CD56+/CD16− Natural Killer cells expressing the inflammatory protease granzyme A are enriched in synovial fluid from patients with osteoarthritis P.

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CD56+/CD16− Natural Killer cells expressing the inflammatory protease granzyme A are enriched in synovial fluid from patients with osteoarthritis P. Jaime, N. García-Guerrero, R. Estella, J. Pardo, F. García-Álvarez, L. Martinez-Lostao Osteoarthritis and Cartilage Volume 25, Issue 10, Pages 1708-1718 (October 2017) DOI: 10.1016/j.joca.2017.06.007 Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Lymphocyte populations in peripheral blood and synovial fluid from OA patients. (A). Lymphocyte populations according the expression of the surface markers: CD3, CD56 and CD16 both in peripheral blood and synovial fluid from all patients included in the study are represented. (B). Percentage of CD16-positive and CD16-negative cells in CD3−/CD56+ cells both in peripheral blood and synovial fluid from all patients included in the study are represented. (C). Dot plots obtained after flow cytometry analysis from a representative OA patient are showed. Exact P-values are indicated in the graph. Osteoarthritis and Cartilage 2017 25, 1708-1718DOI: (10.1016/j.joca.2017.06.007) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Cytotoxicity assay of NK cells from peripheral blood and synovial fluid from OA patients. (A). Flow cytometry analysis of cytotoxic activity of NK cells of a representative patient. Cell Tracker Green labeled NK cells were incubated with target cells (K562 cells) at E:T ratio of 3:1 for 4 h at 37°C. Left panel shows dot plot from flow cytometry analysis in which Cell Tracker Green negative cells are gated. Right panels show dot plots from flow cytometry analysis annexin V and 7-AAD positive cells were quantified in the CellTrackerGreen negative cells. (B). Percentage of apoptotic (annexin V and 7-AAD positive) K562 cells in the cytotoxicity assay carried out with NK cells from peripheral blood and synovial fluid from OA patients are represented. Percentage of apoptotic K562 cells after performing cytotoxicity assays using NK cells from peripheral blood from healthy donors also has been represented. Exact P-values are indicated in the graph. Osteoarthritis and Cartilage 2017 25, 1708-1718DOI: (10.1016/j.joca.2017.06.007) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Percentage of NK cells from peripheral blood and synovial fluid from OA patients expressing granzyme A, granzyme B and perforin. (A). Data represent the mean ± s.e.m. of the percentage of positive cells for granzyme A, granzyme B and perforin in the CD3−/CD56+/CD16+ cell population (white bars) and in the CD3−/CD56+/CD16− (gray bars) from peripheral blood and synovial fluid from OA patients. Exact P-values are indicated in the graph. (B). Dot plots after flow cytometry showing granzyme A, granzyme B and perforin staining from representative OA patients both in peripheral blood and synovial fluid are showed. Osteoarthritis and Cartilage 2017 25, 1708-1718DOI: (10.1016/j.joca.2017.06.007) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Amount of granzyme A, granzyme B and perforin in NK cells from peripheral blood and synovial fluid from OA patients. (A). Data represent the mean ± s.e.m. of the mean fluorescence intensity (MFI) for granzyme A, granzyme B and perforin staining in NK cells from peripheral blood (white bars) and from synovial fluid (gray bars) in the CD3−/CD56+/CD16+ and in the CD3−/CD56+/CD16− cell populations from OA patients. Exact P-values are indicated in the graph. (B). Histograms after flow cytometry showing granzyme A, granzyme B and perforin staining from representative OA patients both in peripheral blood and synovial fluid are showed. Osteoarthritis and Cartilage 2017 25, 1708-1718DOI: (10.1016/j.joca.2017.06.007) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Study of cytokines in plasma and synovial fluid from OA patients. Quantification of the following cytokine levels was performed using Milliplex Hcytomag-60K-Human-cytokine (Millipore): TNF-α, IL-6, IFN-γ, IL-17, and IL-10. Graphs show level of the cytokines from all patients included in the study. Detection limit for cytokine measurement was: 0.4 pg/mL for TNF-α and IL-6; 0.2 pg/mL for IFN-γ; and 0.1 pg/mL for IL-17 and IL-10. Osteoarthritis and Cartilage 2017 25, 1708-1718DOI: (10.1016/j.joca.2017.06.007) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Principal Component Analysis (PCA) of the levels of IL-6 and TNF-α and NK cell populations from synovial fluid from OA patients. Analysis was performed by calculating Pearson's correlation coefficients. PCA and the Pearson's method were applied using the statistical software XLSTAT, versionPro 7.5 (XLSTAT, 2004) and GrandPath Prism 6.0 (GrandPath Software Inc.). Osteoarthritis and Cartilage 2017 25, 1708-1718DOI: (10.1016/j.joca.2017.06.007) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions