cd20 dl594  (Novus Biologicals)

Journal: Nature Communications

Article Title: Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis

doi: 10.1038/s41467-024-46564-6

Figure Lengend Snippet: A Axl and MerTK gene expression (regularised-log normalised reads) in synovial tissue of early arthritis treatment-naive RA patients ( n = 87) according to the histological pathotype defined as Pauci-Immune (P-I, in green), Diffuse-Myeloid (D-M, in red), and Lympho-Myeloid (L-M, in blue). Data are represented as mean ±SEM. p values indicated were calculated using the Kruskal–Wallis test with Dunn’s post hoc test. B Correlation between synovial AXL (top panels) and MERTK (bottom panels) gene expression and semi-quantitative scores (0–4) of B cells (CD20), T cells (CD3), plasma cells (CD138), and sublining macrophages (CD68SL). p values and r -coefficients were calculated using the two-tailed Pearson correlation test. C Regression model analysis with interaction term to estimate the correlation of AXL with MERTK expression in relation to clinical response to conventional synthetic Disease-Modifying-Anti-Rheumatic-Drugs. The clinical response was assessed by EULAR criteria with DAS28(CRP) after 6-months of treatment (good responders in light blue; moderate and non-responders in orange). p-interaction is not significant. The scatter plots show the regression line of the fitted negative binomial generalised mixed effects model with the error bars showing 95% confidence interval (fixed effects). D 2D polar plot of transcript modules containing AXL and MERTK in synovial tissue characterised by lympho-myeloid (L-M), diffuse-myeloid (D-M), and pauci-immune (P-I) pathotypes. Different colours show pairwise comparisons between the three pathotypes: upregulation in one group only (D-M: red, P-I: green and L-M: blue) or in two groups (D-M/P-I: yellow, L-M/P-I: light blue, L-M/D-M: purple). E , F Heatmaps showing the correlation between AXL and MERTK synovial transcript levels at baseline and cytokines and growth factors relevant to the inflammatory response ( E ) and clinical parameters ( F ). The red/blue scale represents the Spearman r coefficient, calculated using the two-tailed Spearman correlation test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CRP, C-Reactive Protein; ESR, erythrocyte sedimentation rate; DAS28 disease activity score 28; TJC/28, tender joints count (0-28); SJC/28, swollen joints count (0-28); VAS GH, Visual Analogue Scale General Health (0–100); HAQ health assessment questionnaire.

Article Snippet: The following fluorescent markers were used to determine the morphology of the tissue: CD68-AF532 (clone KP1, Cat. N. NB100-683 Novus) for macrophages, CD20-DL594 (clone IGEL/773, Cat. N. NBP2-44745, Novus) for B cells, and CD3-AF647 (clone UMAB54, Cat. N. TA807198, Origene) for T cells; nuclei were counterstained with Syto13 (Cat. N. S7575, ThermoFisher).

Techniques: Gene Expression, Clinical Proteomics, Two Tailed Test, Expressing, Sedimentation, Activity Assay

Journal: Nature Communications

Article Title: Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis

doi: 10.1038/s41467-024-46564-6

Figure Lengend Snippet: A , C Representative images of Axl ( A ) and MerTK ( C ) immunohistochemistry (IHC) staining of synovial tissue sections. Scale bar = 100 μm. Representative images of n = 20 samples stained. B Double immunostaining of Axl (red) with CD68 (green, upper panel) and CD55 (yellow, lower panel) in the synovium of RA patients. Nuclei were counterstained with DAPI (blue). White arrows indicate double-positive cells. Scale bar = 50 μm. Representative images of n = 13 samples stained. D Triple immunostaining of Axl (red), MerTK (yellow), and CD68 (green) in the synovium of RA patients showing the presence of both Axl + and MerTK + double-positive CD68 + macrophages (orange arrow) and Axl + or MerTK + single positive CD68 + macrophages (white arrow). Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. Representative images of n = 18 samples stained. E Double immunostaining of Axl (red) with ADAM10 (green) in the synovium of RA patients. Nuclei were counterstained with DAPI (blue). White arrows indicate double-positive cells. Scale bar = 50 μm. Representative images of n = 5 samples stained. F Levels of soluble Axl (sAxl), soluble MerTK (sMerTK) and soluble Gas6 (sGas6) in ng/mL assessed by ELISA in the synovial fluid of RA patients ( n = 18). p values indicated were calculated using the Kruskall–Wallis test, with Dunn’s post hoc test. G Levels of soluble Axl (sAxl) in ng/mL assessed by ELISA in the synovial fluid of RA patients ( n = 18) divided according to synovitis score (low [0–4], high [5-9]). p values indicated were calculated using the two-tailed Mann–Whitney test. F , G Data are represented as mean ±SEM. H Correlation between sAxl synovial fluid levels and the erythrocyte sedimentation rate (ESR) of RA patients ( n = 18). p value and r coefficient were calculated according to the two-tailed Spearman correlation test.

