Autophagy differentially regulates macrophage lipid handling depending on the lipid substrate (oleic acid vs. acetylated-LDL) and inflammatory activation state.

The regulation of lipid droplet (LD) dynamics by autophagy in naïve macrophages is complex: Inhibiting autophagosome initiation steps attenuates oleic acid (OA) induced LD (OA-LD) biogenesis, whereas interfering with later-autophagosome maturation/lysosomal steps accelerates OA-LD biogenesis rate, but not OA-LD degradation. Here we hypothesized that regulation of macrophage lipid handling by autophagy may be lipid-substrate and activation-state-specific. Using automated quantitative live-cell imaging, initial LD biogenesis rate was ~30% slower when the lipid source was acetylated low density lipoprotein (acLDL) compared to OA. Yet, both were similarly affected by triacsin-C, an inhibitor of acyl-CoA synthase, which inhibited, and etomoxir, an inhibitor of acylcarnitine palmitoyl transferase (fatty acid oxidation), which augmented, LD biogenesis rates. An autophagy inducing peptide, Tat-Beclin1, enhanced the degradation, and inhibited (by 37%) the biogenesis of acLDL induced LD (acLDL-LD). Yet, Tat-Beclin1 increased OA-LD biogenesis rate by 70%. When macrophages were pre-activated with LPS + INFG they exhibited increased autophagosome number and area, and reduced BECN1 and ATG14 protein levels, which associated with a markedly attenuated autophagic flux. Concomitantly, OA-LD and acLDL-LD biogenesis rates increased 3 and 7.4-fold, respectively, but could not be further modulated by Tat-Beclin1, as observed in non-activated/naïve macrophages. We propose that macrophage autophagy, and/or components of its machinery, differentially regulate LD/foam-cell biogenesis depending on the lipid-source, and that inflammatory activation uncouples autophagy from LD biogenesis.

α1-Antitrypsin Combines with Plasma Fatty Acids and Induces Angiopoietin-like Protein 4 Expression.

α1-Antitrypsin (A1AT) purified from human plasma upregulates expression and release of angiopoietin-like protein 4 (Angptl4) in adherent human blood monocytes and in human lung microvascular endothelial cells, providing a mechanism for the broad immune-regulatory properties of A1AT independent of its antiprotease activity. In this study, we demonstrate that A1AT (Prolastin), a potent inducer of Angptl4, contains significant quantities of the fatty acids (FA) linoleic acid (C18:2) and oleic acid (C18:1). However, only trace amounts of FAs were present in preparations that failed to increase Angplt4 expression, for example, A1AT (Zemaira) or M-type A1AT purified by affinity chromatography. FA pull-down assays with Western blot analysis revealed a FA-binding ability of A1AT. In human blood-adherent monocytes, A1AT-FA conjugates upregulated expression of Angptl4 (54.9-fold, p < 0.001), FA-binding protein 4 (FABP4) (11.4-fold, p < 0.001), and, to a lesser degree, FA translocase (CD36) (3.1-fold, p < 0.001) relative to A1AT devoid of FA (A1AT-0). These latter effects of A1AT-FA were blocked by inhibitors of peroxisome proliferator-activated receptor (PPAR) ß/δ (ST247) and PPARγ (GW9662). When compared with controls, cell pretreatment with ST247 diminished the effect of A1AT-LA on Angptl4 mRNA (11.6- versus 4.1-fold, p < 0.001) and FABP4 mRNA (5.4- versus 2.8-fold, p < 0.001). Similarly, preincubation of cells with GW9662 inhibited inducing effect of A1AT-LA on Angptl4 mRNA (by 2-fold, p < 0.001) and FABP4 mRNA (by 3-fold, p < 0.001). Thus, A1AT binds to FA, and it is this form of A1AT that induces Angptl4 and FABP4 expression via a PPAR-dependent pathway. These findings provide a mechanism for the unexplored area of A1AT biology independent of its antiprotease properties.

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.

An overview of the modulatory effects of oleic acid in health and disease.

Evidences in the last years have showed the effects of oleic acid (OA) in human health and disease. Olive oil, rich in oleic acid, is supposed to present modulatory effects in a wide physiological functions, while some studies also suggest a beneficial effect on cancer, autoimmune and inflammatory diseases, besides its ability to facilitate wound healing. Although the OA role in immune responses are still controversial, the administration of olive oil containing diets may improve the immune response associated to a more successful elimination of pathogens such as bacteria and fungi, by interfering in many components of this system such as macrophages, lymphocytes and neutrophils. Then, novel putative therapies for inflammatory and infectious diseases could be developed based on the characteristics presented by unsaturated fatty acids like OA. Finally, the purpose of this work was to review some of the modulatory effects of OA on inflammatory diseases and health, aiming at high lightening its potential role on the future establishment of novel therapeutic approaches for infections, inflammatory, immune, cardiovascular diseases or skin repair based on this fatty acid mainly found in the Mediterranean diet.

Acquired resistance to Listeria monocytogenes during a secondary infection in a murine model fed dietary lipids.

