with the emergence of a complex syndrome known as metabolic syndrome (MetS). MetS is defined as a cluster of risk factors for cardiovascular diseases, type 2 diabetes and hepatic steatosis; however, no clear correlations have been established between diet-induced fatty acid redistribution within cellular PLs and the severity/chronology of the symptoms associated with MetS or the function of the targeted organs. To address this issue, in this study we analyzed PL remodeling in rats exposed to a high-fat/high-fructose diet (HFHF) over a 15-week period. PL remodeling was analyzed in several organs, including known MetS targets. We show that fatty acids from the diet can redistribute within PLs in a very selective manner, with phosphatidylcholine being the preferred sink for this redistribution. Moreover, in the HFHF rat model, most organs are protected from this redistribution, at least during the early onset of MetS, at the expense of the liver and skeletal muscles. Interestingly, such a redistribution correlates with clear-cut alterations in the function of these organs.This article has an associated First Person interview with the first author of the paper.

Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases.

Maintaining the equilibrium between saturated and unsaturated fatty acids within membrane phospholipids (PLs) is crucial to sustain the optimal membrane biophysical properties, compatible with selective organelle-based processes. Lipointoxication is a pathological condition under which saturated PLs tend to accumulate within the cell at the expense of unsaturated species, with major impacts on organelle function. Here, we show that human bronchial epithelial cells extracted from lungs of patients with Obstructive Pulmonary Diseases (OPDs), i. e. Cystic Fibrosis (CF) individuals and Smokers, display a characteristic lipointoxication signature, with excessive amounts of saturated PLs. Reconstitution of this signature in cellulo and in silico revealed that such an imbalance results in altered membrane properties and in a dramatic disorganization of the intracellular network of bronchial epithelial cells, in a process which can account for several OPD traits. Such features include Endoplasmic Reticulum-stress, constitutive IL8 secretion, bronchoconstriction and, ultimately, epithelial cell death by apoptosis. We also demonstrate that a recently-identified lipid-like molecule, which has been shown to behave as a "membrane-reshaper", counters all the lipointoxication hallmarks tested. Altogether, these insights highlight the modulation of membrane properties as a potential new strategy to heal and prevent highly detrimental symptoms associated with OPDs.

Transcriptomics and Lipidomics of the Environmental Strain Rhodococcus ruber Point out Consumption Pathways and Potential Metabolic Bottlenecks for Polyethylene Degradation.

Polyethylene (PE), one of the most prominent synthetic polymers used worldwide, is very poorly biodegradable in the natural environment. Consequently, PE represents by itself more than half of all plastic wastes. PE biodegradation is achieved through the combination of abiotic and biotic processes. Several microorganisms have been shown to grow on the surface of PE materials, among which are the species of the Rhodococcus genus, suggesting a potent ability of these microorganisms to use, at least partly, PE as a potent carbon source. However, most of them, if not all, fail to induce a clear-cut degradation of PE samples, showing that bottlenecks to reach optimal biodegradation clearly exist. To identify the pathways involved in PE consumption, we used in the present study a combination of RNA-sequencing and lipidomic strategies. We show that short-term exposure to various forms of PE, displaying different molecular weight distributions and oxidation levels, lead to an increase in the expression of 158 genes in a Rhodococcus representative, R. ruber. Interestingly, one of the most up-regulated pathways is related to alkane degradation and ß-oxidation of fatty acids. This approach also allowed us to identify metabolic limiting steps, which could be fruitfully targeted for optimized PE consumption by R. ruber.

Fate of Eight Different Polymers under Uncontrolled Composting Conditions: Relationships Between Deterioration, Biofilm Formation, and the Material Surface Properties.

With the ever-increasing volume of polymer wastes and their associated detrimental impacts on the environment, the plastic life cycle has drawn increasing attention. Here, eight commercial polymers selected from biodegradable to environmentally persistent materials, all formulated under a credit card format, were incubated in an outdoor compost to evaluate their fate over time and to profile the microbial communities colonizing their surfaces. After 450 days in compost, the samples were all colonized by multispecies biofilms, these latest displaying different amounts of adhered microbial biomass and significantly distinct bacterial and fungal community compositions depending on the substrate. Interestingly, colonization experiments on the eight polymers revealed a large core of shared microbial taxa, predominantly composed of microorganisms previously reported from environments contaminated with petroleum hydrocarbons or plastics debris. These observations suggest that biofilms may contribute to the alteration process of all the polymers studied. Actually, four substrates, independently of their assignment to a polymer group, displayed a significant deterioration, which might be attributed to biologically mediated mechanisms. Relevantly, the deterioration appears strongly associated with the formation of a high-cell density biofilm onto the polymer surfaces. The analysis of various surface properties revealed that roughness and hydrophilicity are likely prominent parameters for driving the biological interactions with the polymers.

Targeting surface voids to counter membrane disorders in lipointoxication-related diseases.

