Pentraxin 3 deficiency exacerbates lipopolysaccharide-induced inflammation in adipose tissue

Abstract

Background/objectives: Pentraxin 3 (PTX3) has been characterized as a soluble and multifunctional pattern recognition protein in the regulation of innate immune response. However, little is known about its role in adipose tissue inflammation and obesity. Herein, we investigated the role of PTX3 in the regulation of lipopolysaccharide (LPS)-induced inflammation in adipocytes and adipose tissue, as well as high-fat diet (HFD)-induced metabolic inflammation in obesity.

Methods: Ptx3 knockdown 3T3-L1 Cells were generated using shRNA for Ptx3 gene and treated with different inflammatory stimuli. For the in vivo studies, Ptx3 knockout mice were treated with 0.3 mg/kg of LPS for 6 h. Adipose tissues were collected for gene and protein expression by qPCR and western blotting, respectively. Ptx3 knockout mice were fed with HFD for 12 week since 6 week of age.

Results: We observed that the expression of PTX3 in adipose tissue and serum PTX3 were markedly increased in response to LPS administration. Knocking down Ptx3 in 3T3-L1 cells reduced adipogenesis and caused a more profound and sustained upregulation of proinflammatory gene expression and signaling pathway activation during LPS-stimulated inflammation in 3T3-L1 adipocytes. In vivo studies showed that PTX3 deficiency significantly exacerbated the LPS-induced upregulation of inflammatory genes and downregulation of adipogeneic genes in visceral and subcutaneous adipose tissue of mice. Accordingly, LPS stimulation elicited increased activation of nuclear factor-κB (NF-κB) and p44/42 MAPK (Erk1/2) signaling pathways in visceral and subcutaneous adipose tissue. The expression of PTX3 in adipose tissue was also induced by HFD, and PTX3 deficiency led to the upregulation of proinflammatory genes in visceral adipose tissue of HFD-induced obese mice.

Conclusions: Our results suggest a protective role of PTX3 in LPS- and HFD-induced sustained inflammation in adipose tissue.

Fig. 1

PTX3 expression during adipocyte differentiation and by inflammatory stimulation in 3T3-L1 adipocytes. a The protein expression and secretion of PTX3 and adiponectin in 3T3-L1 cells during adipocyte differentiation. Induction of Ptx3 mRNA (b) and protein (c) expression by 24 h treatment of IFNγ (10 ng/ml), TNFα (1 nM), IL1β (1 nM), and LPS (1 μg/ml) in 3T3-L1 adipocytes. d Time course of PTX3 protein expression and secretion in 3T3-L1 adipocytes by 1 μg/ml of LPS stimulation. e PTX3 protein expression and secretion in 3T3-L1 adipocytes treated with various doses of LPS for 24 h. For cell culture studies, experiments were repeated 2–3 times yielding similar results. All values are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. basal levels

Fig. 2

Effect of PTX3 silencing on LPS-induced inflammation in 3T3–L1 adipocytes. a Ptx3 mRNA expression in 3T3-L1 adipocytes. b Protein expression of PTX3 in 3T3-L1 adipocytes. c Oil Red-O staining of 3T3-L1 adipocytes on day 8 of differentiation. d Adipogenic gene expression in 3T3-L1 adipocytes on day 8 of differentiation. Gene expression of inflammatory cytokines e and ECM gene expression f in 3T3-L1 adipocytes by LPS stimulation (1 μg/ml) for 24 h. The experiments were repeated three times. For cell culture studies, experiments were repeated 2–3 times yielding similar results. Data are expressed relative to the values for scrambled cells. The values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Scr; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control. Scr: scramble; kd: knockdown

Fig. 3

Effect of PTX3 silencing on LPS-induced expression of cytokines and activation of inflammatory signaling pathways in 3T3-L1 adipocytes. Representative western blots for phosphorylated Erk1/2 (a), NF-κB p65 (b), and Stat3 (c) in 3T3-L1 adipocytes treated with LPS (1 μg/ml) for 30 min, 3 h, and 6 h, respectively. d Time course of gene expression of inflammatory cytokines in 3T3-L1 adipocytes treated with LPS (1 μg/ml). For cell culture studies, experiments were repeated 2–3 times yielding similar results. The values are mean ± SEM for gene expression and mean ± SD for the densitometric quantification of protein expression, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Scr; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control. Scr: scramble; kd: knockdown; Ctrl: control

Fig. 4

Depot- and sex-different expression of PTX3 by LPS stimulation. a Tissue distribution of PTX3 expression from mice without and with LPS treatment (0.3 mg/kg, intraperitoneal injection) for 6 h. b Induction of PTX3 in different tissues by LPS treatment for 6 h in mice (n = 6 per group). Comparison of PTX3 expression in different adipose depots in the basal (c) and 6 h LPS-treated (d) condition between male and female mice (n = 6 per group)

Fig. 5

Effect of PTX3 deficiency on LPS-induced adipose tissue inflammation. The expression of inflammatory genes in epididymal (a) and inguinal adipose tissue (b) from male and female mice treated with LPS for 6 h. The expression of genes involved in adiogenesis in perigonadal (c) and inguinal (d) adipose tissue from female mice upon 6 h of LPS treatment. n = 6 per group. The values were mean ± SEM for gene expression. *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control. WT: wild-type; KO: knockout

Fig. 6

Effect of PTX3 deficiency on LPS-induced activation of inflammatory signaling pathways in adipose tissue. Time course of PTX3 protein expression (a) and activation of inflammatory signaling pathways (b) in adipose tissues treated with LPS in WT mice. Representative western blots for phosphorylated NF-κB p65 and phosphorylated Erk1/2 in epididymal (c) and inguinal (d) adipose tissue upon LPS treatment. n = 6 per group. The values are mean ± SD for the densitometric quantification of protein expression. *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control. WT: wild-type; KO: knockout

Fig. 7

Effect of PTX3 deficiency on HFD-induced adipose inflammation a PTX3 protein expression and quantification in adipose tissues of WT mice by 12 weeks of high-fat diet (HFD) feeding. The age-matched mice fed regular chow diet (RCD) serve as controls. Male, RCD: n = 5, HFD: n = 6; Female, RCD: n = 7, HFD: n = 5. The values were mean ± SD for protein expression. b Body weight in WT and Ptx3-KO male and female mice during 12 weeks of HFD feeding. Male, WT n = 6, Ptx3-KO n = 8; Female, n = 8 per group. c Serum levels of adiponectin and mRNA expression in visceral adipose tissue of WT and Ptx3-KO male and female mice on HFD. Male, WT n = 6, Ptx3-KO n = 8; Female, n = 8 per group. d Proinflammatory (M1 marker) and anti-inflammatory (M2 marker) gene expression in visceral WAT of WT and Ptx3-KO male and female mice on HFD. The values were mean ± SEM for gene expression. *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT; WT: wild-type; KO: knockout; WAT: white adipose tissue