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: Expressing, Two Tailed Test, 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 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: Expressing, Two Tailed Test, Derivative Assay, MANN-WHITNEY, 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 Schematic representation of the Digital Spatial Profiling (DSP) approach, including the selection of three regions of interest (ROI): aggregate (characterised by the presence of CD3 + and CD20 + cells), deep sublining (characterised by the absence of CD3 + and CD20 + cells) and lining with superficial sublining (characterised by the presence of CD68 + cells). Scale bar = 100 μm. DSP was performed on 14 aggregates, 25 lining, and 33 sublining regions. B Three-way radial plot showing differential and overlapping genes across aggregate (green), lining (blue) and sublining (red) regions. AXL and MERTK genes have been labelled showing a significantly higher presence of AXL in both lining and sublining regions and a significant presence of MERTK in the lining region. Significance was internally estimated by the volcano3D package combining significance ( q < 0.05) from both one-way ANOVA and pairwise T test. C Double immunostaining of Axl (red) and MerTK (yellow) in the aggregate, sublining and lining synovial areas of RA patients. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. Representative images of n = 32 samples stained. D Expression of selected individual genes included in the AXL and/or MERTK networks in the aggregate ( n = 14, including 4 non-responder and 10 responder patients), sublining ( n = 33, including 12 non-responder and 21 responder patients) and lining ( n = 25, including 8 non-responder and 17 responder patients) synovial areas of responders (light blue) and non-responders (orange) RA patients to either tocilizumab or rituximab. Boxplots represent the median and first and third quartiles, and whiskers span to the minimum and maximum. A paired Wilcoxon test was undertaken to compare responders and non-responders.

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: Selection, Double Immunostaining, Staining, Expressing