Saturated fatty acids (SFA), which are abundant in the so-called western diet, have been shown to efficiently incorporate within membrane phospholipids and therefore impact on organelle integrity and function in many cell types. In the present study, we have developed a yeast-based two-step assay and a virtual screening strategy to identify new drugs able to counter SFA-mediated lipointoxication. The compounds identified here were effective in relieving lipointoxication in mammalian ß-cells, one of the main targets of SFA toxicity in humans. In vitro reconstitutions and molecular dynamics simulations on bilayers revealed that these molecules, albeit according to different mechanisms, can generate voids at the membrane surface. The resulting surface defects correlate with the recruitment of loose lipid packing or void-sensing proteins required for vesicular budding, a central cellular process that is precluded under SFA accumulation. Taken together, the results presented here point at modulation of surface voids as a central parameter to consider in order to counter the impacts of SFA on cell function.

Non-acidic activation of pain-related Acid-Sensing Ion Channel 3 by lipids.

Extracellular pH variations are seen as the principal endogenous signal that triggers activation of Acid-Sensing Ion Channels (ASICs), which are basically considered as proton sensors, and are involved in various processes associated with tissue acidification. Here, we show that human painful inflammatory exudates, displaying non-acidic pH, induce a slow constitutive activation of human ASIC3 channels. This effect is largely driven by lipids, and we identify lysophosphatidylcholine (LPC) and arachidonic acid (AA) as endogenous activators of ASIC3 in the absence of any extracellular acidification. The combination of LPC and AA evokes robust depolarizing current in DRG neurons at physiological pH 7.4, increases nociceptive C-fiber firing, and induces pain behavior in rats, effects that are all prevented by ASIC3 blockers. Lipid-induced pain is also significantly reduced in ASIC3 knockout mice. These findings open new perspectives on the roles of ASIC3 in the absence of tissue pH variation, as well as on the contribution of those channels to lipid-mediated signaling.

From zero to six double bonds: phospholipid unsaturation and organelle function.

Cellular phospholipids (PLs) differ by the nature of their polar heads as well as by the length and unsaturation level of their fatty acyl chains. We discuss how the ratio between saturated, monounsaturated, and polyunsaturated PLs impacts on the functions of such organelles as the endoplasmic reticulum, synaptic vesicles, and photoreceptor discs. Recent experiments and simulations suggest that polyunsaturated PLs respond differently to mechanical stress, including membrane bending, than monounsaturated PLs owing to their unique conformational plasticity. These findings suggest a rationale for PL acyl chain remodeling by acyltransferases and a molecular explanation for the importance of a balanced fatty acid diet.

Lipid cell biology. Polyunsaturated phospholipids facilitate membrane deformation and fission by endocytic proteins.

Phospholipids (PLs) with polyunsaturated acyl chains are extremely abundant in a few specialized cellular organelles such as synaptic vesicles and photoreceptor discs, but their effect on membrane properties is poorly understood. Here, we found that polyunsaturated PLs increased the ability of dynamin and endophilin to deform and vesiculate synthetic membranes. When cells incorporated polyunsaturated fatty acids into PLs, the plasma membrane became more amenable to deformation by a pulling force and the rate of endocytosis was accelerated, in particular, under conditions in which cholesterol was limiting. Molecular dynamics simulations and biochemical measurements indicated that polyunsaturated PLs adapted their conformation to membrane curvature. Thus, by reducing the energetic cost of membrane bending and fission, polyunsaturated PLs may help to support rapid endocytosis.

Cystic fibrosis bronchial epithelial cells are lipointoxicated by membrane palmitate accumulation.

The F508del-CFTR mutation, responsible for Cystic Fibrosis (CF), leads to the retention of the protein in the endoplasmic reticulum (ER). The mistrafficking of this mutant form can be corrected by pharmacological chaperones, but these molecules showed limitations in clinical trials. We therefore hypothesized that important factors in CF patients may have not been considered in the in vitro assays. CF has also been associated with an altered lipid homeostasis, i. e. a decrease in polyunsaturated fatty acid levels in plasma and tissues. However, the precise fatty acyl content of membrane phospholipids from human CF bronchial epithelial cells had not been studied to date. Since the saturation level of phospholipids can modulate crucial membrane properties, with potential impacts on membrane protein folding/trafficking, we analyzed this parameter for freshly isolated bronchial epithelial cells from CF patients. Interestingly, we could show that Palmitate, a saturated fatty acid, accumulates within Phosphatidylcholine (PC) in CF freshly isolated cells, in a process that could result from hypoxia. The observed PC pattern can be recapitulated in the CFBE41o(-) cell line by incubation with 100 µM Palmitate. At this concentration, Palmitate induces an ER stress, impacts calcium homeostasis and leads to a decrease in the activity of the corrected F508del-CFTR. Overall, these data suggest that bronchial epithelial cells are lipointoxicated by hypoxia-related Palmitate accumulation in CF patients. We propose that this phenomenon could be an important bottleneck for F508del-CFTR trafficking correction by pharmacological agents in clinical trials.

Saturated fatty acids alter the late secretory pathway by modulating membrane properties.

