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

Abstract

The TAM tyrosine kinases, Axl and MerTK, play an important role in rheumatoid arthritis (RA). Here, using a unique synovial tissue bioresource of patients with RA matched for disease stage and treatment exposure, we assessed how Axl and MerTK relate to synovial histopathology and disease activity, and their topographical expression and longitudinal modulation by targeted treatments. We show that in treatment-naive patients, high AXL levels are associated with pauci-immune histology and low disease activity and inversely correlate with the expression levels of pro-inflammatory genes. We define the location of Axl/MerTK in rheumatoid synovium using immunohistochemistry/fluorescence and digital spatial profiling and show that Axl is preferentially expressed in the lining layer. Moreover, its ectodomain, released in the synovial fluid, is associated with synovial histopathology. We also show that Toll-like-receptor 4-stimulated synovial fibroblasts from patients with RA modulate MerTK shedding by macrophages. Lastly, Axl/MerTK synovial expression is influenced by disease stage and therapeutic intervention, notably by IL-6 inhibition. These findings suggest that Axl/MerTK are a dynamic axis modulated by synovial cellular features, disease stage and treatment.

Fig. 1. Synovial AXL and MERTK have distinct molecular expression patterns in early untreated RA patients.

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.

Fig. 2. AXL and MERTK STRING networks similarly correlate with synovial histology.

A, C Heatmaps showing regularised-log-transformed expression for all genes in the Axl module (A) and the MerTK module (C), (B, D) Axl module (B) and MerTK module (D) expression in synovial tissue of early arthritis treatment-naive RA patients (n = 81) 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. E, F Enriched ontology pathways for the Axl module (E) and MerTK module (F). Nominal p values from gene set enrichment analysis are shown.

Fig. 3. Axl and MerTK have distinct and characteristic expression patterns in RA synovium, and Axl ectodomain can be cleaved and released in the synovial fluid.

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.

Fig. 4. AXL and MERTK synovial expression and relationship with clinical features are influenced by the disease stage and treatment exposure.

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.

Fig. 5. AXL is modulated by IL-6 pathway inhibition.

A, B Linear regression model analysis with interaction term to estimate the correlation of AXL with MERTK expression in relation to clinical response to tocilizumab (A) and rituximab (B) in anti-TNF inadequate responder RA patients (R4RA cohort, n = 133, including 65 tocilizumab-treated and 68 rituximab-treated patients). The clinical response was assessed by EULAR criteria with DAS28(CRP) after 16 weeks of treatment (good responders in light blue; moderate and non-responders in orange). p-interaction is significant (0.018) in the tocilizumab-treated patient group and not significant in the rituximab-treated patient group. 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). C, D AXL (C) and MERTK (D) normalised gene expression levels assessed at baseline and 16 weeks following tocilizumab (left panels, n = 15 matched samples) or rituximab (right panels, n = 29 matched samples) treatment. CDAI 50% improvement was used to assess the clinical response (responders in light blue, non-responders in orange). Statistical analysis was performed by negative binomial generalised mixed effects model. FDR: false discovery rate. Data are shown as mean ±95% confidence interval. E IL-6/AXL expression ratio in the synovial tissue of tocilizumab- (n = 15) or rituximab- (n = 29) treated patients. Data are represented as mean ±SEM. p values indicated were calculated using the two-tailed Wilcoxon test for paired data. F Pie chart showing the percentage of Axl or MerTK single positive, MerTK and Axl double-positive and negative synovial tissue of anti-TNF inadequate responder RA patients (R4RA cohort) at baseline pre- (n = 24) and post-treatment with either tocilizumab or rituximab (RTX-treated n = 3, TOC-treated n = 5) (left panels) and representative images of double immunofluorescence staining for Axl (red) and MerTK (yellow) (right panels). Scale bar = 50 μm.

Fig. 6. Digital spatial profiling (DSP) confirms the positional identity of AXL and MERTK.

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.