Stimulation with Peptidoglycan induces interleukin 6 and TLR2 expression and a concomitant downregulation of expression of adiponectin receptors 1 and 2 in 3T3-L1 adipocytes

Abstract

Background: Inflammation is a major component of obesity and diabetes, and toll-like receptors (TLRs) play critical roles in the regulation of inflammation and response to pathogen associated molecular patterns (PAMPs) and fatty acids in. Although immune cells such as macrophages are primarily responsible for recognition and clearance of pathogens, adipocytes are also closely involved in the regulation of innate immunity and inflammation. Whereas it has been demonstrated that adipocytes respond to TLR4 stimulation with lipopolysacccharide, very little is known about their response to the TLR2 agonist, peptidoglycan.

Methods: We investigated the response to peptidoglycan from Staphylococcus aureus in differentiated 3T3-L1 adipocytes. Real-time PCR analysis was used to quantify the expression of interleukin 6 (IL6), adiponectin receptors (adipoR1 and adipoR2), toll-like receptor 2 (TLR2) and 4 (TLR2 4). Media level of IL6 was determined with ELISA.

Results: Adipocyte stimulation peptidoglycan induces IL6 expression (P < 0.01). Both siRNA mediated suppression of TLR2 and immunoneutralization of TLR2 with a TLR2 specific antibody inhibited response to peptidoglycan (P < 0.05). We also examined the regulation of TLR2 and TLR4 mRNA in peptidoglycan treated cells. Both peptidoglycan and lipopolysaccharide (LPS) robustly induce TLR2 mRNA expression, whereas TLR4 mRNA is weakly induced by LPS only (P < 0.05). Additionally, peptidoglycan downregulates the mRNA expression of adiponectin receptors, adipoR1 and adipoR2 (P < 0.05).

Conclusion: Obesity and type 2 diabetes are associated with increased expression of TLR2, this receptor could play a significant but previously unrecognized role in the establishment of chronic inflammation in adipose tissue in obesity.

Figure 1

Lipopolysaccharide (LPS) and peptidoglycan (PEP) induce IL6 mRNA expression and protein secretion. Cells were treated with 100 ng/ml LPS and 10 μg/ml peptidoglycan (PEP) for 3, 6 and 24 hours. Media and mRNA were recovered for each time period and assayed for IL6 gene expression using RT-PCR (1A) and protein secretion with ELISA (1B). mRNA and media concentration of IL6 were increased by LPS and PEP. Bars represent means and ± SEM of this normalized expression from 4 different replicates. Bars with different superscripts are different (P < 0.05).

Figure 2

Polymyxin B inhibits induction of IL6 mRNA by LPS, but had no effect against peptidoglycan. Cells were treated with 100 μg/ml polymyxin B for 30 minutes before treatment with LPS (100 ng/ml) and PEP (10 μg/ml PEP) for 6 hours. Induction of IL6 gene expression by LPS treatment was significantly suppressed in the presence of polymyxin, but not PEP induction of IL6. Bars represent means and ± SEM of this normalized expression from 4 different replicates. Bars with different superscripts are different (P < 0.05).

Figure 3

Effect of immunoneutralization of TLR4 and TLR2 with their respective antibodies and siRNA mediated suppression of TLR2 and TLR4 on IL6 expression. Cells were treated with 5 μg/ml neutralizing antibodies against TLR2 (TLR2ab) and TLR4 (TLR4ab) for 1 hour before treatment with LPS (100 ng/ml) and PEP (10 μg/ml) for 6 hours (3A). Both antibodies inhibit (P < 0.05) the induction of IL6 mRNA by their respective receptors. In cells treated with TLR2 and TLR4 specific siRNAs expression of TLR2 and TLR4 was reduced by their respective siRNAs (3B). Induction of IL6 expression by treatment with peptidoglycan was attenuated in cells treated with TLR2 siRNA, but not the negative siRNA (vesirna) or TLR4 siRNA (3C). Bars represent means and ± SEM of this normalized expression from 4 different replicates. Bars with different superscripts are different (P < 0.05).

Figure 4

Inhibition of extracellular signal regulated kinase (ERK) and JNK inhibits IL6 induction by peptidoglycan. Both ERK and JNK were inhibited by treating cells with 10 μM of U0126 and SP600125(SP) and 50 μg/ml of the NFkB inhibitory peptide (SN50) for 30 minutes before treating with LPS (100 ng/ml) and PEP (10 μg/ml) for 6 hours. Inhibition of ERK and JNK, but not NFκB, inhibited IL6 induction by peptidoglycan. Bars represent means and ± SEM of this normalized expression from 4 different replicates. Bars with different superscripts are different (P < 0.05).

Figure 5

Regulation of TLR2 and TLR4 mRNA expression by LPS, peptidoglycan and linoleic acid. Expression of both TLR2 (5A) and TLR4 (5B) was determined in cells treated with LPS (100 ng/ml), peptidoglycan (10 μg/ml) and linoleic (500 μM) acid for 6 hours. Significant induction of TLR2 (5A) (approx.7 fold) and TLR4 (5B) (1.5 times) was obtained. Bars represent means and ± SEM of this normalized expression from 4 different replicates. Bars with different superscripts are different (P < 0.05).

Figure 6

Regulation of TLR2 induction peptidoglycan by fatty acids and pharmacological inhibitors of ERK (p44/42 MAPK), JNK and NFκB. In cells pretreated with 500 μM linoleic acid (LIN), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and then treated with peptidoglycan (6A), TLR2 mRNA expression was further additively upregulated in the presence of the fatty acids and peptidoglycan. Additionally, in cells were pretreated with the specific inhibitors, 10 μM U0126 and SP600125 (SP) and 50 μg/ml SN50 before peptidoglycan treatment for 6 hours. Inhibition of ERK and JNK, but not NFκB (6B), additively upregulated peptidoglycan induced TLR2 expression. Bars represent means and ± SEM of this normalized expression from 4 different replicates. Bars with different superscripts are different (P < 0.05).

Figure 7

Regulation of adiponectin receptors, adipoR1 and adipoR2, by peptidoglycan. Acute treatment of adipocytes with peptidoglycan (10 μg/ml) for 3 hours leads to a significant downregulation of adipoR1 (7A) and adipoR2 (7B). Bars represent means and ± SEM of this normalized expression from 3 different replicates. Bars with different superscripts are different (P < 0.05).