The role of IL-6R/IL-6 axis in metabolic inflammation remains controversial. We determined the changes in adipose tissue expression of IL-6R and IL-6 in obese, overweight, and lean non-diabetic individuals. Subcutaneous adipose tissue biopsies were collected from 33 obese, 22 overweight, and 10 lean individuals and the expression of IL-6R, IL-6, TNF-α, MCP-1, IP-10, CD11b, CD163, and CD68 was detected by immunohistochemistry; results were also confirmed by real-time RT-PCR and confocal microscopy. The data were compared using unpaired t-test and the dependence between two variables was assessed by Pearson’s correlation test. Obese individuals showed higher IL-6R expression (103.8±4.807) in the adipose tissue as compared with lean/overweight (68.06±4.179) subjects (P<0.0001). The elevated IL-6R expression correlated positively with body mass index (BMI) (r=0.80 P<0.0001) and percent body fat (r=0.69 P=0.003). The increased IL-6R expression in obesity was also confirmed by RT-PCR (Obese: 3.921±0.712 fold; Lean/Overweight: 2.191±0.445 fold; P=0.0453) and confocal microscopy. IL-6 expression was also enhanced in obese adipose tissue (127.0±15.91) as compared with lean/overweight (86.69±5.25) individuals (P=0.03) which correlated positively with BMI (r=0.58 P=0.008). IL-6 mRNA expression was concordantly higher in obese (16.60±2.214 fold) versus lean/overweight (9.376±1.656 fold) individuals (P=0.0108). These changes in the IL-6R/IL-6 expression correlated positively with the adipose tissue expression of CD11b (IL-6R r=0.44 P=0.063; IL-6 r=0.77 P<0.0001), CD163 (IL-6R r=0.45 P=0.045; IL-6 r=0.55 P=0.013), TNF-α (IL-6R r=0.73 P=0.0003; IL-6 r=0.60 P=0.008), MCP-1 (IL-6R r=0.61 P=0.005; IL-6 r=0.63 P=0.004) and IP-10 (IL-6R r=0.41 P=0.08; IL-6 r=0.50 P=0.026). It was, therefore, concluded that obesity was a positive modulator of IL-6R and IL-6 expression in the adipose tissue which might be a contributory mechanism to induce metabolic inflammation.

The chemokine CCL2 (also known as MCP-1) is a key regulator of monocyte infiltration into adipose tissue, which plays a central role in the pathophysiology of obesity-associated inflammation and insulin resistance. It remains unclear how CCL2 production is upregulated in obese humans and rodents. Because elevated levels of the free fatty acid (FFA) palmitate and TNF-α have been reported in obesity, we studied whether these agents interact to trigger CCL2 production. Our data show that combined treatment of THP-1 and primary human monocytic cells with palmitate and TNF-α led to a marked increase in CCL2 production compared to either treatment alone. Mechanistically, we found that cooperative production of CCL2 by palmitate and TNF-α did not require MyD88, but was attenuated by blocking TLR4 or TRIF. IRF3-deficient cells did not show synergistic CCL2 production in response to palmitate/TNF-α. Moreover, IRF3 activation by poly I:C augmented TNF-α induced CCL2 secretion. Interestingly, elevated NF-κB/AP-1 activity resulting from palmitate/TNF-α co-stimulation was attenuated by TRIF/IRF3 inhibition. Diet-induced C57BL/6 obese mice with high FFAs levels showed strong correlation between TNF-α and CCL2 in plasma and adipose tissue and, as expected, also showed increased adipose tissue macrophage accumulation compared to lean mice. Similar results were observed in the adipose tissue samples from obese humans. Overall, our findings support a model in which elevated FFAs in obesity create a milieu for TNF-α to trigger CCL2 production via the TLR4/TRIF/IRF3 signaling cascade, representing a potential contribution of FFAs to metabolic inflammation.