Article Snippet: The following fluorescent markers were used to determine the morphology of the tissue: CD68-AF532 (clone KP1, Cat. N. NB100-683 Novus) for macrophages, CD20-DL594 (clone IGEL/773, Cat. N. NBP2-44745, Novus) for B cells, and CD3-AF647 (clone UMAB54, Cat. N. TA807198, Origene) for T cells; nuclei were counterstained with Syto13 (Cat. N. S7575, ThermoFisher).

Techniques: Immunohistochemistry, Staining, Double Immunostaining, Triple Immunostaining, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY, Sedimentation

Journal: Nature Communications

Article Title: Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis

doi: 10.1038/s41467-024-46564-6

Figure Lengend Snippet: A , C AXL ( A ) and MERTK ( C ) gene expression (vst normalised reads) in synovial tissue of anti-TNF inadequate responder RA patients (R4RA cohort, n = 133) according to the histological pathotype defined as Pauci-Immune (P-I, in green), Diffuse-Myeloid (D-M, in red), and Lympho-Myeloid (L-M, in blue). Data are represented as mean ±SEM. p values were calculated using the Kruskal–Wallis test with Dunn’s post hoc test. B , D Correlation between synovial AXL ( B ) and MERTK ( D ) gene expression and semi-quantitative scores (0–4) of B cells (CD20), T cells (CD3), plasma cells (CD138), and sublining macrophages (CD68SL). p values and r-coefficients were calculated using the two-tailed Pearson correlation test. E Expression of sAxl, sMerTK (pg/mL) or the ratio between sMerTK and sAxl in the supernatant of primary fibroblasts-like synoviocytes (FLS) conditioned with supernatant from M1-polarised THP1 (SN M1) or M2-polarised THP1 (SN M2), or in the respective medium used to condition the cells (SN CT). F Expression of sAxl, sMerTK (pg/mL) or the ratio between sMerTK and sAxl in the supernatant of THP1-derived macrophages conditioned with supernatant from unstimulated RA-FLS (SN FLS) or LPS stimulated FLS (SN FLS Infl.), or in the respective medium used to condition the cells (SN CT). E , F Data are represented as mean ±SEM. p values indicated were calculated using the unpaired two-tailed t test (left and middle panels) or the two-tailed Mann–Whitney test (right panel). Experiments were performed on n = 3 distinct patient-derived FLS. G , H Heatmaps showing the correlation between AXL and MERTK synovial transcript levels at baseline and cytokines and growth factors relevant to the inflammatory response ( G ) and clinical parameters ( H ). The red/blue scale represents the Spearman r coefficient, calculated using the two-tailed Spearman correlation test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CRP C-reactive protein, ESR erythrocyte sedimentation rate, DAS28 disease activity score 28, TJC/28 tender joints count (0–28), SJC/28 swollen joints count (0-28), HAQ health assessment questionnaire.

Article Snippet: The following fluorescent markers were used to determine the morphology of the tissue: CD68-AF532 (clone KP1, Cat. N. NB100-683 Novus) for macrophages, CD20-DL594 (clone IGEL/773, Cat. N. NBP2-44745, Novus) for B cells, and CD3-AF647 (clone UMAB54, Cat. N. TA807198, Origene) for T cells; nuclei were counterstained with Syto13 (Cat. N. S7575, ThermoFisher).

Techniques: Gene Expression, Clinical Proteomics, Two Tailed Test, Expressing, Derivative Assay, MANN-WHITNEY, Sedimentation, Activity Assay