Jc Kwekkeboom

Association between leptin, adiponectin and resistin and long-term progression of hand osteoarthritis

Objective To investigate the association between baseline serum adipokines levels—leptin, adiponectin and resistin—and long-term progression of hand osteoarthritis (HOA). Methods Baseline and 6-year radiographs of 164 patients (mean age 60 years, 81% women) with HOA (defined as a Kellgren and Lawrence score ≥2 in at least two hand joints) were assessed for joint space narrowing (JSN) in 32 hand joints using the Osteoarthritis Research Society International atlas. Progression was defined as a change in the sum of the JSN score above the smallest detectable change of 2, reflecting change above measurement error. Serum adipokines were measured at baseline and patients were categorised by adipokine tertiles. RRs (and 95% CI) of HOA progression for patients in the second and third tertiles were calculated relative to the first tertile, using generalised estimating equations. Adjustments were made for age, sex and body mass index. Results Patients in the two highest tertiles of adiponectin had a decreased risk of 70% (RR=0.3 (0.2 to 0.7)) for HOA progression in comparison with patients in the lowest tertile. Leptin and resistin levels were not associated with progression. Conclusion Adiponectin levels are associated with progression of HOA, suggesting that adiponectin may be involved in the pathophysiology of OA.

Baseline Serum Adipokine Levels Predict Radiographic Progression in Early Rheumatoid Arthritis

OBJECTIVEnAdipose tissue can secrete soluble mediators (adipokines) with potent immune regulatory functions. Some adipokines have been previously associated with radiographic damage in patients with rheumatoid arthritis (RA). In the present study, we investigated the capacity of baseline adipokine levels to predict radiographic progression over a period of 4 years and studied their contribution relative to that of other known risk factors, such as anti-cyclic citrullinated peptide (anti-CCP) antibodies.nnnMETHODSnSerum concentrations of leptin, visfatin, resistin, adiponectin, adipsin, tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) were determined in serum samples obtained at baseline from 253 patients with RA from the Early Arthritis Cohort. The association between levels of these adipokines and radiographic progression was determined using a multivariate normal regression model correcting for age, sex, treatment strategy, body mass index (BMI), and the presence of anti-CCP antibodies.nnnRESULTSnLevels of IL-6, TNFα, visfatin, and adiponectin were positively associated with radiographic progression over 4 years. This association was independent of BMI. However, only adiponectin levels remained significantly associated with radiographic progression when the model was corrected for the presence of anti-CCP antibodies, whereas a trend was observed for IL-6. The association of both TNFα and visfatin with radiographic damage disappeared after correction for the presence of anti-CCP antibodies, which is consistent with the fact that the levels of both cytokines correlated significantly with anti-CCP antibody levels in these patients.nnnCONCLUSIONnOur results indicate that adipokines are predictors of radiographic progression in RA, possibly through distinct underlying biologic mechanisms.

Adipocyte-derived lipids modulate CD4+ T-cell function

Adipose tissue contains several immune cells whose number and phenotype vary depending on the adiposity. In the present study, we show that IFN‐γ+ CD4+ T cells are enriched in human adipose tissue compared with in blood. To gain insight into the underlying mechanisms, we investigated the possibility that human adipocytes modulate CD4+ T‐cell cytokine production and proliferation and show that CD4+ T cells produced increased levels of IFN‐γ when activated in the presence of adipocytes. This effect was mediated by soluble mediators, as shown in transwell and adipocyte‐conditioned medium (ACM) transfer experiments. Additionally, ACM induced increased proliferation of CD4+ T cells upon activation. Intriguingly, the proliferation‐enhancing effect resided mainly in the lipid fraction of ACM, as shown upon separation of the protein and lipid fraction. Further separation of these lipids based on polarity revealed that the modulatory effect is confined to fractions containing free fatty acids. All identified fatty acids were able to individually enhance T‐cell proliferation. These data indicate that adipocytes can modulate CD4+ T‐cell function through the release of lipids. Remarkably, free fatty acids were the most prominent modulators of T‐cell proliferation, possibly leading to an accumulation of these cells in adipose tissue.

