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

Abstract

Background: Obesity 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.

Methods: Patients with knee OA (N = 155, 68% women, mean age 65 years, mean (SD) BMI 29.9 kg/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.

Results: IFP 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 1933 μm) 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.

Conclusions: BMI-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.

Keywords: Inflammation; Infrapatellar fat pad; Macrophages; Obesity; Osteoarthritis.

Fig. 1

Adipocyte volume, and the number of crown-like structures (CLS) and stromal vascular fraction (SVF) cells in the infrapatellar fat pad (IFP) did not correlate with body mass index (BMI). Adipocytes were isolated from the IFP from patients with osteoarthritis (OA), who were undergoing total knee-replacement surgery, adipocyte volume was determined and the correlation with BMI was assessed (N = 56) (a). Adipocyte size was determined upon haematoxylin and eosin (H&E) staining and the correlation with BMI was assessed (N = 18) (b). IFP tissue was stained for CD68 and the number of CLS was quantified. A representative picture of the staining at × 20 (left) and × 40 (right) magnification (c) and the summary of all results is shown (N = 11) (d). The number of SVF cells per gram adipose tissue was determined and the correlation with BMI was assessed (N = 39) (e). Correlation was tested using Spearman’s rank correlation (a, b) or Pearson correlation coefficient (e). Each dot represents one patient. HPF high power field

Fig. 2

Cell infiltrate in adipose tissue. The stromal vascular fraction (SVF) of the infrapatellar fat pad (IFP) was isolated and T cells and macrophages were characterized by flow cytometry (gating strategies were performed as described in Additional file 1: Figure S3). Percentages of CD3⁺ T cells (N = 21) (a), CD4⁺ T cells (N = 29) (b), CD8⁺ T cells (N = 29) (c) and macrophages (N = 37) (d) and their correlation with body mass index (BMI) was determined using Spearman’s rank correlation. A P value <0.05 was considered significant. Each dot represents one patient

Fig. 3

Phenotypic characterization of macrophages in the infrapatellar fat pad (IFP). The stromal vascular fraction (SVF) was isolated from the IFP and macrophages were characterized by flow cytometry. Percentages of CD14⁺ macrophages positive for each specified marker are depicted (N = 6–45) (a). Ex vivo intracellular cytokine production by CD14⁺ macrophages in the IFP is depicted (N = 13) (b). Cytokines were measured in supernatant of unstimulated CD14⁺ from the SVF (N = 2) (c). Median (a and b) or mean (c) is indicated; each dot represents one patient. pt patient

Fig. 4

CD163⁺ macrophages in the infrapatellar fat pad (IFP) are pro-inflammatory. Total macrophage population (left), and CD163⁺ (red) and CD163^- CD14⁺ (green) macrophages (right) are depicted against the forward scatter-area (FSC-a) (a). Differences between CD163⁺ and CD163^- CD14⁺ macrophages in surface marker expression (N = 7–12) (b) and ex vivo intracellular cytokine production (N = 7–8) (c). Each line indicates one patient sample. A P value <0.05, determined by the Wilcoxon signed rank test (b) or paired Student’s t test (c) was considered significant