Fatty and hydroxycarboxylic acid receptors: The missing link of immune response and metabolism in cattle.

Fatty and hydroxycarboxylic acids are one of the main intermediates of energy metabolism in ruminants and critical in the milk production of cattle. High production demands on a dairy farm can induce nutritional imbalances and metabolism disorders, which have been widely associated with the onset of sterile inflammatory processes and increased susceptibility to infections. The literature suggests that short-chain fatty acids (SCFA), long-chain fatty acids (LCFA) and hydroxycarboxylic acids are relevant modulators of the host innate inflammatory response. For instance, increased SCFA and lactate levels are associated with subacute ruminal acidosis (SARA) and the activation of pro-inflammatory processes mediated by diverse leukocyte and vascular endothelial cells. As such, free LCFA and the ketone body ß-hydroxybutyrate are significantly increased in the plasma 1-2 weeks postpartum, coinciding with the time period in which cows are more susceptible to acquiring infectious diseases that the host innate immune system should actively oppose. Today, many of these pro-inflammatory responses can be related to the activation of specific G protein-coupled receptors, including GPR41/FFA3 and GPR43/FFA2 for SCFA; GPR40/FFA1 and GPR120/FFA4 for LCFA, GPR109A/HCA2 for ketone body ß-hydroxybutyrate, and GPR81/HCA1 for lactate, all expressed in different bovine tissues. The activation of these receptors modulates the release of intracellular granules [e.g., metalloproteinase-9 (MMP-9) and lactoferrin], radical oxygen species (ROS) production, chemotaxis, and the production of relevant pro-inflammatory mediators. The article aimed to review the role of natural ligands and receptors and the resulting impact on the host innate immune reaction of cattle and, further, to address the most recent evidence supporting a potential connection to metabolic disorders.

LncRNA uc.48+ is involved in the diabetic immune and inflammatory responses mediated by P2X7 receptor in RAW264.7 macrophages.

High glucose combined with high FFAs can contribute to the unfavorable development of type 2 diabetes mellitus (T2DM) and monocytes/macrophages are important in the occurrence and development of T2DM, which is regarded as a type of low­grade inflammation. Although our previous study demonstrated that increased expression of P2X7 receptor (P2X7R) in peripheral blood monocytes may alter the innate immune system and that long non­coding (lnc)RNA uc.48+ was involved in diabetic neuropathic pain, the involvement of uc.48+ mediated by the P2X7R in monocyte/macrophages during T2DM has not been reported. In the present study, the effectsof uc.48+ small interference RNA (siRNA) on factors, including the mRNA and protein expression of P2X7R, apoptosis and proliferation, levels of reactive oxygen species (ROS), cytokine levels, and expression of phosphorylated (p­) extracellular signal­regulated kinase (ERK)1/2, were examined in RAW264.7 macrophages following exposure to high glucose and high plasma free fatty acids (FFAs). After RAW264.7 cells were transfected with uc.48+ siRNA under high glucose conditions and FFAs treatment, the mRNA expression levels of uc.48+ and P2X7 receptor were detected by reverse transcription­polymerase chain reaction. The protein mass of P2X7 receptor and ERK signaling pathway were assessed by western blotting. ROS and calcium concentrations, and culture supernatant cytokine content [tumor necrosis factor­α, interleukin (IL)­10, IL­1ß] were detected by fluorescent probes and ELISA respectively. Cell viability and apoptosis were determined by MTS test and flow cytometry, respectively. It was found that treatment of RAW264.7 cells with high glucose and FFAs, which exhibited increased expression of uc.48+, evoked P2X7R­mediated immune and inflammatory responses through several means, including cytokine secretion, ROS formation, and activation of the ERK signaling pathway. The uc.48+ siRNA regulated these factors and thus influenced the course and outcome of the immune and inflammatory responses mediated by P2X7R.

Trypanosoma evansi induces detrimental immuno-catabolic alterations and condition like type-2 diabetes in buffaloes.

