Aims: Although toll-like receptors (TLR) are known to mediate the metabolic complications of obesity, the mechanisms underlying its activation remain largely unknown. The present study analyzed a model of dietinduced obesity in mice lacking the TLR4/TLR2 co-receptor CD14. Main methods: Six-week-old male mice lacking CD14 (n = 16) were allocated to either a control diet or a highfat high-simple carbohydrate diet (5.4 kcal/g; 35% fat; 35% sucrose), and compared with C57BL/6 (WT; n = 15) controls. After 12 weeks, body composition, basal sympathetic activity, non-invasive blood pressure and glucose tolerance were evaluated. Hepatic and adipose tissues were collected for mRNA quantification, histology and LPS incubation. Key findings: In both WT and CD14 knockout mice, obesity was accompanied by TLR2 and TLR4 upregulation. However, obese mice lacking CD14 presented decreased lipid and macrophage content in hepatic and adipose tissues, lower urinary levels of noradrenaline, decreased systolic blood pressure, reduced fasting plasma glucose and blunted glucose intolerance, compared with obese WT group. In the presence of exogenous sCD14, adipose tissue incubation with LPS-induced TLR2 and TNF-α upregulation in both WT and CD14 knockout obese mice. Significance: In our model of diet-induced obesity, mice lacking CD14 showed lower adiposity and hepatic steatosis, improved glucose homeostasis, blunted sympathetic overactivity and reduced blood pressure elevation. This was observed in the presence of preserved TLR4 and TLR2 gene expression, and intact TLR4 signaling pathways. These results suggest that CD14-mediated TLR activation might contribute to the cardiovascular and metabolic complications of obesity.

IntroductionA major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response. Specifically, this study aims to demonstrate that the surrogate marker CCP2 can capture ACPAs that bind to autoantigens expressed in vivo in the major inflammatory lesions of RA (that is, in the rheumatoid joint).MethodsPlasma (n = 16) and synovial fluid (n = 26) samples were collected from RA patients with anti-CCP2 IgG levels of above 300 AU/mL. Total IgG was isolated on Protein G columns and subsequently applied to CCP2 affinity columns. Purified anti-CCP2 IgG was analyzed for reactivity and specificity by using the CCPlus® ELISA, in-house peptide ELISAs, Western blot, and immunohisto-/immunocytochemistry.ResultsApproximately 2% of the total IgG pool in both plasma and synovial fluid was CCP2-reactive. Purified anti-CCP2 reactive antibodies from different patients showed differences in binding to CCP2 and differences in binding to citrullinated peptides from α-enolase, vimentin, fibrinogen, and collagen type II, illustrating different ACPA fine-specificity profiles. Furthermore, the purified ACPA bound not only in vitro citrullinated proteins but, more importantly, in vivo-generated epitopes on synovial fluid cells and synovial tissues from patients with RA.ConclusionsWe have isolated ACPAs from plasma and synovial fluid and demonstrated that the CCP2 peptides, frequently used in diagnostic ELISAs, de facto act as surrogate antigens for at least four different, well-characterized, largely non-cross-reactive, ACPA fine specificities. Moreover, we have determined the concentration and proportion of CCP2-reactive IgG molecules in rheumatoid plasma and synovial fluid, and we have shown that the purified ACPAs can be used to detect both in vitro- and in vivo-generated citrullinated epitopes by various techniques. We anticipate that these antibodies will provide us with new opportunities to investigate the potential pathogenic effects of human ACPAs.

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