Regulation of Cathelicidin Antimicrobial Peptide (CAMP) Gene Expression by TNFα and cfDNA in Adipocytes.

Understanding the complex interactions between metabolism and the immune system ("metaflammation") is crucial for the identification of key immunomodulatory factors as potential therapeutic targets in obesity and in cardiovascular diseases. Cathelicidin antimicrobial peptide (CAMP) is an important factor of innate immunity and is expressed in adipocytes. CAMP, therefore, might play a role as an adipokine in metaflammation and adipose inflammation. TNFα, cell-free nucleic acids (cfDNA), and toll-like receptor (TLR) 9 are components of the innate immune system and are functionally active in adipose tissue. The aim of the present study was to investigate the impact of TNFα and cfDNA on CAMP expression in adipocytes. Since cfDNA acts as a physiological TLR9 agonist, we additionally investigated TLR9-mediated CAMP regulation in adipocytes and adipose tissue. CAMP gene expression in murine 3T3-L1 and human SGBS adipocytes and in murine and human adipose tissues was quantified by real-time PCR. Adipocyte inflammation was induced in vitro by TNFα and cfDNA stimulation. Serum CAMP concentrations in TLR9 knockout (KO) and in wildtype mice were quantified by ELISA. In primary adipocytes of wildtype and TLR9 KO mice, CAMP gene expression was quantified by real-time PCR. CAMP gene expression was considerably increased in 3T3-L1 and SGBS adipocytes during differentiation. TNFα significantly induced CAMP gene expression in mature adipocytes, which was effectively antagonized by inhibition of PI3K signaling. Cell-free nucleic acids (cfDNA) significantly impaired CAMP gene expression, whereas synthetic agonistic and antagonistic TLR9 ligands had no effect. CAMP and TLR9 gene expression were correlated positively in murine and human subcutaneous but not in intra-abdominal/visceral adipose tissues. Male TLR9 knockout mice exhibited lower systemic CAMP concentrations than wildtype mice. CAMP gene expression levels in primary adipocytes did not significantly differ between wildtype and TLR9 KO mice. These findings suggest a regulatory role of inflammatory mediators, such as TNFα and cfDNA, in adipocytic CAMP expression as a novel putative molecular mechanism in adipose tissue innate immunity.

Serum Levels and Adipose Tissue Gene Expression of Cathelicidin Antimicrobial Peptide (CAMP) in Obesity and During Weight Loss.

CAMP (Cathelicidin antimicrobial peptide) is synthesized and secreted by adipocytes and involved in adipose tissue (AT) innate immune response and host defense of subcutaneous AT against Gram positive bacteria. Data on the regulation of CAMP in obesity and during weight loss are scarce and reference values do not exist. Serum CAMP levels (ELISA) and AT gene expression levels (quantitative real time PCR) were investigated in two large and longitudinal (12 months) cohorts of severely obese patients undergoing either a low calorie diet (LCD; n=79) or bariatric surgery (BS; n=156). The impact of metabolic factors on CAMP expression in vitro was investigated in differentiated 3T3-L1 adipocytes. CAMP serum levels significantly increased after BS but not during LCD. Females had lower CAMP serum levels and lower gene expression levels in subcutaneous AT. CAMP was positively correlated to unfavorable metabolic factors/adipokines and negatively to favorable factors/adipokines. CAMP gene expression was higher in subcutaneous than in visceral AT but serum CAMP levels were not correlated to levels of AT gene expression. While certain bile acids upregulated CAMP expression in vitro, high glucose/insulin as well as GLP-1 had an inhibitory effect. There exist gender-specific and AT compartment-specific effects on the regulation of CAMP gene expression. Weight loss induced by BS (but not by LCD) upregulated CAMP serum levels suggesting the involvement of weight loss-independent mechanisms in CAMP regulation such as bile acids, incretins and metabolic factors. CAMP might represent an adipokine at the interface between metabolism and innate immune response.

Downregulation of CTRP-3 by Weight Loss In Vivo and by Bile Acids and Incretins in Adipocytes In Vitro.

The adipokine CTRP-3 (C1q/TNF-related protein-3) exerts anti-inflammatory and anti-diabetic effects. Its regulation in obesity and during weight loss is unknown. Serum and adipose tissue (AT) samples were obtained from patients (n = 179) undergoing bariatric surgery (BS). Moreover, patients (n = 131) participating in a low-calorie diet (LCD) program were studied. CTRP 3 levels were quantified by ELISA and mRNA expression was analyzed in AT and in 3T3-L1 adipocytes treated with bile acids and incretins. There was a persistent downregulation of CTRP-3 serum levels during weight loss. CTRP-3 expression was higher in subcutaneous than in visceral AT and serum levels of CTRP-3 were positively related to AT expression levels. A rapid decrease of circulating CTRP-3 was observed immediately upon BS, suggesting weight loss-independent regulatory mechanisms. Adipocytes CTRP-3 expression was inhibited by primary bile acid species and GLP 1. Adipocyte-specific CTRP-3 deficiency increased bile acid receptor expression. Circulating CTRP-3 levels are downregulated during weight loss, with a considerable decline occurring immediately upon BS. Mechanisms dependent and independent of weight loss cause the post-surgical decline of CTRP-3. The data strongly argue for regulatory interrelations of CTRP-3 with bile acids and incretin system.

Role of progranulin in adipose tissue innate immunity.

Regulation of progranulin in adipocytes and its role in inflammation is poorly understood. AIM: (i) to investigate regulation of progranulin in adipocyte differentiation and adipose tissue compartments, (ii) to address progranulin expression in two murine (C57BL/6) models of inflammation. RESULTS: Progranulin expression was induced during adipocyte differentiation. Neither estradiol nor testosterone or metabolic stimuli such as glucose and insulin modified progranulin synthesis. Fatty acids, bile acids and incretins GLP-1 and GIP-1 exerted potent and differential effects on progranulin secretion. LPS, TNF and IL6 significantly increased progranulin secretion. TLR9 agonists decreased and TLR1/2, TLR3, TLR5, and TLR2/6 ligands increased progranulin expression. TLR3-mediated progranulin induction was abrogated by inhibitors of NF-κB and PI3K pathways. Progranulin expression between murine epididymal and subcutaneous adipose tissue did not differ in total adipose tissue, in isolated adipocytes or in the stromal-vascular cell fraction (SVC). However, SVC expressed significantly higher levels of progranulin than adipocytes at all sites. In adipocytes, female mice had significantly higher progranulin expression at all sites. An intra-peritoneal LPS challenge in mice did not affect adipose tissue progranulin expression, whereas peritoneal infection by S. aureus increased progranulin expression after 24 h. CONCLUSIONS: There are relevant sex-, site- and cell-specific effects on progranulin gene expression that is induced during adipocyte differentiation and modulated by various inflammatory and metabolic factors. Most importantly, ligands for TLR1/2 and TLR2/6 (recognizing S. aureus) in vitro and infection by S. aureus in vivo induce progranulin expression suggesting a role of adipocytes in protection against infection by gram-positive bacteria.