Innate immunity in long-term adipogenesis of PIP cell line: ligand specific activation of Toll-like receptors modulates triacylglyceride, cytokine and chemokine secretion in adipocytes

MASAHIKO SUZUKI; KENJI MIYAZAWA; WAKAKO IKEDA-OHTSUBO; HARUKI KITAZAWA; HUMAYUN KOBER; PAULRAJ KANMANI; KAZUTOYO YODA; TADAO SAITO; ASUKA TADA; HISASHI ASO; FANG HE; JULIO VILLENA

Toronto

Congreso; 66th Annual Conference of the Canadian Society of Microbiologists; 2016

Canadian Society of Microbiologists

Previously, we have tested the effects of tumor necrosis factor (TNF)-α to porcine intramuscular adipocytes (PIP) in early stage of differentiation. However, the responsiveness to specific Toll-like receptors (TLRs) ligands in porcine adipocytes remain extensively unclear and only sparse data exist regarding this aspect. The aim of this study was to analyze TLRs expression in adipocytes and to investigate whether TLR ligands influence the release of triacylglyceride, cytokines and chemokines. PIP cells were cultured at a density of 2.5×104/ cm2 in 6 well or 12 well plates. The 4-day post-confluent PIP cells were differentiated and stimulated by innate immune legends for 20 days. The effects of TNF-α 2.5 ng/ml, Pam3csk4(synthetic triacylated lipopeptide; TLR2 ligand) 10 ng/ml, Polyinosinic-polycytidylic acid (Poly(I:C); TLR3 ligand) 0.1 µg/ml or lipopolysaccharide(LPS; TLR4 ligand) 0.1 µg/ml on the release of cytosolic triacylglyceride (TG), chemokine ligand 2 (CCL2), interleukin-6 (IL-6), interleukin-8 (IL-8) and interleukin-10 (IL-10) were determined by enzyme-linked immunosorbent assay (ELISA). Cytosolic TG was measured by using the cytosolic TG assay kit. The cytosolic TG was significantly increased in adipocyte with time dependent manner by Poly (I:C) and LPS. In contrast, TNF-α and Pam3csk4 didn?t show the significant differences compared to control. On the other hands, TNF-α induced CCL2, IL-6 and IL-8 production remarkably with time dependent manner but IL-10 was decrease gradually. Pam3csk4, CCL2, IL-6 and IL-8 were also showed a tendency of increasing. In contrast, IL-10 deceased in the later part of stimulation. Poly (I:C) induced CCL2 production in the early stage of stimulation, its concentration decreased in the later part while the others (IL-6 and IL-8) induced later stage. Besides IL-10 was decreased with time dependent manner. LPS showed stronger inducing of CCL2 production later part and also IL-8 was dramatically increased later part. IL-6 was shown a tendency of increasing, whereas IL-10 dramatically reduced with time dependent manner. These results suggest that innate immune ligands have different effects on adipogenesis of PIP cell line. Especially, Poly (I: C) and LPS induced lipid accumulation. Furthermore, results indicate the existing a TLR pathway in adipocytes that directly connects innate immunity with adipocyte function. As a consequence, the role of the adipose tissue in both immunity and metabolism has to be investigated in future research. The results presented in this work could be useful for explaining the obesity-related metabolic complications, such as insulin resistance observed during infection and the immunological phenomena such as macrophage infiltration of adipose tissue caused by obesity.