S. Andersen

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.

Functional and phenotypical analysis of IL-6-secreting CD4+ T cells in human adipose tissue

Emerging evidence indicates that a dynamic interplay between the immune system and adipocytes contributes to the disturbed homeostasis in adipose tissue of obese subjects. Recently, we observed IL‐6‐secretion by CD4+ T cells from the stromal vascular fraction (SVF) of the infrapatellar fat pad (IFP) of knee osteoarthritis patients directly ex vivo. Here we show that human IL‐6+CD4+ T cells from SVF display a more activated phenotype than the IL‐6− T cells, as evidenced by the expression of the activation marker CD69. Analysis of cytokines secretion, as well as expression of chemokine receptors and transcription factors associated with different Th subsets (Treg, Th1, Th2, Th17 and Tfh) revealed that IL‐6‐secreting CD4+ T cells cannot be assigned to a conventional Th subset. TCRβ gene analysis revealed that IL‐6+ and IL‐6−CD4+ T cells appear clonally unrelated to each other, suggesting a different specificity of these cells. In line with these observations, adipocytes are capable of enhancing IL‐6 production by CD4+ T cells. Thus, IL‐6+CD4+ T cells are TCRαβ T cells expressing an activated phenotype potentially resulting from an interplay with adipocytes that could be involved in the inflammatory processes in the OA joint.

02.40 Lack of obesity-related features in adipocytes and inflammatory cells in the infrapatellar fat pad (ifp) of oa patients

Background Obesity is associated with the development and progression of osteoarthritis (OA). The infrapatellar fat pad (IFP) could contribute to this association due to its localization in the knee joint and secretion of inflammatory mediators. However, little is known about the effects of obesity on the IFP. Therefore, the aim of this study was to investigate the presence of obesity-related features in adipocytes and infiltrating immune cells in the IFP of OA patients. Materials and methods IFP volume was determined by MRI in 79 knee OA patients. IFP and subcutaneous adipose tissue (SCAT) were obtained from 106 knee OA patients (total n=155: 68% women, mean age 65 years, mean (SD) body mass index (BMI) 29.9 kg/m2 (5.7)) undergoing joint replacement surgery. Crown-like structures (CLS) were determined using immunohistochemistry. Adipocyte size was determined by light microscopy and histology. Stromal vascular fraction (SVF) cells were characterised by flow cytometry. Results IFP volume (mean(SD) 23.6 (5.4) mm3) associated with gender and height, but not with BMI. Likewise, volume and size of IFP adipocytes (mean 271 pl, mean 1933 μm) was not correlated with BMI. Few CLS were observed in IFP and the number did not correlate with BMI. Moreover, high BMI was not associated with higher SVF immune cell numbers in IFP, nor with changes in their phenotype. No molecular differences were observed with BMI, besides an increase in TNFα expression. Extensive characterisation of IFP macrophages revealed that CD206 and CD163, usually associated with an anti-inflammatory phenotype were the most abundantly expressed surface markers on macrophages (81% and 41% respectively), while macrophages produced predominantly IL-6 and TNFα, but little IL-10. Interestingly, surface marker and cytokine expression revealed that CD163+ macrophages had an activated and pro-inflammatory phenotype. Conclusions Obesity-related differences usually observed in SCAT and visceral adipose tissue could not be detected in IFP of OA patients, a fat depot implicated in OA pathogenesis.

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

AB0041 Number of Mast Cell Are Higher in End Stage Knee Osteoarthritis

Background Mast cells could be important in osteoarthritis (OA), as it is the only type of immune cell whose numbers were shown to be as high or sometimes higher compared to rheumatoid arthritis (RA). To assess their role in OA, we investigated whether the number and activation status of mast cells varies with the disease stage in OA. Objectives To compare number of mast cells and their degranulation status in synovial tissue between different stages of OA. Methods 56 patients with symptomatic knee OA attending the rheumatology or orthopaedic outpatient clinic were included. 22 patients with mild to established knee OA (mean (±SD) age 60 (7) yrs, 73% women, median (range) BMI 29 (24-44) kg/mm2, median (range) 2(1-4) KL grade)) underwent arthroscopy of the knee and synovial biopsies were obtained. 34 patients with end-stage knee OA (mean (±SD) age 63 (9) yrs, 59% women, median (range) BMI 29 (21-50) kg/mm2, median (range) KL grade 3.0 (1-4)) underwent arthroplasty and synovial tissues were collected. After haematoxylin and eosin staining, samples were microscopically scored on following features: synovial lining layer hyperplasia (0-3), activation of resident cells/stroma (0-3) and inflammatory infiltrates (0-3). Mean total scores (0-9) by 3 observers was used. Synovial tissue biopsies were investigated by double immunofluorescence for both CD117 and tryptase. Number of total mast cells CD117+ and degranulated (CD117+tryptase- or CD117+with visible granules outside cell) and non-degranulated mast cells (CD117+tryptase+ with no granules seen outside cell) were counted in 10 sub sequential high-power fields (HPF) at two locations in synovial tissue by 3 blinded observers. Mean number of mast cells by 3 observers per patient per 10 HPF were used for analysis. Mann-Witney U and Kruskal-Wallis tests were used for comparisons. Results A mean (±SD) total histology grade of 2.5 (1.6) was observed for all patients. Mast cells were readily observed and were mainly located in the sublining layer. Median (range) of mast cells per patient per 10 HPF was 21 (1-116) and was significantly higher in the end-stage (26 (1-116)) than in the mild to established (12 (1-48)) knee OA (p-value=0.007)) (Fig. 1a). Furthermore, total number of mast cells significantly increased with increasing KL grade (p-value=0.001). A relatively small proportion of the mast cells was degranulated in both groups (Fig. 1b). A trend towards a higher percentage of degranulated mast cells in mild to established OA compared to end-stage OA was observed, although it did not reach significance (Fig. 1b) Conclusions Our data indicate that there is an accumulation of mast cells in end-stage OA compared to mild/established OA. Furthermore a less activated state of mast cells was observed in end-stage OA. This suggests that mast cells might have different biological functions in different phases of the disease. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4290