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Supplemental material, sj-pdf-1-tae-10.1177_2042018820930902 for Adipose tissue gene expression of CXCL10 and CXCL11 modulates inflammatory markers in obesity: implications for metabolic inflammation and insulin resistance by Shihab Kochumon, Ashraf Al Madhoun, Fatema Al-Rashed, Rafaat Azim, Ebaa Al-Ozairi, Fahd Al-Mulla and Rasheed Ahmad in Therapeutic Advances in Endocrinology and Metabolism

Supplemental material, sj-pdf-1-tae-10.1177_2042018820930902 for Adipose tissue gene expression of CXCL10 and CXCL11 modulates inflammatory markers in obesity: implications for metabolic inflammation and insulin resistance by Shihab Kochumon, Ashraf Al Madhoun, Fatema Al-Rashed, Rafaat Azim, Ebaa Al-Ozairi, Fahd Al-Mulla and Rasheed Ahmad in Therapeutic Advances in Endocrinology and Metabolism

Correlation between macrophage markers expression in the adipose tissue and body mass index. The adipose tissue gene expression of various monocyte/macrophage markers including CD68, CD86, and CD163 in 94 individuals (49 non-diabetic and 45 type-2 diabetic) was determined by using quantitative real-time RT-PCR as described in Methods. The data show significant positive associations between body mass index (BMI) and adipose tissue gene expression of (A) CD68 (r = 0.26, P = 0.007), (B) CD86 (r = 0.20, P = 0.04), and (C) CD163 (r = 0.27, P = 0.005) macrophage markers. (PDF 115 kb)

Correlation between TLR8 gene and protein expression in the adipose tissue. The adipose tissue TLR8 gene and protein expression was determined in 21 individuals (15 non-diabetic and 6 diabetic) classified as five lean, eight overweight and eight obese by using quantitative real-time RT-PCR and immunohistochemistry, respectively. TLR8 relative mRNA expression was measured as fold change over the average of control gene expression assumed as one. TLR8 protein expression was measured as staining intensity which was quantified using Aperio-positive pixel count algorithm (version 9). The number of positive pixels was normalized to the number of total (positive and negative) pixels to account for variations in the size of the regions sampled. Color and intensity thresholds were established to detect the immunostaining as positive and background staining as negative pixels. The positive correlation between TLR8 gene and protein expression was found to be highly significant (r = 0.86, P

Correlation between TLR8 gene expression in the adipose tissue and glycemia/HbA1c levels. The adipose tissue TLR8 gene expression in 45 type-2 diabetic individuals (32 obese, 10 overweight, and three lean) was determined by quantitative real-time RT-PCR and fasting blood glucose (mmol/L) and glycated hemoglobin (HbA1c) (%) levels were determined using commercial kits as recommended by the manufacturers. The data show that TLR8 gene expression correlated positively with (A) glycemia (r = 0.31, P = 0.04) and (B) HbA1c levels (r = 0.34, P = 0.02). (PDF 15 kb)

Within-group correlations of TLR8 expression with other inflammatory markers in the adipose tissue. (DOCX 22Â kb)

Matrix metalloproteinase-9 (MMP-9) is known to be implicated in the pathogenesis of many inflammatory disorders. FSL-1 (fibroblast-stimulating lipopeptide-1) induces cytokine production by monocytes/macrophages. However, it is unclear whether FSL-1 is also able to induce MMP-9 production. Herein, we determined whether FSL-1 could induce MMP-9 production, and if so, which signal transduction pathway(s) were involved.MMP-9 expression was assessed with real-time qPCR and ELISA. Signaling pathways were studied by using THP1-XBlue™ cells, THP1-XBlue™-defMyD cells, anti-TLR2 mAb and pharmacological inhibitors. Phospho and total proteins were determined by Western blotting.FSL-1 induces MMP-9 expression (P0.001) at both mRNA and protein levels in human monocytic THP-1 cells. Elevated activity (P0.001) of NF-κB/AP-1 was also observed in FSL-1-treated THP-1 cells. FSL-1-induced MMP-9 secretion was markedly suppressed either by neutralizing anti-TLR-2 antibody or by inhibiting clathrin-dependent endocytosis. Furthermore, MyD88(-/-) THP-1 cells did not express MMP-9 in response to FSL-1 treatment. By small interfering RNA-mediated knockdown, we also show that FSL-1-induced up-regulation of MMP-9 requires MyD88. Pre-treatment of THP-1 cells with inhibitors of JNK (SP600125), MEK/ERK (U0126; PD98056; XMD 8-92), p38 MAPK (SB203580) and NF-κB (BAY11-7085, Triptolide, Resveratrol) significantly suppressed (P0.05) MMP-9 gene expression and NF-κB/AP-1 transcription factors activity.These findings provide the first evidence that FSL-1 induces TLR-2-dependent MMP-9 gene expression which requires the recruitment of MyD88 and leads to activation of MEK1/2 /ERK 1/2, MEK5/ERK5, JNK, p38 MAPK and NF-κB/AP-1.