A. Ioan-Facsinay

Epitope spreading of the anti-citrullinated protein antibody response occurs before disease onset and is associated with the disease course of early arthritis

Background Anti-citrullinated protein antibodies (ACPA) are the most predictive factor for the development of rheumatoid arthritis (RA). Objective To investigate whether the recognition of citrullinated epitopes changes during disease onset or progression, by studying the fine specificity of ACPA in serum samples collected throughout the disease course, from before the onset of arthritis to longstanding RA. Methods Antibodies recognising five distinct citrullinated antigens were determined by enzyme-linked immunosorbent assay. Serum samples from 36 individuals who had donated blood before and after disease manifestation were used to investigate the development of citrullinated antigen recognition before disease onset. The association of ACPA reactivities with disease outcome was studied using sera from anti-cyclic citrullinated peptide-2 (CCP2)-positive patients with undifferentiated arthritis (UA) who did or did not progress to RA (UA–RA n=81, or UA–UA n=35). To investigate the ACPA recognition profile in patients with RA over a prolonged period of time, baseline serum samples from 68 patients were compared with samples obtained 7 years later. Results The number of recognised citrullinated peptides increased in the period preceding disease onset. At the time of disease manifestation, patients with UA who later developed RA recognised significantly more peptides than UA–UA patients. At later stages of the disease course, the ACPA fine specificity did not change. Conclusion Epitope spreading with an increase in the recognition of citrullinated antigens occurs before the onset of RA. Immunological differences in ACPA fine specificity between UA–UA patients and UA–RA patients are present at baseline and are associated with the future disease course.

The infrapatellar fat pad of patients with osteoarthritis has an inflammatory phenotype

Objectives Obesity is a risk factor for the development of osteoarthritis (OA) in hands and knees. Adipose tissue can secrete different adipokines with powerful immunomodulatory effects. The infrapatellar fat pad (IFP) is an intra-articular organ in the vicinity of the synovium and cartilage. It is hypothesised that IFP-derived soluble factors could contribute to pathological processes in the knee joint. A study was therefore undertaken to compare the release of inflammatory mediators in the IFP and subcutaneous adipose tissue (ScAT) and to characterise the adipocytes and immune cell infiltrate in these tissues. Methods Paired IFP and ScAT samples were obtained from 27 patients with primary OA. The stromal vascular cell fraction (SVF) was isolated and characterised by fluorescence activated cell sorting. Cytokine and adipokine release in fat- and adipocyte-conditioned media was measured by luminex. Results IFP secreted higher levels of inflammatory mediators such as interleukin 6 (IL-6), adipsin, adiponectin and visfatin than ScAT. This could be due to differences in the phenotype of adipocytes and/or in the composition and phenotype of the SVF cells. IFP adipocyte-conditioned media showed a trend towards more IL-6 and adipsin than ScAT. Moreover, the SVF fraction of IFP contained more cells/g tissue, a lower percentage of T cells and a higher percentage of mast cells than ScAT. In addition, T cells had a predominantly pro-inflammatory phenotype while macrophages had a mixed pro- and anti-inflammatory phenotype in the IFP. Conclusion There are profound differences in secreted inflammatory factors and immune cell composition between the IFP and ScAT. These data indicate that IFP-derived soluble mediators could contribute to pathophysiological processes in the OA knee joint.

The Devil in the Details: The Emerging Role of Anticitrulline Autoimmunity in Rheumatoid Arthritis

Rheumatoid arthritis is a chronic inflammatory autoimmune disease of unknown cause. The immune response against citrullinated Ags has recently become the prime suspect for disease pathogenesis. Immunity against citrullinated Ags is thought to play a pivotal role in the disease for several reasons: 1) citrullinated Ags are expressed in the target organ, the inflamed joint; 2) anti-citrullinated protein Abs are present before the disease becomes manifest; and 3) these Abs are highly specific for rheumatoid arthritis. In this review, data from clinical, genetic, biochemical, and animal studies is combined to create a profile of this remarkable autoantibody response. Moreover, a model is proposed of how the anti-citrullinated proteins response is generated and how it could eventually lead to chronic inflammation.

