CD200 as a Potential New Player in Inflammation during Rotator Cuff Tendon Injury/Repair: An In Vitro Model

Abstract

Rotator cuff tendon (RCT) disease results from multifactorial mechanisms, in which inflammation plays a key role. Pro-inflammatory cytokines and tendon stem cell/progenitor cells (TSPCs) have been shown to participate in the inflammatory response. However, the underlying molecular mechanism is still not clear. In this study, flow cytometry analyses of different subpopulations of RCT-derived TSPCs demonstrate that after three days of administration, TNFα alone or in combination with IFNγ significantly decreases the percentage of CD146+CD49d+ and CD146+CD49f+ but not CD146+CD109+ TSPCs populations. In parallel, the same pro-inflammatory cytokines upregulate the expression of CD200 in the CD146+ TSPCs population. Additionally, the TNFα/IFNγ combination modulates the protein expression of STAT1, STAT3, and MMP9, but not fibromodulin. At the gene level, IRF1, CAAT (CAAT/EBPbeta), and DOK2 but not NF-κb, TGRF2 (TGFBR2), and RAS-GAP are modulated. In conclusion, although our study has several important limitations, the results highlight a new potential role of CD200 in regulating inflammation during tendon injuries. In addition, the genes analyzed here might be new potential players in the inflammatory response of TSPCs.

Keywords: C/EBPbeta; CD146; CD200; DOK2; IFNγ; IRF1; TNFα; inflammation; rotator cuff disease; tendon stem cells; tendons.

Figure 1

Immunophenotypic profile of human rotator-cuff-tendon-derived cells (RCTCs) by flow cytometry. (A) Cells were stained for a panel of the cluster of designation (CD): CD146, CD90, CD44, CD49d, CD49f, CD109, CD200, and CD45. Peaks of fluorescence emission were obtained by flow cytometry, and their right-shifted peak (blue) represents the positivity for the marker analyzed with respect to the isotype negative control (grey peak). CD146 (74% ± 16.1), CD90 (99% ± 0.3), CD44 (100% ± 0.4), CD49d (58% ± 26.6), CD49f (74% ± 8.8), and CD109 (73% ± 19.8). (B) Microscopic analysis of cell morphology after 3 days of in vitro stimulation of proinflammatory cytokines. Representative images of cells exposed to treatments were acquired by phase-contrast microscopy. 100× magnification. (C) Gating strategy. The SSC (side scatter)/FSC (forward scatter) dot plot allows the gating of the cell population by means of its morphological parameters. Cells were afterward stained with the 7-AAD (7-aminoactinomycin) to exclude dead cells from further analyses (CD146). No altered expression of CD146 was promoted after stimulation with single or combined pro-inflammatory cytokines. (D) Graphs represent the percentage of CD146 expression and the MFI (mean fluorescence intensity) of cells exposed to treatments. Fluorescence emission peaks related to CD146 were obtained by flow cytometry. CTRL = untreated cells.

Figure 2

Analysis of inflammation markers in the CD146+TSPCs population. (A) RCTCs, stained positive for CD146, were afterward co-stained for the cluster of designation (CD)49d, CD49f, CD109, and CD200. Representative dot plots show the distribution of the cell population in response to treatments. (B) Graphs represent the percentage of marker expression and the MFI (mean fluorescence intensity) related to CD49d, CD49f, CD109, and CD200. Relative emission peaks were obtained by flow cytometry. The right shift of peaks represents a higher positivity for markers. (C) Migration of RCT-derived cells in response to the various treatments immediately after the stimulus (T0) and after 24 h. Representative images obtained by phase-contrast microscopy. The bar graph represents the percentage of cells covering the gap (empty) area. 100× magnification. (D) Protein expression of MMP (metalloproteinase)-9, STAT (Signal transducer and activator of transcription)1, STAT3, and fibromodulin detected by Western blotting after 24 h. β-actin is used as a loading control. Bar graphs display densitometric values normalized on the ones of the loading control (relative expression). (E) Graphs represent relative gene expressions of IRF (Interferon Regulatory Factor)-1, NFkb (Nuclear factor kappa subunit b), TGRF2 (TGFBR2-Transforming growth receptor factor 2), CAAT (CAAT/Enhancer-binding protein beta), DOK2 (docking protein 2), and RAS-GAP (Ras GTPase activating protein). * = p < 0.05; ** = p < 0.01; and **** = p < 0.0001 between CTRL and treated cells. # = p < 0.05; ## = p < 0.01; ### = p < 0.001 and #### = p < 0.0001 between cells treated with TNFα and cells in the presence of other treatments. δ = p < 0.05; δδ = p < 0.01; δδδ = p < 0.001 and δδδδ = p < 0.0001 between cells treated with IFNγ and cells in the presence of other treatments.