Saturated fatty acids can activate Toll-like receptor 2 (TLR2) and TLR4 but polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA) inhibit the activation. Lipopolysaccharides (LPS) and ...lipopetides, ligands for TLR4 and TLR2, respectively, are acylated by saturated fatty acids. Removal of these fatty acids results in loss of their ligand activity suggesting that the saturated fatty acyl moieties are required for the receptor activation. X-ray crystallographic studies revealed that these saturated fatty acyl groups of the ligands directly occupy hydrophobic lipid binding domains of the receptors (or co-receptor) and induce the dimerization which is prerequisite for the receptor activation. Saturated fatty acids also induce the dimerization and translocation of TLR4 and TLR2 into lipid rafts in plasma membrane and this process is inhibited by DHA. Whether saturated fatty acids induce the dimerization of the receptors by interacting with these lipid binding domains is not known. Many experimental results suggest that saturated fatty acids promote the formation of lipid rafts and recruitment of TLRs into lipid rafts leading to ligand independent dimerization of the receptors. Such a mode of ligand independent receptor activation defies the conventional concept of ligand induced receptor activation; however, this may enable diverse non-microbial molecules with endogenous and dietary origins to modulate TLR-mediated immune responses. Emerging experimental evidence reveals that TLRs play a key role in bridging diet-induced endocrine and metabolic changes to immune responses.
Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report suggested that SFA-induced TLR ...activation in cell culture systems is due to contaminants in BSA used for solubilizing fatty acids. This report raised doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA solubilization induced phosphorylation of inhibitor of nuclear factor-κB α, c-Jun N-terminal kinase (JNK), p44/42 mitogen-activated-kinase (ERK), and nuclear factor-κB subunit p65, and TLR target gene expression in THP1 monocytes or RAW264.7 macrophages, respectively, when cultured in low FBS (0.25%) medium. C12:0 induced NFκB activation through TLR2 dimerized with TLR1 or TLR6, and through TLR4. Because BSA was not used in these experiments, contaminants in BSA have no relevance. Unlike in suspension cells (THP-1), BSA-solubilized C16:0 instead of sodium C16:0 is required to induce TLR target gene expression in adherent cells (RAW264.7). C16:0-BSA transactivated TLR2 dimerized with TLR1 or TLR6 and through TLR4 as seen with C12:0. These results and additional studies with the LPS sequester polymixin B and in MyD88−/− macrophages indicated that SFA-induced activation of TLR2 or TLR4 is a fatty acid-specific effect, but not due to contaminants in BSA or fatty acid preparations.
Acylcarnitines activate proinflammatory signaling pathways Rutkowsky, Jennifer M; Knotts, Trina A; Ono-Moore, Kikumi D ...
American journal of physiology: endocrinology and metabolism,
06/2014, Volume:
306, Issue:
12
Journal Article
Peer reviewed
Open access
Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and ...long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed D,L isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25 μM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.
Integration of sensory and molecular inputs from the environment shapes animal behaviour. A major site of exposure to environmental molecules is the gastrointestinal tract, in which dietary ...components are chemically transformed by the microbiota
and gut-derived metabolites are disseminated to all organs, including the brain
. In mice, the gut microbiota impacts behaviour
, modulates neurotransmitter production in the gut and brain
, and influences brain development and myelination patterns
. The mechanisms that mediate the gut-brain interactions remain poorly defined, although they broadly involve humoral or neuronal connections. We previously reported that the levels of the microbial metabolite 4-ethylphenyl sulfate (4EPS) were increased in a mouse model of atypical neurodevelopment
. Here we identified biosynthetic genes from the gut microbiome that mediate the conversion of dietary tyrosine to 4-ethylphenol (4EP), and bioengineered gut bacteria to selectively produce 4EPS in mice. 4EPS entered the brain and was associated with changes in region-specific activity and functional connectivity. Gene expression signatures revealed altered oligodendrocyte function in the brain, and 4EPS impaired oligodendrocyte maturation in mice and decreased oligodendrocyte-neuron interactions in ex vivo brain cultures. Mice colonized with 4EP-producing bacteria exhibited reduced myelination of neuronal axons. Altered myelination dynamics in the brain have been associated with behavioural outcomes
. Accordingly, we observed that mice exposed to 4EPS displayed anxiety-like behaviours, and pharmacological treatments that promote oligodendrocyte differentiation prevented the behavioural effects of 4EPS. These findings reveal that a gut-derived molecule influences complex behaviours in mice through effects on oligodendrocyte function and myelin patterning in the brain.