Fatty Acids, Lipid Mediators, and T-Cell Function

Research toward the mechanisms underlying obesity-linked complications has intensified during the last years. As a consequence, it has become clear that metabolism and immunity are intimately linked. Free fatty acids and other lipids acquired in excess by current feeding patterns have been proposed to mediate this link due to their immune modulatory capacity. The functional differences between saturated and unsaturated fatty acids, in combination with their dietary intake are believed to modulate the outcome of immune responses. Moreover, unsaturated fatty acids can be oxidized in a tightly regulated and specific manner to generate either potent pro-inflammatory or pro-resolving lipid mediators. These oxidative derivatives of fatty acids have received detailed attention during the last years, as they have proven to have strong immune modulatory capacity, even in pM ranges. Both fatty acids and oxidized fatty acids have been studied especially in relation to macrophage and T-cells functions. In this review, we propose to focus on the effect of fatty acids and their oxidative derivatives on T-cells, as it is an active area of research during the past 5u2009years. The effect of fatty acids and their derivatives on activation and proliferation of T-cells, as well as the delicate balance between stimulation and lipotoxicity will be discussed. Moreover, the receptors involved in the interaction between free fatty acids and their derivatives with T-cells will be summarized. Finally, the mechanisms involved in modulation of T-cells by fatty acids will be addressed, including cellular signaling and metabolism of T-cells. The in vitro results will be placed in context of in vivo studies both in humans and mice. In this review, we summarize the latest findings on the immune modulatory function of lipids on T-cells and will point out novel directions for future research.

Inflammatory Cells in Patients with Endstage Knee Osteoarthritis: A Comparison between the Synovium and the Infrapatellar Fat Pad

Objective. To get a better understanding of inflammatory pathways active in the osteoarthritic (OA) joint, we characterized and compared inflammatory cells in the synovium and the infrapatellar fat pad (IFP) of patients with knee OA. Methods. Infiltrating immune cells were characterized by flow cytometry in 76 patients with knee OA (mean age 63.3, 52% women, median body mass index 28.9) from whom synovial tissue (n = 40) and IFP (n = 68) samples were obtained. Pain was assessed by the visual analog scale (VAS; 0–100 mm). Spearman rank correlations and linear regression analyses adjusted for sex and age were performed. Results. Macrophages and T cells, followed by mast cells, were the most predominant immune cells in the synovium and IFP, and were equally abundant in these tissues. Macrophages and T cells secreted mostly proinflammatory cytokines even without additional stimulation, indicating their activated state. Accordingly, most CD4+ T cells had a memory phenotype and contained a significant population of cells expressing activation markers (CD25+, CD69+). Interestingly, the percent of CD69+ T cells was higher in synovial than IFP CD4+ T cells. Preliminary analyses indicated that the number of synovial CD4+ T cells were associated with VAS pain (β 0.51, 95% CI 0.09–1.02, p = 0.02). Conclusion. Our data suggest that the immune cell composition of the synovium and the IFP is similar, and includes activated cells that could contribute to inflammation through secretion of proinflammatory cytokines. Moreover, preliminary analyses indicate that synovial CD4+ T cells might associate with pain in patients with endstage OA of the knee.

Evolution of synovitis in osteoarthritic knees and its association with clinical features

OBJECTIVEnTo investigate the course of synovitis on contrast-enhanced magnetic resonance images (CE-MRI) in osteoarthritic knees over 2 years, and its association with pain and cartilage deterioration.nnnDESIGNnConsecutive patients (nxa0=xa039, mean age 61 years, 79% woman, median (range) body mass index (BMI) 29 (24-48)xa0kg/mm2) with clinical osteoarthritis (OA) were included. Baseline and follow-up CE-MRI (3xa0T) were scored paired in chronological order for synovitis (semi-quantitatively at 11 sites (range 0-22)), cartilage deterioration and bone marrow lesions (BMLs) (semi-quantitatively according to Knee Osteoarthritis Scoring System (KOSS)). Changes in sum scores were calculated. Cartilage deterioration was defined as change of ≥2 above the smallest detectable change (SDC). Pain was assessed by standardized questionnaires. Analysis of covariance (ANCOVA) and linear regression models were used to investigate association between synovitis change and cartilage deterioration and between synovitis change or cartilage deterioration and change in pain.nnnRESULTSnThe total synovitis score did not change over 2 years (mean change 0.2 (standard deviation (SD) 3.2)), although changes in individual patients were observed. Cartilage deterioration was observed in 51% of patients. Synovitis change score was lower in patients without compared to patients with cartilage deterioration, taking BML change in account (mean difference -2.1 (-4.1 toxa0-0.1)). Change in synovitis was not associated with change in pain, whereas cartilage deterioration was associated with change in Intermittent and Constant OsteoArthritis Pain (ICOAP) constant pain in adjusted models (unstandardised coefficient (B) (95% confidence interval (CI)) 2.8 (0.4-5.3)).nnnCONCLUSIONSnIn individual patients synovitis fluctuates during disease course. Synovitis change was not associated with change in pain. Increase in synovitis is associated with cartilage deterioration, suggesting a role for synovitis as a target for disease-modifying treatment.

