TNF-α/Stearate Induced H3K9/18 Histone Acetylation Amplifies IL-6 Expression in 3T3-L1 Mouse Adipocytes.

Extensive evidence supports the connection between obesity-induced inflammation and the heightened expression of IL-6 adipose tissues. However, the mechanism underlying the IL-6 exacerbation in the adipose tissue remains unclear. There is general agreement that TNF-α and stearate concentrations are mildly elevated in adipose tissue in the state of obesity. We hypothesize that TNF-α and stearate co-treatment induce the increased expression of IL-6 in mouse adipocytes. We therefore aimed to determine IL-6 gene expression and protein production by TNF-α/stearate treated adipocytes and investigated the mechanism involved. To test our hypothesis, 3T3-L1 mouse preadipocytes were treated with TNF-α, stearate, or TNF-α/stearate. IL-6 gene expression was assessed by quantitative real-time qPCR. IL-6 protein production secreted in the cell culture media was determined by ELISA. Acetylation of histone was analyzed by Western blotting. Il6 region-associated histone H3 lysine 9/18 acetylation (H3K9/18Ac) was determined by ChIP-qPCR. 3T3-L1 mouse preadipocytes were co-challenged with TNF-α and stearate for 24 h, which led to significantly increased IL-6 gene expression (81 ± 2.1 Fold) compared to controls stimulated with either TNF-α (38 ± 0.5 Fold; p = 0.002) or stearate (56 ± 2.0 Fold; p = 0.013). As expected, co-treatment of adipocytes with TNF-α and stearate significantly increased protein production (338 ± 11 pg/mL) compared to controls stimulated with either TNF-α (28 ± 0.60 pg/mL; p = 0.001) or stearate (53 ± 0.20 pg/mL, p = 0.0015). Inhibition of histone acetyltransferases (HATs) with anacardic acid or curcumin significantly reduced the IL-6 gene expression and protein production by adipocytes. Conversely, TSA-induced acetylation substituted the stimulatory effect of TNF-α or stearate in their synergistic interaction for driving IL-6 gene expression and protein production. Mechanistically, TNF-α/stearate co-stimulation increased the promoter-associated histone H3 lysine 9/18 acetylation (H3K9/18Ac), rendering a transcriptionally permissive state that favored IL-6 expression at the transcriptional and translational levels. Our data represent a TNF-α/stearate cooperativity model driving IL-6 expression in 3T3-L1 cells via the H3K9/18Ac-dependent mechanism, with implications for adipose IL-6 exacerbations in obesity.

Endoplasmic Reticulum Stress Promotes the Expression of TNF-α in THP-1 Cells by Mechanisms Involving ROS/CHOP/HIF-1α and MAPK/NF-κB Pathways.

Obesity and metabolic syndrome involve chronic low-grade inflammation called metabolic inflammation as well as metabolic derangements from increased endotoxin and free fatty acids. It is debated whether the endoplasmic reticulum (ER) stress in monocytic cells can contribute to amplify metabolic inflammation; if so, by which mechanism(s). To test this, metabolic stress was induced in THP-1 cells and primary human monocytes by treatments with lipopolysaccharide (LPS), palmitic acid (PA), or oleic acid (OA), in the presence or absence of the ER stressor thapsigargin (TG). Gene expression of tumor necrosis factor (TNF)-α and markers of ER/oxidative stress were determined by qRT-PCR, TNF-α protein by ELISA, reactive oxygen species (ROS) by DCFH-DA assay, hypoxia-inducible factor 1-alpha (HIF-1α), p38, extracellular signal-regulated kinase (ERK)-1,2, and nuclear factor kappa B (NF-κB) phosphorylation by immunoblotting, and insulin sensitivity by glucose-uptake assay. Regarding clinical analyses, adipose TNF-α was assessed using qRT-PCR/IHC and plasma TNF-α, high-sensitivity C-reactive protein (hs-CRP), malondialdehyde (MDA), and oxidized low-density lipoprotein (OX-LDL) via ELISA. We found that the cooperative interaction between metabolic and ER stresses promoted TNF-α, ROS, CCAAT-enhancer-binding protein homologous protein (CHOP), activating transcription factor 6 (ATF6), superoxide dismutase 2 (SOD2), and nuclear factor erythroid 2-related factor 2 (NRF2) expression (p ≤ 0.0183),. However, glucose uptake was not impaired. TNF-α amplification was dependent on HIF-1α stabilization and p38 MAPK/p65 NF-κB phosphorylation, while the MAPK/NF-κB pathway inhibitors and antioxidants/ROS scavengers such as curcumin, allopurinol, and apocynin attenuated the TNF-α production (p ≤ 0.05). Individuals with obesity displayed increased adipose TNF-α gene/protein expression as well as elevated plasma levels of TNF-α, CRP, MDA, and OX-LDL (p ≤ 0.05). Our findings support a metabolic-ER stress cooperativity model, favoring inflammation by triggering TNF-α production via the ROS/CHOP/HIF-1α and MAPK/NF-κB dependent mechanisms. This study also highlights the therapeutic potential of antioxidants in inflammatory conditions involving metabolic/ER stresses.