The present study aimed to investigate the perturbations in immuno-metabolic and redox status of buffaloes with trypanosomosis. Thirteen buffaloes suffering from clinical trypanosomosis and eight apparently healthy buffaloes were included in the present study. Buffaloes with trypanosomosis found to have markedly elevated levels of interleukin-10 (IL-10), nonesterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) in comparison with healthy controls. Whereas, total antioxidant capacity (TAC) and haemoglobin levels of buffaloes with trypanosomosis were significantly lower than the healthy controls. Remarkable elevation in malondialdehyde (MDA) and protein carbonyls (PC) levels were also observed in the diseased buffaloes. Moreover, buffaloes with trypanosomosis were found to have markedly elevated levels of serum glucose, total proteins, globulins, urea and aspartate aminotransferase (AST) and markedly lowered levels of serum calcium, total cholesterol levels and albumin/globulin (A/G) ratio as compared to the controls. Findings of our study evidently suggest that Trypanosoma evansi induces remarkable immunosuppressive and pro-oxidative status with an increased catabolic activity and hyperglycemic condition like type-2 diabetes in naturally infected buffaloes. Therefore, immuno-metabolic and pro-oxidative predicaments should be addressed by the veterinary clinician while managing the clinical cases of trypanosomosis in buffaloes.

A highly efficient 2D exfoliated metal dichalcogenide for the on-farm rapid monitoring of non-esterified fatty acids.

Herein, we report on the development of an electrochemically active liquid exfoliated 2D MoS2 nanosheet based biointerface for the on-farm monitoring of non-esterified fatty acid (NEFA) biomarkers.

Global assessment of oxidized free fatty acids in brain reveals an enzymatic predominance to oxidative signaling after trauma.

Traumatic brain injury (TBI) is a major health problem associated with significant morbidity and mortality. The pathophysiology of TBI is complex involving signaling through multiple cascades, including lipid peroxidation. Oxidized free fatty acids, a prominent product of lipid peroxidation, are potent cellular mediators involved in induction and resolution of inflammation and modulation of vasomotor tone. While previous studies have assessed lipid peroxidation after TBI, to our knowledge no studies have used a systematic approach to quantify the global oxidative changes in free fatty acids. In this study, we identified and quantified 244 free fatty acid oxidation products using a newly developed global liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. This methodology was used to follow the time course of these lipid species in the contusional cortex of our pediatric rat model of TBI. We show that oxidation peaked at 1h after controlled cortical impact and was progressively attenuated at 4 and 24h time points. While enzymatic and non-enzymatic pathways were activated at 1h post-TBI, enzymatic lipid peroxidation was the predominant mechanism with 15-lipoxygenase (LOX) contributing to the majority of total oxidized fatty acid content. Pro-inflammatory lipid mediators were significantly increased at 1 and 4h after TBI with return to basal levels by 24h. Anti-inflammatory lipid mediators remained significantly increased across all three time points, indicating an elevated and sustained anti-inflammatory response following TBI.

Metabolism meets immunity: The role of free fatty acid receptors in the immune system.

There are significant numbers of nutrient sensing G protein-coupled receptors (GPCRs) that can be found in cells of the immune system and in tissues that are involved in metabolic function, such as the pancreas or the intestinal epithelium. The family of free fatty acid receptors (FFAR1-4, GPR84), plus a few other metabolite sensing receptors (GPR109A, GPR91, GPR35) have been for this reason the focus of studies linking the effects of nutrients with immunological responses. A number of the beneficial anti-inflammatory effects credited to dietary fats such as omega-3 fatty acids are attributed to their actions on FFAR4.This might play an important protective role in the development of obesity, insulin resistance or asthma. The role of the short-chain fatty acids resulting from fermentation of fibre by the intestinal microbiota in regulating acute inflammatory responses is also discussed. Finally we assess the therapeutic potential of this family of receptors to treat pathologies where inflammation is a major factor such as type 2 diabetes, whether by the use of novel synthetic molecules or by the modulation of the individual's diet.

Free fatty acids: potential proinflammatory mediators in rheumatic diseases.

OBJECTIVES: Due to their role in inflammatory metabolic diseases, we hypothesised that free fatty acids (FFA) are also involved in inflammatory joint diseases. To test this hypothesis, we analysed the effect of FFA on synovial fibroblasts (SF), human chondrocytes and endothelial cells. We also investigated whether the toll-like receptor 4 (TLR4), which can contribute to driving arthritis, is involved in FFA signalling. METHODS: Rheumatoid arthritis SF, osteoarthritis SF, psoriatic arthritis SF, human chondrocytes and endothelial cells were stimulated in vitro with different FFA. Immunoassays were used to quantify FFA-induced protein secretion. TLR4 signalling was inhibited extracellularly and intracellularly. Fatty acid translocase (CD36), responsible for transporting long-chain FFA into the cell, was also inhibited. RESULTS: In rheumatoid arthritis synovial fibroblasts (RASF), FFA dose-dependently enhanced the secretion of the proinflammatory cytokine IL-6, the chemokines IL-8 and MCP-1, as well as the matrix-degrading enzymes pro-MMP1 and MMP3. The intensity of the response was mainly dependent on the patient rather than on the type of disease. Both saturated and unsaturated FFA showed similar effects on RASF, while responses to the different FFA varied for human chondrocytes and endothelial cells. Extracellular and intracellular TLR4 inhibition as well as fatty acid transport inhibition blocked the palmitic acid-induced IL-6 secretion of RASF. CONCLUSIONS: The data show that FFA are not only metabolic substrates but may also directly contribute to articular inflammation and degradation in inflammatory joint diseases. Moreover, the data suggest that, in RASF, FFA exert their effects via TLR4 and require extracellular and intracellular access to the TLR4 receptor complex.