An Advanced LC–MS/MS Platform for the Analysis of Specialized Pro-Resolving Lipid Mediators

Here we present an advanced platform for the analysis of specialized pro-resolving mediators (SPM), including a set of pro-inflammatory lipids and respective biochemical pathway markers. The platform is characterized by a largely simplified sample preparation protocol, employing methanol protein-precipitation instead of solid phase extraction. Sample preparation and analysis can be carried out in 96-well format, ensuring higher throughput. In addition, faster analysis times and higher sensitivities have been achieved when compared to other platforms. In total 36 lipid mediators—SPM, their pathway markers and a set of pro-inflammatory lipids including three internal standards—are analyzed within an 11xa0min LC–MS/MS run. The method was validated using human plasma, including repeatability, linearity, recovery, matrix effects, and accuracy. The presented platform was applied in the analysis of synovial fluid post-mortem samples and cellular extracts from polymorphonuclear cells as well as whole blood treated with ionophore in the presence of the polyunsaturated fatty acid eicosapentaenoic acid.

Inflammatory features of infrapatellar fat pad in rheumatoid arthritis versus osteoarthritis reveal mostly qualitative differences

Rheumatoid arthritis (RA) and osteoarthritis (OA) are characterised by joint destruction. In both diseases, inflammation is implicated; however, RA is generally associated with more inflammation in synovium1 and synovial fluid (SF)2–4xa0compared with OA. The infrapatellar fat pad (IFP), an adipose tissue located in the knee joint, has been proposed to contribute to disease progression in OA. Although IFP of OA has been extensively characterised and identified as a source of inflammation in the joint,5–7 only scarce information is available on IFP in patients with RA. Therefore, our aim was to get a first insight into the possible contribution of IFP to the inflammatory processes in the RA joint.nnTo this end, we compared leftover IFP and synovium from patients with RA (n=20: 80% women, mean (SD) age 64 (10.7) years, median (range) body mass index (BMI) 26.9 (18) kg/m2) and patients with primary knee OA (n=51: 62% women, mean (SD) age 66 (8.9) years, median (range) BMI 28.9 (15) kg/m2); all were undergoing knee joint replacement surgery. The study was approved by the local medical ethical committee. Fat-conditioned medium (FCM) and adipocyte-conditioned medium (ACM) were generated as previously described.5 Adipokines and cytokines were measured in FCM …

Adipocytes from the infrapatellar fat pad of OA patients modulate CD4+ T cell cytokine production

Aim As shown in previous studies, the infrapatellar fat pad (IFP) of OA patients has an inflammatory phenotype and is infiltrated with immune cells, including CD4+ T cells. Because several studies have shown that the phenotype of CD4+ T cells in adipose tissue is changing with the adiposity of the individual, we hypothesized that this effect could be due to the modulation of infiltrating T cells by the tissue-resident adipocytes. Therefore, we investigated whether adipocytes from IFP can modulate CD4+ T cell cytokine production.

SAT0001 Lack of obesity-related features in adipocytes and inflammatory cells in the infrapatellar fat pad (IFP)