ROS/TNF-α Crosstalk Triggers the Expression of IL-8 and MCP-1 in Human Monocytic THP-1 Cells via the NF-κB and ERK1/2 Mediated Signaling.

IL-8/MCP-1 act as neutrophil/monocyte chemoattractants, respectively. Oxidative stress emerges as a key player in the pathophysiology of obesity. However, it remains unclear whether the TNF-α/oxidative stress interplay can trigger IL-8/MCP-1 expression and, if so, by which mechanism(s). IL-8/MCP-1 adipose expression was detected in lean, overweight, and obese individuals, 15 each, using immunohistochemistry. To detect the role of reactive oxygen species (ROS)/TNF-α synergy as a chemokine driver, THP-1 cells were stimulated with TNF-α, with/without H2O2 or hypoxia. Target gene expression was measured by qRT-PCR, proteins by flow cytometry/confocal microscopy, ROS by DCFH-DA assay, and signaling pathways by immunoblotting. IL-8/MCP-1 adipose expression was significantly higher in obese/overweight. Furthermore, IL-8/MCP-1 mRNA/protein was amplified in monocytic cells following stimulation with TNF-α in the presence of H2O2 or hypoxia (p ˂ 0.0001). Synergistic chemokine upregulation was related to the ROS levels, while pre-treatments with NAC suppressed this chemokine elevation (p ≤ 0.01). The ROS/TNF-α crosstalk involved upregulation of CHOP, ERN1, HIF1A, and NF-κB/ERK-1,2 mediated signaling. In conclusion, IL-8/MCP-1 adipose expression is elevated in obesity. Mechanistically, ROS/TNF-α crosstalk may drive expression of these chemokines in monocytic cells by inducing ER stress, HIF1A stabilization, and signaling via NF-κB/ERK-1,2. NAC had inhibitory effect on oxidative stress-driven IL-8/MCP-1 expression, which may have therapeutic significance regarding meta-inflammation.

Short Chain Fatty Acid Acetate Increases TNFα-Induced MCP-1 Production in Monocytic Cells via ACSL1/MAPK/NF-κB Axis.

Short-chain fatty acid (SCFA) acetate, a byproduct of dietary fiber metabolism by gut bacteria, has multiple immunomodulatory functions. The anti-inflammatory role of acetate is well documented; however, its effect on monocyte chemoattractant protein-1 (MCP-1) production is unknown. Similarly, the comparative effect of SCFA on MCP-1 expression in monocytes and macrophages remains unclear. We investigated whether acetate modulates TNFα-mediated MCP-1/CCL2 production in monocytes/macrophages and, if so, by which mechanism(s). Monocytic cells were exposed to acetate with/without TNFα for 24 h, and MCP-1 expression was measured. Monocytes treated with acetate in combination with TNFα resulted in significantly greater MCP-1 production compared to TNFα treatment alone, indicating a synergistic effect. On the contrary, treatment with acetate in combination with TNFα suppressed MCP-1 production in macrophages. The synergistic upregulation of MCP-1 was mediated through the activation of long-chain fatty acyl-CoA synthetase 1 (ACSL1). However, the inhibition of other bioactive lipid enzymes [carnitine palmitoyltransferase I (CPT I) or serine palmitoyltransferase (SPT)] did not affect this synergy. Moreover, MCP-1 expression was significantly reduced by the inhibition of p38 MAPK, ERK1/2, and NF-κB signaling. The inhibition of ACSL1 attenuated the acetate/TNFα-mediated phosphorylation of p38 MAPK, ERK1/2, and NF-κB. Increased NF-κB/AP-1 activity, resulting from acetate/TNFα co-stimulation, was decreased by ACSL1 inhibition. In conclusion, this study demonstrates the proinflammatory effects of acetate on TNF-α-mediated MCP-1 production via the ACSL1/MAPK/NF-κB axis in monocytic cells, while a paradoxical effect was observed in THP-1-derived macrophages.

MIP-1α Induction by Palmitate in the Human Monocytic Cells Implicates TLR4 Signaling Mechanism.