OBJECTIVE: The ω-3 polyunsaturated fatty acids can suppress immune system functions. This property may cause adverse effects by impairing host resistance to infection. The present study focused on estimating the impact of different dietary lipids on the immune system of mice after a secondary infection with Listeria monocytogenes. METHODS: BALB/c mice were divided into five dietary groups of olive oil, fish oil, sunflower oil, high-oleic sunflower oil, or low fat that was administered for 8 wk. The mice were immunized with 10(3) colony-forming units. Thirty-eight days later, each mouse was challenged with 10(4) colony-forming units. Mice survival and bacterial clearance from livers and spleens were determined. In addition, cytokine, chemokine, and adhesion molecule productions were quantified from the sera. RESULTS: Survival percentage in mice fed a fish oil diet was 100% and bacterial numbers from spleen were decreased at 72 h. Interleukin-12, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 productions were decreased. Levels of tumor necrosis factor-α and interferon-γ were increased, whereas macrophage inflammatory protein-1α (MIP-1α) production was unaltered. CONCLUSION: Immune defense in mice fed a fish oil diet was improved after secondary exposure, acquiring an adequate resistance. This result could be attributable to an increase of a T-helper type 1 response.

Humanized monoclonal antibody against the chemokine CXCL-8 (IL-8) effectively prevents acute lung injury.

As one of the most important endogenous chemotactic factors for neutrophils, the chemokine CXCL8 (IL-8) is involved in the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), characterized by massive neutrophil infiltration in the lung. Since neutralization of CXCL8 with polyclonal antibody has been shown to reduce the severity of ALI/ARDS in animal models, we explored the potential of humanized anti-CXCL8 antibody as a preventive or therapeutic agent for ALI. We used a 'two-hit' protocol to induce ALI in rabbits that showed extensive edema in the alveolar lumina, marked infiltration of neutrophils in the lung tissue, fibrin deposition in alveolar space, and destruction of pulmonary architecture, culminating in severe hypoxemia. Concomitant challenge with endotoxin after priming with oleic acid (OA) induced a marked elevation of CXCL8 level in bronchoalveolar lavage fluid. Treatment of the rabbits with a humanized anti-CXCL8 antibody prevented neutrophil infiltration in the lung in association with alleviated ALI syndrome. Our results indicate a promising future for utilization of humanized anti-CXCL8 antibody in the prevention and treatment of ALI and ARDS in human.

Loss of Stearoyl-CoA desaturase-1 attenuates adipocyte inflammation: effects of adipocyte-derived oleate.

BACKGROUND AND PURPOSE: Adipose inflammation is crucial to the pathogenesis of metabolic disorders. This study aimed at identify the effects of stearoyl-CoA desaturase-1 (SCD1) on the inflammatory response of a paracrine network involving adipocytes, macrophages, and endothelial cells. METHODS AND RESULTS: Loss of SCD1 in both genetic (Agouti) and diet-induced obesity (high-fat diet) mouse models prevented inflammation in white adipose tissue and improved its basal insulin signaling. In SCD1-deficient mice, white adipose tissue exhibited lower inflammation, with a reduced response to lipopolysaccharide in isolated adipocytes, but not in peritoneal macrophages. Mimicking the in vivo paracrine regulation of white adipose tissue inflammation, SCD1-deficient adipocyte-conditioned medium attenuated the induction of tumor necrosis factor (TNF) alpha/interleukin 1beta gene expression in RAW264.7 macrophages and reduced the adhesion response in endothelial cells. We further demonstrated that the adipocyte-derived oleate (18:1n9), but not palmitoleate (16:1n7), mediated the inflammation in macrophages and adhesion responses in endothelial cells. CONCLUSIONS: Loss of SCD1 attenuates adipocyte inflammation and its paracrine regulation of inflammation in macrophages and endothelial cells. The reduced oleate level is linked to the inflammation-modulating effects of SCD1 deficiency.

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.

Evaluation of irritancy and sensitization potential of metalworking fluid mixtures and components.

There are approximately 1.2 million workers exposed to metalworking fluids (MWF), which are used to reduce the heat and friction associated with industrial machining and grinding operations. Irritancy and sensitization potential of 9 National Toxicology Program (NTP) nominated MWFs (TRIM 229, TRIM VX, TRIM SC210, CIMTECH 310, CIMPERIAL 1070, CIMSTAR 3800, SYNTILO 1023, SUPEREDGE 6768, and CLEAREDGE 6584) were examined in a combined local lymph node assay (LLNA). BALB/c mice were dermally exposed to each MWF at concentrations up to 50%. Significant irritation was observed after dermal exposure to all MWFs except CIMTECH 310 and SYNTILO 1023. Of the 9 MWFs, 6 induced greater than a 3-fold increase in lymphocyte proliferation and 7 tested positive in the irritancy assay. TRIM VX yielded the lowest EC3 value (6.9%) with respect to lymphocyte proliferation. Chemical components of TRIM VX identified using HPLC were screened for sensitization potential using structural activity relationship (SAR) modeling and the LLNA. TOPKAT predicted triethanolamine (TEA) as a sensitizer while Derek for Windows predicted only 4-chloro-3-methylphenol (CMP) to be positive for sensitization. When tested in the LLNA only CMP (EC3 = 11.6%) and oleic acid (OA) (EC3 = 29.7%) were identified as sensitizers. Exposure to all tested TRIM VX components resulted in statistically significant irritation. An additive proliferative response was observed when mixtures of the two identified sensitizing TRIM VX components, OA and CMP, were tested in the LLNA. This is one explanation of why the EC3 value of TRIM VX, with respect to lymphocyte proliferation, is lower than those assigned to its sensitizing components.