Background Obesity is associated with the development and progression of osteoarthritis (OA), both for weight-bearing and non-weight bearing joints. Several lines of research indicate that obesity-related systemic factors, such as adipose tissue-derived factors, could be involved in this association. The infrapatellar fat pad (IFP) is an adipose tissue depot localized in the knee joint. and could mediate obesity-associated effects. However, it is currently unknown whether and how obesity affects IFP. Objectives To investigate the presence of obesity-related features in adipocytes and infiltrating immune cells in the IFP of OA patients. Methods Knee OA patients (N=155: 68% women, mean age 65 years, mean (SD) BMI 29.9 kg/m2 (5.7)) were recruited: IFP volume was determined by MRI in 79 knee OA patients, while IFP and subcutaneous adipose tissue (SCAT) were obtained from 106 patients undergoing arthroplasty. Crown-like structures (CLS) were determined using immunohistochemistry. Adipocyte size was determined by light microscopy and histology. Stromal vascular fraction (SVF) cells were characterized by flow cytometry. Results IFP volume (mean (SD) 23.6 (5.4) mm3) was associated with height, but not with BMI or other obesity-related features such as waist circumference, fat percentage and waist to hip ratio. The volume of IFP adipocytes did not correlate with BMI, in contrast to SCAT adipocytes. Few CLS were observed in IFP and their number did not differ between individuals with high and low BMI. Moreover, high BMI was not associated with higher infiltrating immune cell numbers in IFP, nor with changes in immune cell populations. Likewise, no molecular differences were observed in FCM-secreted factors between high and low BMI, except for an increased TNFa secretion in obesity. Since obesity is usually associated with a shift towards pro-inflammatory macrophages in conventional adipose tissue, we have extensively characterized IFP macrophages. Surprisingly, CD206 and CD163, usually associated with an anti-inflammatory phenotype were the most abundantly expressed surface markers on macrophages (81% and 41% respectively). In contrast, cytokine profiles revealed a pro-inflammatory phenotype of the total macrophage population, with cells producing predominantly IL-6 and TNFα, but little IL-10. Interestingly, the CD163+ macrophages were bigger and had a more activated and pro-inflammatory phenotype than their CD163- counterparts. However, no association with BMI could be observed for different macrophage populations or their cytokines. Conclusions BMI-related features usually observed in SCAT and visceral adipose tissue could not be detected in IFP of OA patients, a fat depot implicated in OA pathogenesis. Disclosure of Interest None declared

A6.22 Specialised pro-resolving lipid mediators in chronic inflammation: a comparison between rheumatoid arthritis and osteoarthritis

Background and objectives Inflammation is usually a self-resolving process. The resolution phase involves several cells and molecules that act in a coordinated manner to suppress inflammation, help clearance of apoptotic cells and restore tissue homeostasis. Among these molecules, the pro-resolving specialised lipid mediators (SPM) have emerged as biomarkers and potent mediators of the resolution process. We hypothesised that the underlying cause of chronic inflammation in Rheumatoid Arthritis (RA) and Osteoarthritis (OA)could be the failure of activating pro-resolving mechanisms, involving poly-unsaturated fatty acids (PUFA) and their biologically active metabolites. Methods To investigate this, we have collected synovial fluid from 20 RA and 33 OA patients. We measured more than 50 analytes in the cell-free supernatant, including SPM, their pathway markers and accompanying PUFA, using liquid chromatography combined with mass spectrometry (LC-MS/MS). To this end, we have set-up a novel analytical platform, characterised by a simplified work-up protocol and high-throughput capabilities, making it particularly suitable for clinical studies. Moreover, we have quantified and characterised the cellular infiltrate using flow cytometry and the appropriate combination of antibodies directed against cell surface markers. Results By using as little as 40 µL of synovial fluid, we could demonstrate the presence of significant amounts of 5-HETE; 12-HETE; 15-HETE; 10S,17S-diHDHA; PGE2; 17-HDHA, a series of other hydroxylated analytes and a number of PUFA in synovial fluid of both patient groups. Remarkably, analyses of lipid mediator concentrations revealed higher levels of 12-HETE, 17-HDHA and the pro-resolving 10S,17S-diHDHA in OA versus RA patient samples, indicating a stronger activation of pro-resolving pathways in OA than in RA. Analyses of the cellular infiltrate indicated that in general, less infiltrating cells were found in OA than in RA and the majority of the infiltrating cells were monocytes and neutrophils. The number of monocytes correlated with the number of neutrophils in both diseases, suggesting no specific recruitment of a certain cell-type. Interestingly, we found a strong negative correlation between numbers of infiltrating cells and the concentration of 17-HDHA in the pooled RA and OA samples, as well as the individual disease samples, suggesting a possible biological role for this lipid in cell migration blockage. Conclusion These data indicate that both inflammatory and pro-resolving pathways are activated in RA and OA synovial fluid. However, the level of inflammation is less in OA than in RA and this could be explained by the presence of higher levels of SPM in OA compared to RA.