BACKGROUND/AIMS: MIP-1α (macrophage inflammatory protein 1α)/CCL3 chemokine is associated with the adipose tissue inflammation in obesity. Both MIP-1α and free fatty acids are elevated in obesity/T2D. We asked if free fatty acid palmitate could modulate MIP1α expression in the human monocytic cells. METHODS: Human monocytic THP-1 cells and macrophages were stimulated with palmitate and TNF-α (positive control). MIP-1α expression was measured with real time RT-PCR, Flow Cytometry and ELISA. Signaling pathways were identified by using THP-1-XBlue™ cells, THP-1-XBlue™-defMyD cells, anti-TLR4 mAb and TLR4 siRNA. RESULTS: Our data show that palmitate induced significant increase in MIP1α production in monocytic THP-1 cells/macrophages. MIP-1α induction was significantly suppressed when cells were treated with anti-TLR4 antibody prior stimulation with palmitate. Using TLR4 siRNA, we further demonstrate that palmitate-induced MIP-1α expression in monocytic cells requires TLR4. Moreover, THP1 cells defective in MyD88, a major adaptor protein involved in TLR4 signaling, were unable to induce MIP-1α production in response to palmitate. Palmitate-induced MIP-1α expression was suppressed by inhibition of MAPK, NFkB and PI3K signaling pathways. In addition, palmitate-induced NF-κB/AP-1 activation was observed while production of MIP-1α. However, this activation of NF-κB/AP-1 was abrogated in MyD88 deficient cells. CONCLUSION: Overall, these results show that palmitate induces TLR4dependent MIP-1α expression requiring the MyD88 recruitment and activation of MAPK, NF-κB/AP-1 and PI3K signaling. It implies that the increased systemic levels of free fatty acid palmitate in obesity/T2D may contribute to metabolic inflammation through excessive production of MIP-1a.

Oxidative Stress Induces Expression of the Toll-Like Receptors (TLRs) 2 and 4 in the Human Peripheral Blood Mononuclear Cells: Implications for Metabolic Inflammation.

BACKGROUND/AIMS: Innate immune toll-like receptors (TLRs) are emerging as nutrient sensors. Oxidative stress in the adipose tissue in obesity acts as a critical early trigger of altered pathophysiology. TLR2/TLR4 adipose upregulation has been associated with insulin resistance in humans; however, it remains unclear whether oxidative stress can modulate expression of TLR2/4 and related immune-metabolic regulators (IRF3/5) in immune cells. We, therefore, assessed their expression along with proinflammatory cytokines in the human PBMC following induction of oxidative stress. METHODS: PBMC were isolated from blood of healthy donors using Ficoll-Paque method and cells were treated with H2O2 to induce oxidative stress. ROS was measured by DCFH-DA assay. Target gene and protein expression was determined using real-time RT-PCR and flow cytometry/confocal microscopy, respectively. TLR2/4 expression by H2O2 in presence of ROS-inhibitors or leptin/LPS/fatty acids was also assessed. Expression of phosphorylated/total ERK1/2, c-Jun, p38, and NF-κB was determined by western blotting. The data (mean±SEM) were compared using unpaired student's t-test or ANOVA; all P-values <0.05 were considered significant. RESULTS: TLR2/4 mRNA/protein expression was elevated by oxidative stress in PBMC compared to controls (P<0.001). This induction was abrogated by apocynin/N-acetyl cysteine treatments (P<0.01). H2O2-induced TLR2/4 gene expression was further enhanced by leptin, LPS, oleate, or palmitate (P<0.05). Oxidative stress also promoted expression of IRF3/5 and proinflammatory cytokines including IFN-γ, IL-1ß, IL-6, TNF-α, and MCP-1/CCL2. This oxidative stress in PBMC involved MAPK/NF-κB dependent signaling. CONCLUSION: Taken together, oxidative stress upregulates expression of TLR2/4, IRF3/5 and signature proinflammatory cytokines in PBMC, involving MAPK/NF-κB dependent signaling, all of which may have implications for metabolic inflammation.

The Cooperative Induction of CCL4 in Human Monocytic Cells by TNF-α and Palmitate Requires MyD88 and Involves MAPK/NF-κB Signaling Pathways.

Chronic low-grade inflammation, also known as metabolic inflammation, is a hallmark of obesity and parallels with the presence of elevated circulatory levels of free fatty acids and inflammatory cytokines/chemokines. CCL4/MIP-1ß chemokine plays a key role in the adipose tissue monocyte recruitment. Increased circulatory levels of TNF-α, palmitate and CCL4 are co-expressed in obesity. We asked if the TNF-α/palmitate could interact cooperatively to augment the CCL4 production in human monocytic cells and macrophages. THP-1 cells/primary macrophages were co-treated with TNF-α/palmitate and CCL4 mRNA/protein expression was assessed using qRT-PCR/ELISA. TLR4 siRNA, a TLR4 receptor-blocking antibody, XBlue™-defMyD cells and pathway inhibitors were used to decipher the signaling mechanisms. We found that TNF-α/palmitate co-stimulation augmented the CCL4 expression in monocytic cells and macrophages compared to controls (p < 0.05). TLR4 suppression or neutralization abrogated the CCL4 expression in monocytic cells. Notably, CCL4 cooperative induction in monocytic cells was: (1) Markedly less in MyD88-deficient cells, (2) IRF3 independent, (3) clathrin dependent and (4) associated with the signaling mechanism involving ERK1/2, c-Jun, JNK and NF-κB. In conclusion, TNF-α/palmitate co-stimulation promotes the CCL4 expression in human monocytic cells through the mechanism involving a TLR4-MyD88 axis and MAPK/NF-κB pathways. These findings unravel a novel mechanism of the cooperative induction of CCL4 by TNF-α and palmitate which could be relevant to metabolic inflammation.