Palmitate has proapoptotic and proinflammatory effects on articular cartilage and synergizes with interleukin-1.

OBJECTIVE: Obesity is a major risk factor for the development of osteoarthritis (OA) that is associated with a state of low-grade inflammation and increased circulating levels of adipokines and free fatty acids (FFAs). The aim of this study was to analyze the effects of saturated (palmitate) and monounsaturated (oleate) FFAs on articular chondrocytes, synoviocytes, and cartilage. METHODS: Human articular chondrocytes and fibroblast-like synoviocytes obtained from young healthy donors and OA chondrocytes from patients undergoing total knee replacement surgery were treated with palmitate or oleate alone or in combination with interleukin-1ß (IL-1ß). Cell viability, caspase activation, and gene expression of proinflammatory factors, extracellular matrix (ECM) proteins, and proteases were studied. In addition, chondrocyte viability, IL-6 production, and matrix damage were assessed in bovine and human articular cartilage explants cultured with FFAs in the presence or absence of IL-1ß. RESULTS: Palmitate, but not oleate, induced caspase activation and cell death in IL-1ß-stimulated normal chondrocytes, and up-regulated the expression of IL-6 and cyclooxygenase 2 in chondrocytes and fibroblast-like synoviocytes through Toll-like receptor 4 (TLR-4) signaling. In cartilage explants, palmitate induced chondrocyte death, IL-6 release, and ECM degradation. Palmitate synergized with IL-1ß in stimulating proapoptotic and proinflammatory cellular responses. Pharmacologic inhibition of caspases or TLR-4 signaling reduced palmitate and IL-1ß induced cartilage damage. CONCLUSION: Palmitate acts as a proinflammatory and catabolic factor that, in synergy with IL-1ß, induces chondrocyte apoptosis and articular cartilage breakdown. Collectively, our data suggest that elevated levels of saturated FFAs that are often found in patients who are obese may contribute to the pathogenesis of OA.

Adipocyte-derived lipids modulate CD4+ T-cell function.

Adipose tissue contains several immune cells whose number and phenotype vary depending on the adiposity. In the present study, we show that IFN-γ(+) CD4(+) T cells are enriched in human adipose tissue compared with in blood. To gain insight into the underlying mechanisms, we investigated the possibility that human adipocytes modulate CD4(+) T-cell cytokine production and proliferation and show that CD4(+) T cells produced increased levels of IFN-γ when activated in the presence of adipocytes. This effect was mediated by soluble mediators, as shown in transwell and adipocyte-conditioned medium (ACM) transfer experiments. Additionally, ACM induced increased proliferation of CD4(+) T cells upon activation. Intriguingly, the proliferation-enhancing effect resided mainly in the lipid fraction of ACM, as shown upon separation of the protein and lipid fraction. Further separation of these lipids based on polarity revealed that the modulatory effect is confined to fractions containing free fatty acids. All identified fatty acids were able to individually enhance T-cell proliferation. These data indicate that adipocytes can modulate CD4(+) T-cell function through the release of lipids. Remarkably, free fatty acids were the most prominent modulators of T-cell proliferation, possibly leading to an accumulation of these cells in adipose tissue.

Sebum free fatty acids enhance the innate immune defense of human sebocytes by upregulating beta-defensin-2 expression.