Saturated fatty acids (SFA) have been reported to alter organelle integrity and function in many cell types, including muscle and pancreatic ß-cells, adipocytes, hepatocytes and cardiomyocytes. SFA accumulation results in increased amounts of ceramides/sphingolipids and saturated phospholipids (PL). In this study, using a yeast-based model that recapitulates most of the trademarks of SFA-induced lipotoxicity in mammalian cells, we demonstrate that these lipid species act at different levels of the secretory pathway. Ceramides mostly appear to modulate the induction of the unfolded protein response and the transcription of nutrient transporters destined to the cell surface. On the other hand, saturated PL, by altering membrane properties, directly impact vesicular budding at later steps in the secretory pathway, i.e. at the trans-Golgi Network level. They appear to do so by increasing lipid order within intracellular membranes which, in turn, alters the recruitment of loose lipid packing-sensing proteins, required for optimal budding, to nascent vesicles. We propose that this latter general mechanism could account for the well-documented deleterious impacts of fatty acids on the last steps of the secretory pathway in several cell types.

Ergosterol biosynthesis: a fungal pathway for life on land?

Sterols, essential lipids of most eukaryotic cells, ensure important structural and signaling functions. The selection pressure that has led to different dominant sterols in the three eukaryotic kingdoms remains unknown. Here, we investigated the influence of the progression in the different steps of the ergosterol biosynthetic pathway (EBP) on the yeast resistance to transitions from aqueous to aerial media, typical perturbations of the higher fungi habitats. Five mutants of the EBP (ergΔ), accumulating different sterol intermediates in the EBP, and the wild-type (WT) strain were exposed to drying under atmospheric air or nitrogen and wetting. Results show that the progression in the EBP parallels an increase in the yeast resistance to air-drying with a maximal survival rate for the WT strain. When drying/wetting was performed under nitrogen, yeast survival was higher, particularly for the earlier mutants of the EBP. Thus, ergosterol, through its protective role against mechanical and oxidative stress, might have been selected by the pressure induced by drying/wetting cycles occurring in the fungi habitats. These results support the Bloch hypothesis, which postulates that the properties of sterols are gradually optimized for function along the biosynthetic pathway and provide a response to the enduring question "why ergosterol in fungi?".

Is fat so bad? Modulation of endoplasmic reticulum stress by lipid droplet formation.

LDs (lipid droplets) have long been considered as inert particles used by the cells to store fatty acids and sterols as esterified non-toxic lipid species (i.e. triacylglycerols and steryl esters). However, accumulating evidence suggests that LDs behave as a dynamic compartment, which is involved in the regulation of several aspects of the homoeostasis of their originating organelle, namely the ER (endoplasmic reticulum). The ER is particularly sensitive to physiological/pathological stimuli, which can ultimately induce ER stress. In the present review, after considering the basic mechanisms of LD formation and the signal cascades leading to ER stress, we focus on the connections between these two pathways. Taking into consideration recent data from the literature, we will try to draw possible mechanisms for the role of LDs in the regulation of ER homoeostasis and in ER-stress-related diseases.

Nature of sterols affects plasma membrane behavior and yeast survival during dehydration.

The plasma membrane (PM) is a main site of injury during osmotic perturbation. Sterols, major lipids of the PM structure in eukaryotes, are thought to play a role in ensuring the stability of the lipid bilayer during physicochemical perturbations. Here, we investigated the relationship between the nature of PM sterols and resistance of the yeast Saccharomyces cerevisiae to hyperosmotic treatment. We compared the responses to osmotic dehydration (viability, sterol quantification, ultrastructure, cell volume, and membrane permeability) in the wild-type (WT) strain and the ergosterol mutant erg6Δ strain. Our main results suggest that the nature of membrane sterols governs the mechanical behavior of the PM during hyperosmotic perturbation. The mutant strain, which accumulates ergosterol precursors, was more sensitive to osmotic fluctuations than the WT, which accumulates ergosterol. The hypersensitivity of erg6Δ was linked to modifications of the membrane properties, such as stretching resistance and deformation, which led to PM permeabilization during the volume variation during the dehydration-rehydration cycles. Anaerobic growth of erg6Δ strain with ergosterol supplementation restored resistance to osmotic treatment. These results suggest a relationship between hydric stress resistance and the nature of PM sterols. We discuss this relationship in the context of the evolution of the ergosterol biosynthetic pathway.

Modulation of lipid-induced ER stress by fatty acid shape.

Exposure of pancreatic ß cells to long-chain saturated fatty acids (SFA) induces a so-called endoplasmic reticulum (ER) stress that can ultimately lead to cell death. This process is believed to participate in insulin deficiency associated with type 2 diabetes, via a decrease in ß-cell mass. By contrast, some unsaturated fatty acid species appear less toxic to the cells and can even alleviate SFA-induced ER stress. In the present study, we took advantage of a simple yeast-based model, which brings together most of the trademarks of lipotoxicity in human cells, to screen fatty acids of various structures for their capacity to counter ER stress. Here we demonstrate that the tendency of a free fatty acid (FFA) to reduce SFA toxicity depends on a complex conjunction of parameters, including chain length, level of unsaturation, position of the double bonds and nature of the isomers (cis or trans). Interestingly, potent FFA act as building blocks for phospholipid synthesis and help to restore an optimal membrane organization, compatible with ER function and normal protein trafficking.