TNF-α in Combination with Palmitate Enhances IL-8 Production via The MyD88- Independent TLR4 Signaling Pathway: Potential Relevance to Metabolic Inflammation.

Elevated levels of IL-8 (CXCL8) in obesity have been linked with insulin resistance and type 2 diabetes (T2D). The mechanisms that lead to the profound production of IL-8 in obesity remains to be understood. TNF-α and saturated free fatty acids (FFAs) are increased in obese humans and correlate with insulin resistance. Hence, we sought to investigate whether the cooccurrence of TNF-α and FFAs led to increase the production of IL-8 by human monocytes. We found that co-stimulation of human monocytes with palmitate and TNF-α led to increased IL-8 production as compared to those stimulated with palmitate or TNF-α alone. The synergistic production of IL-8 by TNF-α/palmitate was suppressed by neutralizing anti- Toll like receptor 4 (TLR4) antibody and by genetic silencing of TLR4. Both MyD88-deficient and MyD88-competent cells responded comparably to TNF-α/Palmitate. However, TIR-domain-containing adapter-inducing interferon (TRIF) inhibition or interferon regulatory transcription factor 3 (IRF3) knockdown partly blocked the synergistic production of IL-8. Our human data show that increased adipose tissue TNF-α expression correlated positively with IL-8 expression (r = 0.49, P = 0.001). IL-8 and TNF-α correlated positively with macrophage markers including CD68, CD163 and CD86 in adipose tissue. These findings suggest that the signaling cross-talk between saturated fatty acid palmitate and TNF-α may be a key driver in obesity-associated chronic inflammation via an excessive production of IL-8.

Palmitate Activates CCL4 Expression in Human Monocytic Cells via TLR4/MyD88 Dependent Activation of NF-κB/MAPK/ PI3K Signaling Systems.

BACKGROUND/AIMS: Obesity is associated with adipose tissue inflammation which plays a key role in the development of insulin resistance and type 2 diabetes (T2D). Saturated free fatty acids (SFAs) levels are found to be elevated in obesity and T2D. Chemokines are known to have potent inflammatory functions in a wide range of biological processes linked to immunological disorders. Since CCL4 (Chemokine (C-C motif) ligand 4), also known as macrophage inflammatory protein-1ß (MIP-1ß), plays an important role in the migration of monocytes into the adipose tissue, we investigated the expression of CCL4 in monocytic cells/macrophages following activation with free fatty acid palmitate. METHODS: Human monocytic cell line THP-1 and macrophages derived from THP-1 and primary monocytes were stimulated with palmitate and LPS (positive control). CCL4 expression and secretion were measured with real time RT-PCR and ELISA respectively. Signaling pathways were identified by using THP-1-XBlueTM cells, THP-1-XBlueTM-defMyD cells, anti-TLR4 mAb and TLR4 siRNA. RESULTS: Palmitate induces CCL4 expression at both mRNA and protein levels in human monocytic cells. Palmitate-induced CCL4 production was markedly suppressed by neutralizing anti-TLR-4 antibody. Additionally, silencing of TLR4 by siRNA also significantly suppressed the palmitate-induced up-regulation of CCL4. MyD88-deficient cells did not express CCL4 in response to palmitate treatment. Inhibition of NF-kB and MAPK pathways suppressed the palmitate mediated induction of CCL4. Moreover, induction of CCL4 was blocked by PI3 Kinase inhibitors LY294002 and wortmannin. CONCLUSION: Collectively, our results show that palmitate induces CCL4 expression via activation of the TLR4-MyD88/NF-kB/MAPK/ PI3K signaling cascade. Thus, our findings suggest that the palmitate-induced CCL4 production might be an underlying mechanism of metabolic inflammation.

Increased Expression of the Innate Immune Receptor TLR10 in Obesity and Type-2 Diabetes: Association with ROS-Mediated Oxidative Stress.