Background/Aims: Obese individuals are known to have increased Matrix metalloproteinase (MMP)-9 plasma levels and MMP-9 is reported to play an important role in obesity-associated adipose tissue inflammation. Since in obesity, the levels of circulatory saturated free fatty acid (FFA) palmitate (palimitic acid) are increased and modulate the expression of inflammatory mediators, the role of palmitate in the regulation of MMP-9 remains unclear. Methods: Human monocytic cell line THP-1 and primary monocytes were stimulated with palmitate and TNF-α (positive control). MMP-9 expression was assessed with real time RT-PCR and ELISA. Signaling pathways were studied by using THP-1-XBlue™ cells, THP-1-XBlue™-defMyD cells, anti-TLR4 mAb and TLR4 siRNA. Phosphorylation of NF-kB and c-Jun was analyzed by Western blotting. Results: Here, we provide the evidence that palmitate induces MMP-9 expression at both mRNA (THP-1: 6.8 ± 1.2 Fold; P = 0.01; Primary monocytes: 5.9 ± 0.7 Fold; P = 0.0003) and protein (THP1: 1116 ±14 pg/ml; P<0.001; Primary monocytes: 1426 ± 13.8; P = 0.0005) levels in human monocytic cells. Palmitate-induced MMP-9 secretion was markedly suppressed by neutralizing anti-TLR-4 antibody (P < 0.05). Furthermore, genetic silencing of TLR4 by siRNA also significantly abrogated the palmitate-induced up-regulation of MMP-9. Additionally, MyD88^-/- THP-1 cells did not express MMP-9 in response to palmitate treatment. Increased NF-κB/AP-1 activity (P<0.05) was also observed in palmitate-treated THP-1 cells. Conclusion: Altogether, these results show that palmitate induces TLR4-dependent activation of MMP-9 gene expression, which requires the recruitment of MyD88 leading to activation of NF-kB/AP-1 transcription factors. Thus, our findings suggest that the palmitate-induced MMP-9 secretion might be an underlying mechanism of its increased levels in obesity and related metabolic inflammation.

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.

Obesity is associated with elevated levels of TNF-α and proinflammatory CD11c monocytes/macrophages. TNF-α mediated dysregulation in the plasticity of monocytes/macrophages is concomitant with pathogenesis of several inflammatory diseases, including metabolic syndrome, but the underlying mechanisms are incompletely understood. Since neutral sphingomyelinase-2 (nSMase2: SMPD3) is a key enzyme for ceramide production involved in inflammation, we investigated whether nSMase2 contributed to the inflammatory changes in the monocytes/macrophages induced by TNF-α. In this study, we demonstrate that the disruption of nSMase activity in monocytes/macrophages either by chemical inhibitor GW4869 or small interfering RNA (siRNA) against SMPD3 results in defects in the TNF-α mediated expression of CD11c. Furthermore, blockage of nSMase in monocytes/macrophages inhibited the secretion of inflammatory mediators IL-1β and MCP-1. In contrast, inhibition of acid SMase (aSMase) activity did not attenuate CD11c expression or secretion of IL-1β and MCP-1. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was also attenuated by the inhibition of nSMase2. Moreover, NF-kB/AP-1 activity was blocked by the inhibition of nSMase2. SMPD3 was elevated in PBMCs from obese individuals and positively corelated with TNF-α gene expression. These findings indicate that nSMase2 acts, at least in part, as a master switch in the TNF-α mediated inflammatory responses in monocytes/macrophages.

IntroductionThe proinflammatory cytokine IL‐18 is involved in the pathogenesis of metabolic syndrome. While the changes in IL‐18 are known, IL‐18R expression and relationship with IL‐18 and other inflammatory markers in the adipose tissue in obesity/type‐2 diabetes (T2D) remain unclear.MethodsWe, therefore, determined the adipose tissue expression of IL‐18R and IL‐18 mRNA/protein in lean, overweight, and obese individuals with and without T2D, 15 each, using qRT‐PCR, immunohistochemistry, and confocal microscopy. Data (mean ± SEM) were analyzed using unpaired t‐test and Pearson's correlation (r); all P values ≤0.05 were considered statistically significant.ResultsWe found the upregulated gene/protein expression of IL‐18R and IL‐18 in non‐diabetic obese/overweight as compared with lean individuals (P < 0.05). BMI correlated positively (P < 0.05) with the adipose tissue expression of IL‐18R (mRNA: r = 0.90 protein: r = 0.84) and IL‐18 (mRNA: r = 0.84 protein: r = 0.80). Similarly, in T2D individuals, gene and protein expression of IL‐18R/IL‐18 was significantly higher in obese as compared with overweight/lean individuals. The BMI was associated with the changes in both IL‐18R (mRNA: r = 0.55 protein: r = 0.50) and IL‐18 (mRNA: r = 0.53 protein: r = 0.57) expression. IL‐18R/IL‐18 gene expression in the adipose tissue was positively associated (P < 0.05) with local gene expression of other inflammatory markers including CD11c, CD86, CD68, CD163, TNF‐α, and CCL5. Homeostatic model assessment of insulin resistance (HOMA‐IR) was higher in diabetic/non‐diabetic obese and it correlated with BMI (P < 0.05). IL‐18R and IL‐18 mRNA/protein expression in obesity was associated with HOMA‐IR only in non‐diabetics.ConclusionsThe adipose tissue IL‐18R/IL‐18 expression is enhanced in obesity which associates with proinflammatory gene signature and insulin resistance in these individuals.

