Inducible Toll-like receptor and NF-kappaB regulatory pathway expression in human adipose tissue

Abstract

Objective: Inflammatory activity in fat tissue has recently been implicated in mechanisms of insulin resistance and obesity-related metabolic dysfunction. Toll-like receptors (TLRs) play a key role in innate immune responses and recent studies implicate the TLR pathway in mechanisms of inflammation and atherosclerosis. The aim of this study was to examine differential TLR expression and function in human adipose tissue.

Methods and procedures: We biopsied subcutaneous abdominal fat from 16 obese subjects (age 39+/-11 years, BMI 49+/-14 kg/m2) and characterized TLR expression using quantitative real-time PCR and confocal immunofluorescence imaging. In tissue culture, we stimulated isolated human adipocytes with Pam3CSK4 and lipopolysaccharide (LPS) (TLR2 and TLR4 agonists, respectively) and quantified TLR activity, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) production, and nuclear factor-kappaB (NF-kappaB) p65 nuclear activation using real-time PCR, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence.

Results: TLR1, 2, and 4 protein colocalized with adiponectin in human adipocytes with TLR4 exhibiting the highest immunohistochemical expression. Using real-time PCR, we confirmed higher level of gene expression for TLR4 as compared to other members of the TLR family (TLR1, 2, 7, 8) in human adipose depots (P<0.001). In tissue culture, adipocyte TLR2/TLR4 mRNA expression and protein increased significantly following Pam3CSK4 and LPS (P<0.001). TLR2/TLR4 stimulation was associated with NF-kappaB p65 nuclear translocation and proinflammatory cytokine production.

Discussion: The findings demonstrate that TLRs are inducible in adipose tissue and linked with downstream NF-kappaB activation and cytokine release. Adipose stores may play a dynamic role in the regulation of inflammation and innate immunity in human subjects via modulation of the TLR/NF-kappaB regulatory pathway.

Figure 1

High power double immunofluorescence confocal imaging demonstrating presence of Toll-like receptor 2 (TLR2) (left upper panel, green), TLR4 (right upper panel, green), adiponectin (left lower panel, red), and TLR4-adiponectin colocalization (right lower panel, yellow) in human abdominal subcutaneous adipose tissue.

Figure 2

Immunofluorescence intensity for Toll-like receptor (TLR) protein subtypes 1, 2, and 4 in human adipose tissue. Fluorescence intensity was greatest for TLR4 (*P < 0.05). Human blood monocytes (n = 3) were used as positive control (**P < 0.001). Data are presented as mean ± s.e. a.u., arbitrary unit.

Figure 3

Toll-like receptor (TLR) expression in human adipose tissue using quantitative real-time PCR. As shown, the highest level of gene expression was for TLR4 (*P < 0.001 compared to other members of the TLR family). TLR7 expression was greater than TLR8 (^†P = 0.04). Data are presented as mean ± s.e. a.u., arbitrary unit.

Figure 4

Toll-like receptor 2 (TLR2)/TLR4 mRNA and protein expression in response to Pam3CSK4 and lipopolysaccharide (LPS) stimulation of adipocytes. (a) TLR2 /TLR4 gene expression increased significantly in treated adipocytes as compared to untreated control (*P < 0.01, n = 3). (b,c) TLR2/TLR4 protein expression quantified by immunofluorescence increased significantly in stimulated adipocytes compared to control. (b) Upper panels show basal expression of (A) TLR2 and (B) TLR4 in untreated cells; lower panels demonstrate (C) expression of TLR2 in Pam3CSK4-treated cells; (D) expression of TLR4 in LPS-treated cells (**P < 0.005; ***P < 0.001, respectively). Data are presented as mean ± s.e. a.u., arbitrary unit.

Figure 5

Activation and nuclear translocation of nuclear factor-κB (NF-κB) p65 following Pam3CSK4 and lipopolysaccharide (LPS) stimulation of adipocytes. (a) NF-κB p65 mRNA expression increased significantly in treated adipocytes (*P < 0.005; **P < 0.001, respectively, n = 3) as compared to untreated control. (b, ELISA) Phosphorylated NF-κB p65 in whole-cell extract increased significantly in treated adipocytes as compared to control (*P < 0.005, n = 3). (c, ELISA; d, confocal microscope) Panels depict expression of NF-κB p65 in (A) untreated cells followed by nuclear translocation of NF-κB p65 in (B) Pam3CSK4-treated cells and in (C) LPS-treated cells. Jurkat nuclear extract was used as a positive control (n = 3). Data are presented as mean ± s.e. ELISA, enzyme-linked immunosorbent assay.

Figure 6

(a) Tumor necrosis factor-α (TNF-α) and (b) interleukin-6 (IL-6) secretion in response to Pam3CSK4 and lipopolysaccharide (LPS) treatment of adipocytes. Cytokine release from treated adipocytes were significantly increased compared to control (*P < 0.005; **P < 0.001, n = 3). Data are presented as mean ± s.e.