BACKGROUND/AIMS: Metabolic diseases such as obesity and type-2 diabetes (T2D) are known to be associated with chronic low-grade inflammation called metabolic inflammation together with an oxidative stress milieu found in the expanding adipose tissue. The innate immune Toll-like receptors (TLR) such as TLR2 and TLR4 have emerged as key players in metabolic inflammation; nonetheless, TLR10 expression in the adipose tissue and its significance in obesity/T2D remain unclear. METHODS: TLR10 gene expression was determined in the adipose tissue samples from healthy non-diabetic and T2D individuals, 13 each, using real-time RT-PCR. TLR10 protein expression was determined by immunohistochemistry, confocal microscopy, and flow cytometry. Regarding in vitro studies, THP-1 cells, peripheral blood mononuclear cells (PBMC), or primary monocytes were treated with hydrogen peroxide (H2O2) for induction of reactive oxygen species (ROS)-mediated oxidative stress. Superoxide dismutase (SOD) activity was measured using a commercial kit. Data (mean±SEM) were compared using unpaired student's t-test and P<0.05 was considered significant. RESULTS: The adipose tissue TLR10 gene/protein expression was found to be significantly upregulated in obesity as well as T2D which correlated with body mass index (BMI). ROS-mediated oxidative stress induced high levels of TLR10 gene/protein expression in monocytic cells and PBMC. In these cells, oxidative stress induced a time-dependent increase in SOD activity. Pre-treatment of cells with anti-oxidants/ROS scavengers diminished the expression of TLR10. ROS-induced TLR10 expression involved the nuclear factor-kappaB (NF-κB)/mitogen activated protein kinase (MAPK) signaling as well as endoplasmic reticulum (ER) stress. H2O2-induced oxidative stress interacted synergistically with palmitate to trigger the expression of TLR10 which associated with enhanced expression of proinflammatory cytokines/chemokine. CONCLUSION: Oxidative stress induces the expression of TLR10 which may represent an immune marker for metabolic inflammation.

Plasma fetuin-A/α2-HS-glycoprotein correlates negatively with inflammatory cytokines, chemokines and activation biomarkers in individuals with type-2 diabetes.

BACKGROUND: Fetuin-A/AHSH is a novel hepatokine that acts as a vascular calcification inhibitor and as an endogenous TLR-4 ligand. Fetuin-A may act as a positive or negative acute phase protein (APP) in disease conditions. The relationship between circulatory fetuin-A and inflammatory biomarkers in type-2 diabetes (T2D) remains controversial. Therefore, the purpose of this study was to determine the plasma fetuin-A levels in 53 T2D (BMI = 29.7 ± 4.5 kg/m2) and 72 non-diabetic individuals (BMI = 28.2 ± 5.8 kg/m2) using premixed 38-plex MAP human cytokine/chemokine magnetic bead immunoassays and the data (mean ± SEM) were statistically analyzed to determine Pearson's correlation (r) between fetuin-A and detected analytes; P-values ≤0.05 were considered significant. RESULTS: The data show that plasma fetuin-A levels were comparable in both groups (P = 0.27) and in T2D individuals, fetuin-A associated negatively (P ≤ 0.05) with a large number of proinflammatory cytokines/chemokines and activation biomarkers including TNF-α, IFN-α2, IFN-γ, IL-1α, IL-1ß, IL-1RA, IL-3, IL-4, IL-7, IL-9, IL-12p40/p70, IL-15, CCL-2, CCL-4, CCL-11, CCL-22, CXCL-8, CX3CL-1, EFF-2, EGF, G-CSF, GM-CSF, GRO, sCD40L, and VEGF. In non-diabetics, fetuin-A also correlated positively with certain TH2 cytokines (IL-5, IL-13) and chemokines (CCL-3, CCL-5, CCL-7). Notably, in vitro fetuin-A production was significantly suppressed in HepG2 cells treated with TNF-α, IL-1ß, and IFN-γ which supported the clinical findings of a negative association between fetuin A and inflammatory mediators. CONCLUSIONS: The negative association between circulatory fetuin-A and systemic inflammatory mediators in T2D patients suggests that plasma fetuin-A may have predictive significance as a negative APP in metabolic disease.

Gut Dysbiosis Shaped by Cocoa Butter-Based Sucrose-Free HFD Leads to Steatohepatitis, and Insulin Resistance in Mice.