Association analysis of copy numbers of FC-gamma receptor genes for rheumatoid arthritis and other immune-mediated phenotypes

Segmental duplications (SDs) comprise about 5% of the human genome and are enriched for immune genes. SD loci often show copy numbers variations (CNV), which are difficult to tag with genotyping methods. CNV in the Fcγ receptor region (FCGR) has been suggested to be associated with rheumatic diseases. The objective of this study was to delineate association of FCGR-CNV with rheumatoid arthritis (RA), coeliac disease and Inflammatory bowel disease incidence. We developed a method to accurately quantify CNV in SD loci based on the intensity values from the Immunochip platform and applied it to the FCGR locus. We determined the method’s validity using three independent assays: segregation analysis in families, arrayCGH, and whole genome sequencing. Our data showed the presence of two separate CNVs in the FCGR locus. The first region encodes FCGR2A, FCGR3A and part of FCGR2C gene, the second encodes another part of FCGR2C, FCGR3B and FCGR2B. Analysis of CNV status in 4578 individuals with RA and 5457 controls indicated association of duplications in the FCGR3B gene in antibody-negative RA (P=0.002, OR=1.43). Deletion in FCGR3B was associated with increased risk of antibody-positive RA, consistently with previous reports (P=0.023, OR=1.23). A clear genotype–phenotype relationship was observed: CNV polymorphisms of the FCGR3A gene correlated to CD16A expression (encoded by FCGR3A) on CD8 T-cells. In conclusion, our method allows determining the CNV status of the FCGR locus, we identified association of CNV in FCGR3B to RA and showed a functional relationship between CNV in the FCGR3A gene and CD16A expression.

Radiographic progression of knee osteoarthritis is associated with MRI abnormalities in both the patellofemoral and tibiofemoral joint

OBJECTIVEnTo investigate patterns of MRI abnormalities in the patellofemoral (PFJ) and tibiofemoral joint (TFJ) and their association with radiographic progression, using hypothesis free analyses.nnnDESIGNn205 patients from the GARP study with symptomatic OA at multiple sites (mean age 60 years, 80% woman, median BMI 26 kg/m(2)), underwent knee MRI at baseline. Cartilage damage, osteophytes, cysts, bone marrow lesions (BMLs) and effusion/synovitis were scored according to a validated scoring method. Baseline and 6-year TFJ and PFJ radiographs were scored (0-3) for JSN and osteophytes according to OARSI and Burnett atlases, respectively; progression was defined as ≥1 point increase. Baseline patterns of MRI abnormalities derived from principal component analysis (PCA) were associated with progression using adjusted generalized estimating equations (GEE).nnnRESULTSnPCA resulted in extraction of six components, explaining 69% of variance. In 29% and 29% of 133 patients with follow-up the TFJ progressed, whereas in 15% and 9% the PFJ progressed for osteophytes and JSN, respectively. Component 1 (cartilage damage of the PFJ and osteophytes of both joints) was statistically significant associated with TFJ JSN progression and PFJ osteophyte progression. Component 2 (all lateral PFJ abnormalities except osteophytes) was associated with JSN/osteophyte progression in the PFJ alone, whereas component 3 (all medial TFJ abnormalities except osteophytes) was associated with JSN and osteophyte progression in both PFJ and TFJ.nnnCONCLUSIONnBaseline structural damage and bone turnover activity, as reflected by BMLs, seem to be involved in knee OA progression. Moreover, progression in PFJ and TFJ seems to be related.