Various sebum free fatty acids (FFAs) have shown antibacterial activity against a broad range of gram-positive bacteria, resulting in the suggestion that they are accountable, at least partially, for the direct antimicrobial activity of the skin surface. In this study, we examined the effects of sebum FFAs on the antimicrobial peptide (AMP)-mediated innate immune defense of human sebocytes. Incubation of lauric acid, palmitic acid, or oleic acid (OA) with human sebocytes dramatically enhanced their expression of human beta-defensin (hBD)-2, one of the predominant AMPs found in the skin, whereas remarkable increases in hBD-1, hBD-3, and human cathelicidin LL-37 were not observed. Secreted hBD-2 was detectable by western blotting in the supernatant of sebocyte culture incubated with each FFA, but not with a vehicle control. The supernatant of FFA-incubated sebocyte culture showed antimicrobial activity against Propionibacterium acnes, whereas the enhanced antimicrobial activity of human sebocytes was neutralized by anti-hBD-2 IgG. In addition, the FFA-induced hBD-2 expression was suppressed by blocking the cluster of differentiation (CD)36 fatty acid translocase on the surface of sebocytes with anti-human CD36 IgG or blocking the NF-kappaB signaling pathway with BMS-345541, a highly selective inhibitor of inhibitory kappaB kinase. These data suggest that sebum FFAs upregulate the expression of hBD-2 in human sebocytes, which may enhance the disinfecting activity of the human sebaceous gland. The FFA-induced upregulation of hBD-2 is facilitated by CD36-mediated FFA uptake and NF-kappaB-mediated transactivation. The upregulation of mouse beta-defensin 4, a mouse ortholog for hBD-2, was also observed in the hair follicle sebaceous glands of mouse ear skin after an epicutaneous application of OA, the most hBD-2-inducible FFA tested. This report highlights the potential of using FFAs as a multifunctional antimicrobial therapy agent for acne vulgaris treatment; FFAs may provide direct antibacterial activities against P. acnes and enhance the skin's innate antibacterial defense by inducing the expression of hBD-2 in sebocytes as well.

Glucocorticoids and thiazolidinediones interfere with adipocyte-mediated macrophage chemotaxis and recruitment.

The link between intra-abdominal adiposity and type II diabetes has been known for decades, and adipose tissue macrophage (ATM)-associated inflammation has recently been linked to insulin resistance. However, the mechanisms associated with ATM recruitment remain ill defined. Herein, we describe in vitro chemotaxis studies, in which adipocyte conditioned medium was used to stimulate macrophage migration. We demonstrate that tumor necrosis factor alpha and free fatty acids, key inflammatory stimuli involved in obesity-associated autocrine/paracrine inflammatory signaling, stimulate adipocyte expression and secretion of macrophage chemoattractants. Pharmacological studies showed that peroxisome proliferator-activated receptor gamma agonists and glucocorticoids potently inhibit adipocyte- induced recruitment of macrophages. This latter effect was mediated by the glucocorticoid receptor, which led to decreased chemokine secretion and expression. In vivo results were quite comparable; treatment of high fat diet-fed mice with dexamethasone prevented ATM accumulation in epididymal fat. This decrease in ATM was most pronounced for the proinflammatory F4/80(+), CD11b(+), CD11c(+) M-1-like ATM subset. Overall, our results elucidate a beneficial function of peroxisome proliferator-activated receptor gamma activation and glucocorticoid receptor/glucocorticoids in adipose tissue and indicate that pharmacologic prevention of ATM accumulation could be beneficial.

Further study on the roles of the effector molecules of immunosuppressive macrophages induced by mycobacterial infection in expression of their suppressor function against mitogen-stimulated T cell proliferation.

Previously, we found that phospholipids and reactive nitrogen intermediates (RNI) collaborated in expression of the T cell mitogenesis-inhibitory activity of immunosuppressive macrophages induced by Mycobacterium avium-intracellulare complex (MAIC) infection. In this study, we examined the roles of free fatty acids (FFA) and prostaglandins (PG) as effectors of MAIC-induced macrophages, and moreover, their collaborating effects with RNI. First, treatment of MAIC-induced macrophages with quinacrine (phospholipase A2 (PLA2) inhibitor), dexamethasone (inhibitor of PLA2 and PG synthesis) or indomethacin (PG synthesis inhibitor) attenuated their suppressor activity against concanavalin A (Con A)-induced mitogenesis of splenocytes (SPC), indicating important roles of FFA liberated from membrane phospholipids and PG, as effectors. Second, oleic acid, PGE2, RNI generated from NOR 4 (a new nitric oxide (NO) donor), and phosphatidylserine (PS) exhibited suppressor activity against SPC mitogenesis without showing significant cytotoxicity, in an irreversible manner. Third, the suppressor activities of RNI and PGE2 were potentiated by combined use with oleic acid in a synergistic manner. Fourth, a dual-chamber experiment in which target SPC were separated from MAIC-induced macrophages by a Millipore filter revealed a requirement for cell-to-cell contact for expression of the suppressor function of MAIC-induced macrophages. These findings indicate that RNI, FFA, PG, and phospholipids (presumably PS) and their collaboration play central roles in expression of the T cell mitogenesis-inhibitory function of MAIC-induced suppressor macrophages.