BACKGROUND: High-fat diets cause gut dysbiosis and promote triglyceride accumulation, obesity, gut permeability changes, inflammation, and insulin resistance. Both cocoa butter and fish oil are considered to be a part of healthy diets. However, their differential effects on gut microbiome perturbations in mice fed high concentrations of these fats, in the absence of sucrose, remains to be elucidated. The aim of the study was to test whether the sucrose-free cocoa butter-based high-fat diet (C-HFD) feeding in mice leads to gut dysbiosis that associates with a pathologic phenotype marked by hepatic steatosis, low-grade inflammation, perturbed glucose homeostasis, and insulin resistance, compared with control mice fed the fish oil based high-fat diet (F-HFD). RESULTS: C57BL/6 mice (5-6 mice/group) were fed two types of high fat diets (C-HFD and F-HFD) for 24 weeks. No significant difference was found in the liver weight or total body weight between the two groups. The 16S rRNA sequencing of gut bacterial samples displayed gut dysbiosis in C-HFD group, with differentially-altered microbial diversity or relative abundances. Bacteroidetes, Firmicutes, and Proteobacteria were highly abundant in C-HFD group, while the Verrucomicrobia, Saccharibacteria (TM7), Actinobacteria, and Tenericutes were more abundant in F-HFD group. Other taxa in C-HFD group included the Bacteroides, Odoribacter, Sutterella, Firmicutes bacterium (AF12), Anaeroplasma, Roseburia, and Parabacteroides distasonis. An increased Firmicutes/Bacteroidetes (F/B) ratio in C-HFD group, compared with F-HFD group, indicated the gut dysbiosis. These gut bacterial changes in C-HFD group had predicted associations with fatty liver disease and with lipogenic, inflammatory, glucose metabolic, and insulin signaling pathways. Consistent with its microbiome shift, the C-HFD group showed hepatic inflammation and steatosis, high fasting blood glucose, insulin resistance, increased hepatic de novo lipogenesis (Acetyl CoA carboxylases 1 (Acaca), Fatty acid synthase (Fasn), Stearoyl-CoA desaturase-1 (Scd1), Elongation of long-chain fatty acids family member 6 (Elovl6), Peroxisome proliferator-activated receptor-gamma (Pparg) and cholesterol synthesis (ß-(hydroxy ß-methylglutaryl-CoA reductase (Hmgcr). Non-significant differences were observed regarding fatty acid uptake (Cluster of differentiation 36 (CD36), Fatty acid binding protein-1 (Fabp1) and efflux (ATP-binding cassette G1 (Abcg1), Microsomal TG transfer protein (Mttp) in C-HFD group, compared with F-HFD group. The C-HFD group also displayed increased gene expression of inflammatory markers including Tumor necrosis factor alpha (Tnfa), C-C motif chemokine ligand 2 (Ccl2), and Interleukin-12 (Il12), as well as a tendency for liver fibrosis. CONCLUSION: These findings suggest that the sucrose-free C-HFD feeding in mice induces gut dysbiosis which associates with liver inflammation, steatosis, glucose intolerance and insulin resistance.

TNFα induces matrix metalloproteinase-9 expression in monocytic cells through ACSL1/JNK/ERK/NF-kB signaling pathways.

Studies have established the association between increased plasma levels of matrix metalloproteinase (MMP)-9 and adipose tissue inflammation. Tumor necrosis factor α (TNFα) was elevated in obesity and is involved in the induction of MMP-9 in monocytic cells. However, the underlying molecular mechanism was incompletely understood. As per our recent report, TNFα mediates inflammatory responses through long-chain acyl-CoA synthetase 1 (ACSL1). Therefore, we further investigated the role of ACSL1 in TNFα-mediated MMP-9 secretion in monocytic cells. THP-1 cells and primary monocytes were used to study MMP-9 expression. mRNA and protein levels of MMP-9 were determined by qRT-PCR and ELISA, respectively. Signaling pathways were studied using Western blotting, inhibitors, and NF-kB/AP1 reporter cells. We found that THP-1 cells and primary human monocytes displayed increased MMP-9 mRNA expression and protein secretion after incubation with TNFα. ACSL1 inhibition using triacsin C significantly reduced the expression of MMP-9 in the THP-1 cells. However, the inhibition of ß-oxidation and ceramide biosynthesis did not affect the TNFα-induced MMP-9 production. Using small interfering RNA-mediated ACSL1 knockdown, we further confirmed that TNFα-induced MMP-9 expression/secretion was significantly reduced in ACSL1-deficient cells. TNFα-mediated MMP-9 expression was also significantly reduced by the inhibition of ERK1/ERK2, JNK, and NF-kB. We further observed that TNFα induced phosphorylation of SAPK/JNK (p54/46), ERK1/2 (p44/42 MAPK), and NF-kB p65. ACSL1 inhibition reduced the TNFα-mediated phosphorylation of SAPK/JNK, c-Jun, ERK1/2, and NF-kB. In addition, increased NF-κB/AP-1 activity was inhibited in triacsin C treated cells. Altogether, our findings suggest that ACSL1/JNK/ERK/NF-kB axis plays an important role in the regulation of MMP-9 induced by TNFα in monocytic THP-1 cells.

IFN-γ and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation.

