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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., Competing Interests: The authors declare that they have no competing interests., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Background/aims: Increased circulatory levels of both TNF-α and CCL4/MIP-1β are found in metabolic diseases. However, it is unclear whether TNF-α which is a signature proinflammatory cytokine involved in metabolic inflammation, can induce/promote the expression of CCL4., Methods: THP-1 human monocytic cells and THP-1-derived macrophages were stimulated with TNF-α and LPS-treatment as a positive control. CCL4 mRNA/protein expression was measured using qRT-PCR/ELISA, respectively. Stress-activated protein kinases (SAPK)/ c-Jun N-terminal kinase (JNK) activity was determined using the assay kit. Mechanistic pathways were studied using anti-TNFR1/2 antibodies, pharmacological inhibitors, siRNAs, and NF-κB/AP-1 reporter-expressing THP-1-XBlue cells. Phosphorylation of signaling molecules was assessed by Western blotting., Results: TNF-α induces CCL4 expression at mRNA and protein levels, in both THP-1 monocytic cells and macrophages (P<0.05). TNF-α-driven CCL4 production was markedly abrogated by pre-treatment with anti-TNFR1/2 neutralizing antibodies. TNF-α treatment induced phosphorylation of SAPK/JNK, c-Jun, and NF-κB. Genetic and/or pharmacologic inhibition of SAPK/JNK and NF-κB pathways suppressed the TNF-α-induced CCL4 expression (P<0.05). NF-κB/AP-1 activity was found to be significantly increased in TNFα-treated SEAP reporter-expressing monocytic cells., Conclusion: These data suggest that TNF-α drives CCL4 expression in THP-1 monocytic cells/macrophages via the activation of SAPK/JNK and NF-κB pathways. The findings may provide new insights into understanding the regulatory role of TNF-α in augmenting CCL4 expression during inflammatory conditions., Competing Interests: The authors declare that they have no competing interests., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

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 H 2 O 2 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 H 2 O 2 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). H 2 O 2 -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-1β, 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., Competing Interests: The authors declare that they have no competing interests involved., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

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-1β (MIP-1β), 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., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

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., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background: Matrix metalloproteinase (MMP)-9 is known to degrade the extracellular matrix and increased MMP-9 levels are related with the pathogenesis of many inflammatory conditions including obesity. Pam3CSK4 is a synthetic triacylated lipopeptide (LP) which is a potent activator of immune cells and induces cytokine production. However, it is unclear whether Pam3CSK4 is able to induce MMP-9 expression in monocytic cells. We, therefore, determined MMP-9 production by Pam3CSK4-treated THP-1 cells and also investigated the signal transduction pathway(s) involved., Methods: MMP-9 expression was determined by real-time qPCR and ELISA. MMP-9 activity was assessed by zymography. THP-1 cells, THP1-XBlueTM cells, THP1-XBlueTM-defMyD cells, anti-TLR2 mAb and selective pharmacological inhibitors were used to study signaling pathways involved. Phosphorylated and total proteins were detected by western blotting., Results: Pam3CSK4 induced MMP-9 expression (P<0.05) at both mRNA and protein levels in human monocytic THP-1 cells. Increased NF-κB/AP-1 activity was detected in Pam3CSK4-treated THP-1 cells and MMP-9 production in these cells was significantly suppressed by pre-treatment with anti-TLR2 neutralizing antibody or by inhibition of clathrin-dependent endocytosis. Also, MyD88-/- THP-1 cells did not express MMP-9 following treatment with Pam3CSK4. Inhibition of JNK, MEK/ERK, p38 MAPK and NF-κB significantly suppressed MMP-9 gene expression (P<0.05)., Conclusion: Pam3CSK4 induces MMP-9 production in THP-1 cells through the TLR-2/MyD88-dependent mechanism involving MEK/ERK, JNK, p38 MAPK and NF-κB/AP-1 activation., (© 2017 The Author(s)Published by S. Karger AG, Basel.)