TAR DNA-binding protein 43 (TDP-43) aggregation is the most common pathological hallmark in frontotemporal dementia (FTD) and characterizes nearly all patients with motor neuron disease (MND). The ...earliest stages of TDP-43 pathobiology are not well-characterized, and whether neurodegeneration results from TDP-43 loss-of-function or aggregation remains unclear. In the behavioral variant of FTD (bvFTD), patients undergo selective dropout of von Economo neurons (VENs) and fork cells within the frontoinsular (FI) and anterior cingulate cortices. Here, we examined TDP-43 pathobiology within these vulnerable neurons in the FI across a clinical spectrum including 17 patients with sporadic bvFTD, MND, or both. In an exploratory analysis based on our initial observations, we further assessed ten patients with
C9orf72
-associated bvFTD/MND. VENs and fork cells showed early, disproportionate TDP-43 aggregation that correlated with anatomical and clinical severity, including loss of emotional empathy. The presence of a TDP-43 inclusion was associated with striking nuclear and somatodendritic atrophy. An intriguing minority of neurons lacked detectable nuclear TDP-43 despite the apparent absence of a cytoplasmic TDP-43 inclusion. These cells showed neuronal atrophy comparable to inclusion-bearing neurons, suggesting that the loss of nuclear TDP-43 function promotes neurodegeneration, even when TDP-43 aggregation is inconspicuous or absent.
Selective Laser Sintering (SLS) is an additive manufacturing process that uses a laser to fuse powdered starting materials into solid 3D structures. Despite the potential for fabrication of complex, ...high-resolution structures with SLS using diverse starting materials (including biomaterials), prohibitive costs of commercial SLS systems have hindered the wide adoption of this technology in the scientific community. Here, we developed a low-cost, open-source SLS system (OpenSLS) and demonstrated its capacity to fabricate structures in nylon with sub-millimeter features and overhanging regions. Subsequently, we demonstrated fabrication of polycaprolactone (PCL) into macroporous structures such as a diamond lattice. Widespread interest in using PCL for bone tissue engineering suggests that PCL lattices are relevant model scaffold geometries for engineering bone. SLS of materials with large powder grain size (~500 μm) leads to part surfaces with high roughness, so we further introduced a simple vapor-smoothing technique to reduce the surface roughness of sintered PCL structures which further improves their elastic modulus and yield stress. Vapor-smoothed PCL can also be used for sacrificial templating of perfusable fluidic networks within orthogonal materials such as poly(dimethylsiloxane) silicone. Finally, we demonstrated that human mesenchymal stem cells were able to adhere, survive, and differentiate down an osteogenic lineage on sintered and smoothed PCL surfaces, suggesting that OpenSLS has the potential to produce PCL scaffolds useful for cell studies. OpenSLS provides the scientific community with an accessible platform for the study of laser sintering and the fabrication of complex geometries in diverse materials.
The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) ...and TLR2. The results from our previous studies demonstrated that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4. However, the underlying mechanism has not been understood. Here, we report for the first time that the saturated fatty acid lauric acid induced dimerization and recruitment of TLR4 into lipid rafts, however, dimerization was not observed in non-lipid raft fractions. Similarly, LPS and lauric acid enhanced the association of TLR4 with MD-2 and downstream adaptor molecules, TRIF and MyD88, into lipid rafts leading to the activation of downstream signaling pathways and target gene expression. However, docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, inhibited LPS- or lauric acid-induced dimerization and recruitment of TLR4 into lipid raft fractions. Together, these results demonstrate that lauric acid and DHA reciprocally modulate TLR4 activation by regulation of the dimerization and recruitment of TLR4 into lipid rafts. In addition, we showed that TLR4 recruitment to lipid rafts and dimerization were coupled events mediated at least in part by NADPH oxidase-dependent reactive oxygen species generation. These results provide a new insight in understanding the mechanism by which fatty acids differentially modulate TLR4-mediated signaling pathway and consequent inflammatory responses which are implicated in the development and progression of many chronic diseases.