Background: Overexpression of CCL2 (MCP-1) has been implicated in pathogenesis of metabolic conditions, such as obesity and T2D. However, the mechanisms leading to increased CCL2 expression in obesity are not fully understood. Since both IFN-γ and LPS levels are found to be elevated in obesity and shown to be involved in the regulation of metabolic inflammation and insulin resistance, we investigated whether these two agents could synergistically trigger the expression of CCL2 in obesity. Methods: Monocytes (Human monocytic THP-1 cells) were stimulated with IFN-γ and LPS. CCL2 gene expression was determined by real-time RT-PCR. CCL2 protein was determined by ELISA. Signaling pathways were identified by using epigenetic inhibitors and STAT1 siRNA. Acetylation of H3K27 was analyzed by Western blotting. The acetylation level of histone H3K27 in the transcriptional initiation region of CCL2 gene was determined by ChIP-qPCR. Results: Our results show that the co-incubation of THP-1 monocytes with IFN-γ and LPS significantly enhanced the expression of CCL2, compared to treatment with IFN-γ or LPS alone. Similar results were obtained using primary monocytes and macrophages. Interestingly, IFN-γ priming was found to be more effective than LPS priming in inducing synergistic expression of CCL2. Moreover, STAT1 deficiency significantly suppressed this synergy for CCL2 expression. Mechanistically, we showed that IFN-γ priming induced acetylation of lysine 27 on histone 3 (H3K27ac) in THP-1 cells. Chromatin immunoprecipitation (ChIP) assay followed by qRT-PCR revealed increased H3K27ac at the CCL2 promoter proximal region, resulting in stabilized gene expression. Furthermore, inhibition of histone acetylation with anacardic acid suppressed this synergistic response, whereas trichostatin A (TSA) could substitute IFN-γ in this synergy. Conclusion: Our findings suggest that IFN-γ, in combination with LPS, has the potential to augment inflammation via the H3K27ac-mediated induction of CCL2 in monocytic cells in the setting of obesity.

MIP-1α Expression Induced by Co-Stimulation of Human Monocytic Cells with Palmitate and TNF-α Involves the TLR4-IRF3 Pathway and Is Amplified by Oxidative Stress.

Metabolic inflammation is associated with increased expression of saturated free fatty acids, proinflammatory cytokines, chemokines, and adipose oxidative stress. Macrophage inflammatory protein (MIP)-1α recruits the inflammatory cells such as monocytes, macrophages, and neutrophils in the adipose tissue; however, the mechanisms promoting the MIP-1α expression remain unclear. We hypothesized that MIP-1α co-induced by palmitate and tumor necrosis factor (TNF)-α in monocytic cells/macrophages could be further enhanced in the presence of reactive oxygen species (ROS)-mediated oxidative stress. To investigate this, THP-1 monocytic cells and primary human macrophages were co-stimulated with palmitate and TNF-α and mRNA and protein levels of MIP-1α were measured by using quantitative reverse transcription, polymerase chain reaction (qRT-PCR) and commercial enzyme-linked immunosorbent assays (ELISA), respectively. The cognate receptor of palmitate, toll-like receptor (TLR)-4, was blunted by genetic ablation, neutralization, and chemical inhibition. The involvement of TLR4-downstream pathways, interferon regulatory factor (IRF)-3 or myeloid differentiation (MyD)-88 factor, was determined using IRF3-siRNA or MyD88-deficient cells. Oxidative stress was induced in cells by hydrogen peroxide (H2O2) treatment and ROS induction was measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. The data show that MIP-1α gene/protein expression was upregulated in cells co-stimulated with palmitate/TNF-α compared to those stimulated with either palmitate or TNF-α (P < 0.05). Further, TLR4-IRF3 pathway was implicated in the cooperative induction of MIP-1α in THP-1 cells, and this cooperativity between palmitate and TNF-α was clathrin-dependent and also required signaling through c-Jun and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Notably, ROS itself induced MIP-1α and could further promote MIP-1α secretion together with palmitate and TNF-α. In conclusion, palmitate and TNF-α co-induce MIP-1α in human monocytic cells via the TLR4-IRF3 pathway and signaling involving c-Jun/NF-κB. Importantly, oxidative stress leads to ROS-driven MIP-1α amplification, which may have significance for metabolic inflammation.

Increased Adipose Tissue Expression of Interferon Regulatory Factor (IRF)-5 in Obesity: Association with Metabolic Inflammation.

Interferon regulatory factor (IRF)-5 is known to be involved in M1 macrophage polarization, however, changes in the adipose expression of IRF5 in obesity and their relationship with the local expression of proinflammatory cytokines/chemokines are unknown. Therefore, IRF5 gene expression was determined in the subcutaneous adipose tissue samples from 53 non-diabetic individuals (6 lean, 18 overweight, and 29 obese), using real-time RT-PCR. IRF5 protein expression was also assessed using immunohistochemistry and/or confocal microscopy. Adipose gene expression of signature immune metabolic markers was also determined and compared with adipose IRF5 gene expression. Systemic levels of C-reactive protein and adiponectin were measured by ELISA. The data show that adipose IRF5 gene (P = 0.008) and protein (P = 0.004) expression was upregulated in obese compared with lean individuals. IRF5 expression changes correlated positively with body mass index (BMI; r = 0.37/P = 0.008) and body fat percentage (r = 0.51/P = 0.0004). In obese, IRF5 changes associated positively with HbA1c (r = 0.41/P = 0.02). A good agreement was found between gene and protein expression of IRF5 in obese subjects (r = 0.65/P = 0.001). IRF5 gene expression associated positively with adipose inflammatory signatures including local expression of TNF-α, IL-6, CXCL8, CCL-2/5, IL-1ß, IL-18, CXCL-9/10, CCL7, CCR-1/2/5, TLR-2/7/8/9, IRF3, MyD88, IRAK-1, and inflammatory macrophage markers (P < 0.05). Interestingly, IRF5 gene expression correlated positively with CRP (r = 0.37, P = 0.03) and negatively with adiponectin levels (r = -0.43, P = 0.009). In conclusion, elevated adipose IRF5 expression in obesity concurs with the typical inflammatory signatures, locally and systemically. Hence, the IRF5 upregulation may represent a novel adipose tissue marker for metabolic inflammation.