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

BackgroundObesity is associated with the development and progression of osteoarthritis (OA). Although the infrapatellar fat pad (IFP) could be involved in this association, due to its intracapsular localization in the knee joint, there is currently little known about the effect of obesity on the IFP. Therefore, we investigated cellular and molecular body mass index (BMI)-related features in the IFP of OA patients.MethodsPatients with knee OA (Nu2009=u2009155, 68% women, mean age 65xa0years, mean (SD) BMI 29.9xa0kg/m2 (5.7)) were recruited: IFP volume was determined by magnetic resonance imaging in 79 patients with knee OA, while IFPs and subcutaneous adipose tissue (SCAT) were obtained from 106 patients undergoing arthroplasty. Crown-like structures (CLS) were determined using immunohistochemical analysis. Adipocyte size was determined by light microscopy and histological analysis. Stromal vascular fraction (SVF) cells were characterized by flow cytometry.ResultsIFP volume (mean (SD) 23.6 (5.4) mm3) was associated with height, but not with BMI or other obesity-related features. Likewise, volume and size of IFP adipocytes (mean 271 pl, mean 1933xa0μm) was not correlated with BMI. Few CLS were observed in the IFP, with no differences between overweight/obese and lean individuals. Moreover, high BMI was not associated with higher SVF immune cell numbers in the IFP, nor with changes in their phenotype. No BMI-associated molecular differences were observed, besides an increase in TNFα expression with high BMI. Macrophages in the IFP were mostly pro-inflammatory, producing IL-6 and TNFα, but little IL-10. Interestingly, however, CD206 and CD163 were associated with an anti-inflammatory phenotype, were the most abundantly expressed surface markers on macrophages (81% and 41%, respectively) and CD163+ macrophages had a more activated and pro-inflammatory phenotype than their CD163- counterparts.ConclusionsBMI-related features usually observed in SCAT and visceral adipose tissue could not be detected in the IFP of OA patients, a fat depot implicated in OA pathogenesis.

A1.11 T cells in the infrapatellar fat pad of osteoarthritis patients as a source of IL-6 in the joint

Background and objectives The infrapatellar fat pad (IFP) is an adipose tissue organ present in the knee next to the synovium and cartilage, thereby constituting a potential player in the pathological processes in the osteoarthritic joint. Obesity-associated changes occur in IFP and this supports the hypothesis that IFP could mediate the association between obesity and the development and progression of osteoarthritis (OA). Interestingly, these changes were observed in the stromal vascular fraction (SVF) rather than adipocytes. As this fraction contain many different types of immune cells, we characterised the SVF of the IFP in OA patients phenotypically and functionally. Materials and methods IFP samples were obtained from knee OA patients (N = 43) undergoing joint replacement surgery (58.1% women; mean (SD) age 66.4 years (10.9); mean (SD) BMI 29.2 kg/m2 (5.7)). The SVF was isolated and cells were characterised based on surface markers expression and cytokine production using flow cytometry. Results Characterisation of the SVF of IFP showed the presence of various immune cells in this tissue, whereby macrophages and T cells were most abundant. Interestingly, flow cytometry analyses of ex vivo cytokine production by different cells revealed a subpopulation of CD4+ T cells that were able to produce IL-6 without further stimulation. These IL-6 producing CD4+ T cells expressed CD69, indicating recent activation. Upon polyclonal stimulation, CD4+ T cells were able to secrete IFNγ, TNFα, and IL-4. However, IL-6 producing CD4+ T cells did not secrete these cytokines. Furthermore, chemokine receptor expression revealed that these IL-6 producing T cells could not be categorised as conventional T helper 1 (Th1), Th2, Th17 or Tfh cells. These data indicate that IL-6-secreting T cells are a distinct population of T cells. Finally, we have also studied whether these IL-6 producing T cells are also present in other tissues. Indeed, we have found these cells also in sc adipose tissues and synovium of OA patients, but only at low frequencies in blood. Conclusion In conclusion, we have found a novel population of CD4+ T cells which secrete IL-6 directly ex vivo and are in an activated state, indicating that these CD4+ T cells might recognise adipose tissue antigens and could be involved in the inflammatory processes present in human adipose tissue. Moreover, they are a source of IL-6 in the OA joint, thereby potentially contributing to joint inflammation.