BackgroundThe emerging role of TLR2/4 as immuno-metabolic receptors points to key involvement of TLR/IL-1R/MyD88 pathway in obesity/type-2 diabetes (T2D). IL1R-associated kinase (IRAK)-1 is a critical adapter protein (serine/threonine kinase) of this signaling pathway. The changes in adipose tissue expression of IRAK-1 in obesity/T2D remain unclear. We determined modulations in IRAK-1 gene/protein expression in the subcutaneous adipose tissues from lean, overweight and obese individuals with or without T2D.MethodsA total of 49 non-diabetic (22 obese, 19 overweight and 8 lean) and 42 T2D (31 obese, 9 overweight and 2 lean) adipose tissue samples were obtained by abdominal subcutaneous fat pad biopsy and IRAK-1 expression was determined using real-time RT-PCR, immunohistochemistry, and confocal microscopy. IRAK-1 mRNA expression was compared with adipose tissue proinflammatory mediators (TNF-α, IL-6, IL-18), macrophage markers (CD68, CD11c, CD163), and plasma markers (CCL-5, C-reactive protein, adiponectin, and triglycerides). The data were analyzed using t test, Pearson’s correlation, and multiple stepwise linear regression test.ResultsIn non-diabetics, IRAK-1 gene expression was elevated in obese (P = 0.01) and overweight (P = 0.04) as compared with lean individuals and this increase correlated with body mass index (r = 0.45; P = 0.001) and fat percentage (r = 0.36; P = 0.01). In diabetics, IRAK-1 mRNA expression was also higher in obese as compared with lean subjects (P = 0.012). As also shown by immunohistochemistry/confocal microscopy in non-diabetics and by immunohistochemistry in diabetics, IRAK-1 protein expression was higher in obese than overweight and lean adipose tissues. IRAK-1 gene expression correlated positively/significantly with mRNAs of TNF-α (r = 0.46; P = 0.0008), IL-6 (r = 0.30; P = 0.03) and IL-18 (r = 0.31; P = 0.028) in non-diabetics; and only with TNF-α (r = 0.32; P = 0.03) in diabetics. IRAK-1 expression also correlated positively/significantly with CD68 (r = 0.32; P = 0.02), CD11c (r = 0.30; P = 0.03), and CD163 (r = 0.43; P = 0.001) in non-diabetics; and only with CD163 (r = 0.34; P = 0.02) in diabetics. IRAK-1 mRNA levels also correlated with plasma markers including CCL-5 (r = 0.39; P = 0.02), C-reactive protein (r = 0.48; P = 0.005), adiponectin (r = −0.36; P = 0.04), and triglycerides (r = 0.40; P = 0.02) in non-diabetics; and only with triglycerides (r = −0.36; P = 0.04) in diabetics. IRAK-1 expression related with TLR2 (r = 0.39; P = 0.007) and MyD88 (r = 0.36; P = 0.01) in non-diabetics; and MyD88 (r = 0.52; P = 0.0003) in diabetics.ConclusionsThe elevated IRAK-1 expression in obese adipose tissue showed consensus with local/circulatory inflammatory signatures and represented as a tissue marker for metabolic inflammation. The data have clinical significance as interventions causing IRAK-1 suppression may alleviate meta-inflammation in obesity/T2D.Electronic supplementary materialThe online version of this article (doi:10.1186/s13098-015-0067-7) contains supplementary material, wh...

Adipose tissue (AT) associated cytokines are involved in the development of chronic low-grade inflammation in obese individuals. IL-2, a pleiotropic cytokine, contributes to immune alterations during inflammation. However, the interaction between AT-IL-2 and other inflammatory biomolecules in obesity remains elusive. We investigated whether AT-IL-2 expression was associated with markers of inflammation and insulin resistance in overweight/obese individuals. Subcutaneous fat tissues were collected from 56 individuals (lean/overweight/obese) for RNA extraction. IL-2 and inflammatory mediators were quantified by qRT-PCR and immunohistochemistry. CRP was measured by ELISA. AT-IL-2 expression was higher in obese compared with lean individuals (P < 0.021) and correlated with BMI. IL-2 correlated with interleukins IL-8 and IL-12A (r = 0.333–0.481; p = 0.0001–0.029); as well as with chemokines and their receptors including CCL5, CCL19, CCR2 and CCR5 (r = 0.538–0.677; p < 0.0001). Moreover, IL-2 correlated with toll-like receptors (TLR2, TLR8, TLR10), interferon regulatory factor 5 (IRF5) and cluster of differentiation CD11c (r = 0.282–0.357; p < 0.039). Notably, IL-2 was associated positively with fasting blood glucose (FBG), HbA1c, TGL and CRP (r ≥ 0.423;P ≤ 0.007). In multiple regression analysis, IL-2 is an independent predictor of IL-8, IL-12A, TLR10, TGL and HbA1c. Overall, our data demonstrate that increased expression of the AT-IL-2, in obesity, may represent a novel biomarker for progression of metabolic inflammation and insulin-resistance.