A4.2 Adipocytes Modulate T Cell Function through Release of Lipids

Background and Objectives Obesity is characterised by the presence of inflammation in adipose tissue. Accumulation of several immune cell-types, including CD4+ T cells, has been previously reported in the increasing adipose tissue. This accumulation is also paralleled by changes in cytokine profiles and phenotype of the infiltrating cells. One of the possible mechanisms involved in these changes is the modulation of T cell function by tissue-resident adipocytes. Therefore, we investigated whether adipocytes derived from various adipose tissues can modulate CD4+ T cell cytokine production and proliferation and studied the mechanisms involved in this process. Materials and Methods CD4+ T cells were purified from peripheral blood mononuclear cells using magnetic beads coated with anti-human CD4. Plate-bound anti-CD3 and soluble anti-CD28 antibodies were used to activate T cells. Adipocytes were isolated from IFP of OA patients by collagenase digestion and were either cultured with purified CD4+ T cells or were cultured in vitro for 24 hours in DMEM/F12 medium supplemented with 0.5% bovine serum albumin to generate adipocyte-conditioned medium (ACM). Cytokine/adipokine production was measured by intracellular cytokine staining (ICS), ELISA or cytokine multiplex. Lipids were isolated using hapten and lipid profiling was performed by liquid chromatography combined with mass spectrometry. Results CD4+ T cells produced increased levels of IFNγ when activated in the presence of adipocytes. This effect is mediated by soluble mediators, as shown in transwell and adipocyte-conditioned medium (ACM) transfer experiments. Additionally, ACM induced increased proliferation of CD4+ T cells upon activation. Furthermore, adipose tissue contained more IFNγ-producing CD4+ T cells than peripheral blood of the same individuals, in 3 out of 3 cases tested, which indicates a possible in vivo relevance of our results. To investigate the possible molecular mechanisms involved in this effect, we separated the protein and lipid fraction of ACM. Surprisingly, despite previous data indicating that several adipocyte-derived proteins can modulate T cell function, we have found that the increased proliferation of T cells is mainly due to the lipids isolated from ACM. Further separation of these lipids based on polarity revealed that the modulatory effect is mainly confined to fractions containing free fatty acids. All identified fatty acids were able to individually enhance T cell proliferation. Conclusions These data indicate that adipocytes can modulate CD4+ T cell function through release of soluble mediators. Remarkably, within the soluble mediators identified, lipids and especially free fatty acids are the most prominent modulators of T cell proliferation.

Adipocytes Modulate the Phenotype of Macrophages Through Secreted Lipids

Background Adipose tissue secretes a wide range of soluble factors that can influence whole body metabolism. Previous studies have shown an accumulation of macrophages and an enhanced pro-inflammatory profile of these cells in adipose tissue of obese mice. Modulation of macrophages by soluble mediators released by adipocytes has been proposed as a possible mechanism underlying these changes. In humans, an increased number of macrophages in adipose tissue of obese individuals have been observed, although no clear change in macrophages phenotype could be established. Moreover, no information exists about the interaction between macrophages and adipocytes in humans. Objective In the present study, we explored the possibility that adipocytes modulate the phenotype of macrophages and studied the possible molecular pathways involved in this modulation. Results Treatment of macrophages with adipocyte-conditioned medium (ACM) resulted in a strong reduction in IL-12p40 secretion upon LPS stimulation, whereas TNFα and other cytokines remained largely unaffected. This effect was independent of the source of ACM. Interestingly, the inhibition increased with increase in Body Mass Index (BMI) of the adipocyte donor. Therefore, it was hypothesised that the effect is mediated by a soluble factor whose release is correlated to the BMI of the adipocyte donor. To this end, we measured several cytokines, adipokines and lipids present in ACM. Among these, the release of several free fatty acids (FA) and PGE2 correlated with the BMI of the adipocyte donor. Further tests indicated that oleic and linoleic acid, as well as PGE2 were able to inhibit IL12p40 secretion, whereas palmitic acid could not. Upon separation of ACM protein and lipid fractions, we confirmed that inhibition of IL12p40 resides mainly in the ACM lipid fraction. Conclusions These results provide first evidence that obesity-related changes in macrophage phenotype could be mediated by adipocytes in humans. These effects are mainly mediated through lipids released by adipocytes. Intriguingly, modulation appears different than in murine obesity, indicating that the immunomodulatory effects of obesity could be different in humans and mice.