Human T lymphocytes require lipid as either lipoprotein or nonesterified fatty acid for in vitro activation.

Serum, which is required for activation of T cells in vitro, was fractionated to isolate and identify major supportive components. The serum activity concentrated exclusively with the lipoprotein fractions LDL and HDL. Furthermore, oleic and linoleic acids were equally active supplements, suggesting that resting T cells may require exogenous lipid because they are incapable of synthesizing fatty acid. A survey of fatty acids disclosed that all were capable of some degree of support. Titration delineated the narrow concentration range of lipid supplements successful in supporting T cell proliferation and demonstrated that concentrations must be rigidly controlled to optimize cell proliferation.

Skin reaction to lipids from avirulent strain Shibaura of Leptospira interrogans serovar copenhageni.

Sonically disrupted cells from avirulent strain Shibaura of Leptospira interrogans serovar copenhageni induced a skin reaction characterized by infiltration of polymorphonuclear leukocytes (PMN) associated with some edema in guinea pigs. To determine the substance inducing infiltration of PMN, lipids of avirulent strain Shibaura were extracted with chloroform--methanol--water after washing with acetone. The lipids comprised 28% of the dry weight of the cell. When the lipids were further separated into water--methanol and chloroform fractions, the most severe PMN infiltration of all samples was seen in the skin inoculated with extract recovered from the chloroform fraction. Neutral and polar lipids were detected after thin-layer chromatography of the chloroform extract. Neutral lipids were detected as free fatty acids (FFA). Fatty acids contained in polar lipids were mainly palmitic acid and palmitoleic acid, whereas FFA comprised 66.5% oleic acid. Skin reactions consisting of marked edema with mild infiltration of PMN were elicited by FFA. There was no obvious difference between a commercially available FFA mixture and the FFA from avirulent strain Shibaura. These observations suggest that FFA may play some role in the pathogenesis of leptospirosis.

The role of extracellular bactericidal factors in pulmonary host defense.

Extracellular killing provides an attractive hypothesis to explain the rapid alveolar killing of inhaled bacterial pathogens in the absence of conventional opsonins for phagocytosis. Some evidence of extracellular killing of inhaled pneumococci has been obtained using histologic studies and bronchoalveolar lavage. Although studies of the antimicrobial activity of lung lavage fluid in vitro have given variable results, a variety of antimicrobial factors have been detected in lung lavage fluids. Studies of lysozyme, peptides, iron binding proteins, free fatty acids and other factors that are found free in lung lavage fluid indicate that some of these factors could be a part of extracellular pulmonary host defenses. However, their precise role is not known. A survey of mechanisms of extracellular killing shows that granulocytes, monocytes, macrophages, and T lymphocytes all have the capacity to kill extracellularly in vitro in some circumstances. It remains to be determined which of these diverse mechanisms operate within the lung and how they function in relationship to other host defenses.

Metabolic immunodepression which increases the risk of cancer.

When some metabolic changes occur--namely, when blood levels of free fatty acids (F.F.A.), insulin, cholesterol, or triglycerides rise--cellular immunity becomes depressed. These metabolic changes also promote the division of somatic, non-lymphoid cells, which in turn increases the possibility of malignant transformation. The diametrically opposite effects of these metabolic changes on non-lymphoid cells, on the one hand, and on T-dependent lymphocytes and macrophages on the other, increase an individual's susceptibility to cancer. This normally occurs with pregnancy, rapid growth, stress, and ageing. Many chemical carcinogens raise the hypothalamic threshold to feedback control mechanisms, decrease glucose tolerance, and increase blood-insulin--i.e., all the main changes of "cancer-proneness". Changes in biogenic amine level in the hypothalamus during ageing and stress and after an exposure to some carcinogens are responsible for the elevation of hypothalamic threshold to feedback control.

Free fatty acids, complement activation, and polyclonal B-cell stimulation as factors in the immunopathogenesis of African trypanosomiasis.

Human and animal forms of African trypanosomiasis are characterised by sustained hypocomplementaemia, gross hypergammaglobulinaemia M, and profound immunosuppression. It is suggested that this hypocomplementaemia is probably due to the action of a trypanosome-derived complement-activating factor and that the elevated IgM levels may be the combined result of this decomplementation, together with a subsequent failure of the normal IgM-to-IgG antibody switch mechanism and polyclonal B-lymphocyte activation by a trypanosome-generated mitogen. The immunosuppression in this disease may be a result of the collective immunosuppressive effects of trypanosome-derived immune-modulating free fatty acids, polyclonally stimulating B-cell mitogen, and complement-activating factors.