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.

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.

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Purpose: Changes in plasma levels of T-cell cytokines may result in inflammatory disorders, including metabolic disease. The changes in circulatory IL-5 levels in obesity and type-2 diabetes (T2D) and their relationship with major Th1 and Th2 cytokines are poorly understood. The aim of this study was to determine obesity/T2D-associated perturbation in plasma IL-5 levels and investigate its association with Th1/Th2 cytokine levels in the circulation. Experiments: Plasma samples were collected from 43 diabetic and 22 non-diabetic individuals selected over a wide range of body mass index (BMI), and further classified as obese (BMI=31-40 kg/cm 2), overweight (BMI=26-30 kg/ cm 2), and lean (BMI=18-25 kg/cm 2). Clinical metabolic parameters were determined using commercial kits. IL-5 and Th1/ Th2 cytokines were measured using Luminex X-MAP® technology. The data were compared using unpaired t-test and dependence between two variables was assessed by Pearson's correlation coefficient (r). Results: In the diabetic cohort, circulatory IL-5 levels were significantly lower as compared to non-diabetic counterparts (Diabetic: 1.55 ± 0.23 pg/ml; Non diabetic: 3.31 ± 0.49 pg/ml; P=0.0004). Furthermore, we found that in diabetic individuals, plasma IL-5 levels were found to be significantly higher (P=0.036) in obese group (1.488 ± 0.21 pg/ ml) as compared with lean+overweight group (1.03 ± 0.13 pg/ml). In diabetic individuals, IL-5 levels correlated positively with Th1/Th2 cytokines including IL-12 (r=0.

BackgroundChronic inflammation is a hallmark of type-2 diabetes (T2D) and asthma. Monocyte chemoattractant protein (MCP)-1 or CCL-2 is a key regulator of monocytic infiltration into the sites of inflammation. The changes in systemic MCP-1 levels and its relationship with other inflammatory/immune markers in T2D patients with asthma remain unclear and have been addressed in this study.MethodsPlasma samples from 10 asthmatic T2D patients (Group I: BMI = 37.82 ± 9.75 kg/m2), 13 non-asthmatic T2D patients (Group II: BMI = 32.68 ± 4.63 kg/m2), 23 asthma patients without T2D (Group III: BMI = 30.14 ± 6.74 kg/m2), and 25 non-asthmatic non-diabetic controls (Group IV: BMI = 27.99 ± 5.86 kg/m2) were used to measure levels of MCP-1 and multiple cytokine/chemokine biomarkers with bead-based multiplex assays using Luminex technology. IgE/ECP were measured using commercial ELISA kits. Data (mean ± SEM) were compared using unpaired Student’s t-test and linear dependence between two variables was assessed by Pearson’s correlation coefficient (r) and P ≤ 0.05 was considered as significant.ResultsPlasma MCP-1 levels were significantly higher in Group I (337.95 ± 46.40 pg/mL) as compared with Group II (216.69 ± 17.30 pg/mL), Group III (251.76 ± 19.80 pg/mL), and Group IV (223.52 ± 133.36 pg/mL). MCP-1 showed differential association with tested biomarkers by correlating positively with: (i) IFN-α2, IL-10, fractalkine, and VEGF in T2D patients with asthma; (ii) IL-6 and GRO-α in T2D patients without asthma; (iii) MDC, IP-10, GM-CSF, FGF-2, and PDGF-AA/BB in patients with asthma only; and (iv) FPG and TG in non-asthmatic non-diabetic controls. MCP-1 associated with IL-1RA only in subjects with asthma.ConclusionThe systemic MCP-1 levels were significantly elevated in T2D patients with asthma as compared with those without asthma and/or diabetes while these changes correlated differentially with important biomarkers of inflammation and airway remodeling.

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IL-6 is elevated in obese individuals and participates in the metabolic dysfunction associated with that condition. However, the mechanisms that promote IL-6 expression in obesity are incompletely understood. Because elevated levels of palmitate and LPS have been reported in obesity, we investigated whether these agents interact to potentiate IL-6 production. In this study, we report that LPS induces higher levels of IL-6 in human monocytes in the presence of palmitate. Notably, the priming effect of palmitate is associated with enhanced p300 binding and transcription factor recruitment to Il6 promoter regions. Gene silencing of p300 blocks this action of palmitate. RNA polymerase II recruitment was also enhanced at the Il6 promoter in palmitate/LPS-exposed cells. Acetylation levels of H3K9 and H3K18 were increased in monocytes treated with palmitate. Moreover, LPS stimulation of palmitate-treated cells led to increased levels of the transcriptionally permissive acetylation marks H3K9/H3K18 in the Il6 promoter compared with LPS alone. The effect of palmitate on LPS-induced IL-6 production was suppressed by the inhibition of histone acetyltransferases. Conversely, histone deacetylase inhibitors trichostatin A or sodium butyrate can substitute for palmitate in IL-6 production. Esterification of palmitate with CoA was involved, whereas β-oxidation and ceramide biosynthesis were not required, for the induction of IL-6 and H3K9/H3K18 acetylation. Monocytes of obese individuals showed significantly higher H3K9/H3K18 acetylation and Il6 expression. Overall, our findings support a model in which increased levels of palmitate in obesity create a setting for LPS to potentiate IL-6 production via chromatin remodeling, enabling palmitate to contribute to metabolic inflammation.