Mast Cells Differentiated in Synovial Fluid and Resident in Osteophytes Exalt the Inflammatory Pathology of Osteoarthritis

Abstract

Introduction: Osteophytes are a prominent feature of osteoarthritis (OA) joints and one of the clinical hallmarks of the disease progression. Research on osteophytes is fragmentary and modes of its contribution to OA pathology are obscure.

Aim: To elucidate the role of osteophytes in OA pathology from a perspective of molecular and cellular events.

Methods: RNA-seq of fully grown osteophytes, collected from tibial plateau of six OA patients revealed patterns corresponding to active extracellular matrix re-modulation and prominent participation of mast cells. Presence of mast cells was further confirmed by immunohistochemistry, performed on the sections of the osteophytes using anti-tryptase alpha/beta-1 and anti-FC epsilon RI antibodies and the related key up-regulated genes were validated by qRT-PCR. To test the role of OA synovial fluid (SF) in mast cell maturation as proposed by the authors, hematopoietic stem cells (HSCs) and ThP1 cells were cultured in a media supplemented with 10% SF samples, obtained from various grades of OA patients and were monitored using specific cell surface markers by flow cytometry. Proteomics analysis of SF samples was performed to detect additional markers specific to mast cells and inflammation that drive the cell differentiation and maturation.

Results: Transcriptomics of osteophytes revealed a significant upregulation of mast cells specific genes such as chymase 1 (CMA1; 5-fold) carboxypeptidase A3 (CPA3; 4-fold), MS4A2/FCERI (FCERI; 4.2-fold) and interleukin 1 receptor-like 1 (IL1RL1; 2.5-fold) indicating their prominent involvement. (In IHC, anti-tryptase alpha/beta-1 and anti- FC epsilon RI-stained active mast cells were seen populated in cartilage, subchondral bone, and trabecular bone.) Based on these outcomes and previous learnings, the authors claim a possibility of mast cells invasion into osteophytes is mediated by SF and present in vitro cell differentiation assay results, wherein ThP1 and HSCs showed differentiation into HLA-DR+/CD206+ and FCERI+ phenotype, respectively, after exposing them to medium containing 10% SF for 9 days. Proteomics analysis of these SF samples showed an accumulation of mast cell-specific inflammatory proteins.

Conclusions: RNA-seq analysis followed by IHC study on osteophyte samples showed a population of mast cells resident in them and may further accentuate inflammatory pathology of OA. Besides subchondral bone, the authors propose an alternative passage of mast cells invasion in osteophytes, wherein OA SF was found to be necessary and sufficient for maturation of mast cell precursor into effector cells.

Keywords: RNA-seq; immune cell differentiation; mast cells; osteoarthritis; osteophytes; proteomics; synovial fluid.

Figure 1

Demonstrate an overview of differentially expressed genes in transcriptome analysis performed using osteophyte samples and non-osteophytic control tissue obtained from six knee OA patients (n = 595). (a) Volcano plot of the differentially expressed genes. Here, black dots represent insignificant genes, while blue and red dots represent down-regulated and up-regulated genes, respectively. The plot is generated using LogFC values of the expressed genes. Carboxypeptidase A3 (CPA3), selectin E (SELE), membrane-spanning 4-domains A2/Fc fragment of IgE receptor 1a (MS4A2/FCERI), chymase 1 (CMA1), interleukin 1 receptor-like 1 (IL1RL1), collagen type 1 alpha 1 chain (COL1A1), COL1A2, matrix metalloproteinase 1 (MMP-1), MMP-3 and MMP-13 are among the significantly upregulated genes; (b) Histogram of the highly significant genes, which were obtained after K-means cluster analysis; (c) t-SNE plot of ‘highly differentiated’ genes (generated after K-means cluster analysis). t-SNE is a dimensional reduction technique of presenting large gene data sets; we generated a two-dimensional t-SNE plot to verify the cluster analysis. For this, we considered logFC, logCPR and p-values of the highly significant genes in the osteophyte specimens.

Figure 2

Cluster analysis of differential gene expression between osteophyte and control samples. (a) Denotes elbow method of k-means clustering to find out highly significant up-regulated; the optimum number of clusters is 7. (b) Cluster analysis of up-regulated genes—as k-means clustering for up-regulated is 7, the data is divided into 7 clusters. For up-regulated genes, clusters 1, 5 and 7 showed p < 0.001; 87 genes are highly up-regulated in osteophytes.

Figure 3

qRT-PCR analysis to validate transcriptome results of the osteophyte samples. mRNA levels of CPA3, CMA1, TPSAB1, MMP-1, MMP-3 and MMP-13 were normalized against ACTB, the housekeeping gene. All the values are expressed as mean ± SD. TPSAB1 (20.04-fold), CMA1 (15.57-fold), MMP-1 (16.32-fold) and MMP-3 (21.5-fold) showed a significant up-regulation. CPA3 (6.64-fold) and MMP-13 (4.01-fold) also showed up-regulation, which remained insignificant on statistical scale, however. This qRT-PCR validation is limited to confirm the expression of key upregulated genes from the transcriptomics data in the osteophytes. * p < 0.05, ** p < 0.01 compared to ACTB.

Figure 4

Representative slides of IHC staining with anti-FCERI and anti-TPSAB1 to visualize mast cells in osteophyte sections. Representative areas of osteophytes of study patients are shown as P1, P2 and P3. Antibody-stained mast cells were localized in bone trabeculae, cartilage region, where the columnar chondrocytes can be seen. MC are mast cells at 100X magnification revealing granular features. Bottom panel: histograms—control and osteophytes, denote anti-FCERI and anti-TPSAB1 stained cells (%) respectively; * p < 0.05, ** p < 0.01 compared to control.

Figure 5

In vitro cell differentiation assay, where ThP1 and HSCs were treated with 10% (of culture medium) SF of KL grade I to IV for 9 days; expression of cell surface markers were analysed by flow cytometry on the 10th day. Shown are the representative overlaid histograms of the surface markers on ThP1 and HSCs. Bar graphs are the summary of the surface markers’ expression study, performed in triplicate. Sequence of ThP1 to macrophages differentiation analysed by (a) CD206, and (b) HLA-DR, (a1,b1) represent summary of CD206+ and HLADR+ expression values on ThP1 to macrophages differentiation sequence respectively; (c) sequence of HSCs to mast cells differentiation analysed by FCERI, (c1) represent summary of FCERI+ expression values on HSCs to mast cell differentiation sequence; * p < 0.05, *** p < 0.001 compared to UC.

Figure 6

Proteomics analysis of OA SF. For the proteomics analysis, four patients of each grade (4 × 4 = 16) were analysed through MS/MS. (a–c) represents a grade-wise picture (lattice plots) of different protein subsets that were differentially expressed in OA SFs; differential protein expressions were determined by comparing the fold change of each protein found in KL grade I SFs. In total, 799 proteins were found differentially expressed. These proteins contain mast cell regulatory factors such as light chain immunoglobulins, S100 A12, histones, actins, mitogen-activated protein kinase (MAPK) family and mast cells degranulation proteases such as carboxypeptidase and cathepsins. (d)—functional analysis of the proteomics study in the form of key Reactome pathways with their p-values and FDRs.

Figure 7

An overview of pathogenic dimension around osteophytes pertinent to mast cell action.