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.

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.

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.

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.