TLR4/MyD88 -mediated CCL2 production by lipopolysaccharide (endotoxin): Implications for metabolic inflammation.

BACKGROUND: Obese human and mice were reported to have higher circularity endotoxin (LPS) levels as compared to their lean counter parts. The current study was aimed to reveal the molecular mechanisms underlying the LPS mediated induction of CCL2 in human monocytes/macrophages. METHODS: Human monocytic cell line THP-1, THP-1 cells derived macrophages and primary macrophages were treated with LPS and TNF-α (positive control). CCL2 expression was determined with real-time RT-PCR and ELISA. THP-1-XBlue™ cells, THP-1-XBlue™-defMyD cells, TLR4 neutralization antibody, TLR4 siRNA and inhibitors for NF-kB and MAPK were used to study the signaling pathways. Phosphorylation of NF-kB and c-Jun was analyzed by ELISA. RESULTS: LPS upregulates CCL2 expression at both mRNA (THP-1: 23.40 ± .071 Fold, P < 0.0001; THP-1-derived macrophages: 103 ± 0.56 Fold, < 0.0001; Primary macrophages: 48 ± 1.41 Fold, P < 0.0005) and protein (THP1 monocytes:1048 ± 5.67 pg/ml, P < 0.0001; THP-1-derived macrophages; 2014 ± 2.12, P = 0.0001; Primary macrophages: 859.5 ± 3.54, P < 0.0001) levels in human monocytic cells/macrophages. Neutralization of TLR4 blocked LPS-induced CCL-2 secretion (P < 0.0001). Silencing of TLR4 by siRNA also significantly reduced LPS-induced CCL-2 production. Furthermore, MyD88-Knockout cells treated with LPS did not produce CCL-2. NF-kB and c-Jun phosphorylation was noted in LPS treated cells. CONCLUSION: Overall, our data reveal that LPS induces CCL-2 in monocytes/macrophages via TLR4/MyD88 signaling which leads to the activation of NF-kB/AP-1 transcription factors.

Increased circulatory levels of fractalkine (CX3CL1) are associated with inflammatory chemokines and cytokines in individuals with type-2 diabetes.

BACKGROUND: Fractalkine (CX3CL1) is involved in the development of numerous inflammatory conditions including metabolic diseases. However, changes in the circulatory fractalkine levels in type-2 diabetes (T2D) and their relationship with inflammatory chemokines/cytokines remain unclear. The aim of the study was to determine the T2D-associated modulations in plasma fractalkine levels and investigate their relationship with circulatory chemokines/cytokines. METHODS: A total of 47 plasma samples were collected from 23 T2D and 24 non-diabetic individuals selected over a wide range of body mass index (BMI). Clinical metabolic parameters were determined using standard commercial kits. Fractalkine and chemokines/cytokines were measured using Luminex X-MAP® technology. C-reactive protein (CRP) was measured by ELISA. The data were compared using unpaired t-test and the dependence between two variables was assessed by Pearson's correlation coefficient (r). RESULTS: Plasma fractalkine levels were significantly higher (P = 0.005) in T2D patients (166 ± 14.22 pg/ml) as compared with non-diabetics (118 ± 8.90 pg/ml). In T2D patients, plasma fractalkine levels correlated positively (P ≤ 0.05) with inflammatory chemokines/cytokines including CCL3 (r = 0.52), CCL4 (r = 0.85), CCL11 (r = 0.51), CXCL1 (r = 0.67), G-CSF (r = 0.91), IFN-α2 (r = 0.97), IL-17A (r = 0.79), IL-1ß (r = 0.97), IL-12P70 (r = 0.90), TNF-α (r = 0.58), and IL-6 (r = 0.60). In non-diabetic individuals, fractalkine levels correlated (P ≤ 0.05) with those of CCL4 (r = 0.49), IL-1ß (r = 0.73), IL-12P70 (r = 0.41), and TNF-α (r = 0.50). Notably, plasma fractalkine levels in T2D patients associated with systemic inflammation (CRP) (r = 0.65, P = 0.02). CONCLUSIONS: The altered plasma fractalkine levels associate differentially with inflammatory chemokines/cytokines in T2D patients which may have implications for T2D immunopathogenesis.