Many studies have shown that TLR4- and TLR2-deficient mice are protected from high-fat diet-induced inflammation and insulin resistance, suggesting that saturated fatty acids derived from the ...high-fat diet activate TLR-mediated proinflammatory signaling pathways and induce insulin resistance. However, evidence that palmitic acid, the major dietary saturated fatty acid, can directly activate TLR has not been demonstrated. In this article, we present multiple lines of evidence showing that palmitic acid directly activates TLR2, a major TLR expressed on human monocytes, by inducing heterodimerization with TLR1 in an NADPH oxidase-dependent manner. Dimerization of TLR2 with TLR1 was inhibited by the n-3 fatty acid docosahexaenoic acid. Activation of TLR2 by palmitic acid leads to expression of pro-IL-1β that is cleaved by caspase-1, which is constitutively present in monocytes, to release mature IL-1β. Our results reveal mechanistic insight about how palmitic acid activates TLR2, upregulates NALP3 expression, and induces inflammasome-mediated IL-1β production in human monocytes, which can trigger enhanced inflammation in peripheral tissues, and suggest that these processes are dynamically modulated by the types of dietary fat we consume.
Hyperglycemia-induced inflammation is central in diabetes complications, and monocytes are important in orchestrating these effects. Toll-like receptors (TLRs) play a key role in innate immune ...responses and inflammation. However, there is a paucity of data examining the expression and activity of TLRs in hyperglycemic conditions. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression and mechanism of their induction in monocytic cells under high-glucose conditions.
High glucose (15 mmol/l) significantly induced TLR2 and TLR4 expression in THP-1 cells in a time- and dose-dependent manner (P < 0.05). High glucose increased TLR expression, myeloid differentiation factor 88, interleukin-1 receptor-associated kinase-1, and nuclear factor-kappaB (NF-kappaB) p65-dependent activation in THP-1 cells. THP-1 cell data were further confirmed using freshly isolated monocytes from healthy human volunteers (n = 10).
Pharmacological inhibition of protein kinase C (PKC) activity and NADPH oxidase significantly decreased TLR2 and TLR4 mRNA and protein (P < 0.05). Knocking down both TLR2 and TLR4 in the cells resulted in a 76% (P < 0.05) decrease in high-glucose-induced NF-kappaB activity, suggesting an additive effect. Furthermore, PKC-alpha knockdown decreased TLR2 by 61% (P < 0.05), whereas inhibition of PKC-delta decreased TLR4 under high glucose by 63% (P < 0.05). Small inhibitory RNA to p47Phox in THP-1 cells abrogated high-glucose-induced TLR2 and TLR4 expression. Additional studies revealed that PKC-alpha, PKC-delta, and p47Phox knockdown significantly abrogated high-glucose-induced NF-kappaB activation and inflammatory cytokine secretion.
Collectively, these data suggest that high glucose induces TLR2 and -4 expression via PKC-alpha and PKC-delta, respectively, by stimulating NADPH oxidase in human monocytes.
Impairment of vasodilator action of insulin is associated with endothelial dysfunction and insulin resistance. Activation of Toll-like receptor 4 (TLR4) induces proinflammatory response and ...endoplasmic reticulum (ER) stress. Saturated fatty acids (SFA) activate TLR4, which induces ER stress and endothelial dysfunction. Therefore, we determined whether TLR4-mediated ER stress is an obligatory step mediating SFA-induced endothelial dysfunction. Palmitate stimulated proinflammatory responses and ER stress, and this was suppressed by knockdown of TLR4 in primary human aortic endothelial cells (HAEC). Next, we examined the role of TLR4 in vasodilatory responses in intact vessels isolated from wild-type (WT, C57BL/6) and TLR4-KO mice after feeding high-fat (HFD) or normal chow diet (NCD) for 12 wk. Arterioles isolated from HFD WT mice exhibited impaired insulin-stimulated vasodilation compared with arterioles isolated from NCD WT mice. Deficiency of TLR4 was protective from HFD-induced impairment of insulin-stimulated vasodilation. There were no differences in acetylcholine (Ach)- or sodium nitroprusside (SNP)-stimulated vasodilation between the two groups. Furthermore, we examined whether ER stress is involved in SFA-induced impairment of vasodilator actions of insulin. Infusion of palmitate showed the impairment of vasodilatory response to insulin, which was ameliorated by coinfusion with tauroursodeoxycholic acid (TUDCA), an ER stress suppressor. Taken together, the results suggest that TLR4-induced ER stress may be an obligatory step mediating the SFA-mediated endothelial dysfunction.
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