Elevated Fcy receptor expression augments pro-inflammatory macrophage phagocytosis in systemic sclerosis and associated rheumatic diseases

Abstract

Objectives: To investigate the pro-phagocytic phenotype of macrophages in SSc and other rheumatic diseases by examining their activation, signalling pathways and treatment responses, with the goal of uncovering mechanisms that drive enhanced phagocytosis.

Methods: Single-cell RNA sequencing (scRNA-seq) datasets (GSE138669/GSE212109) from skin and lung macrophages of healthy controls (HC) and SSc patients were analysed. Human monocyte-derived macrophages (hMDMs) were differentiated from CD14+ monocytes from HC, SSc, RA, PsA, and axSpA patients. In selected experiments, hMDMs were pretreated with 0.1 μM nintedanib. Phagocytic activity was quantified using pHrodo bioparticles and flow cytometry. Macrophage surface markers were evaluated by flow cytometry, NF-κB signalling by Western blot and gene expression by RT-qPCR.

Results: Analysis of scRNA-seq datasets revealed a pro-phagocytic signature in SSc-affected organs. SSc macrophages, particularly the FCGR3Ahi cluster in skin, exhibited elevated expression of FCGR genes and enriched FcγR-mediated phagocytosis pathways, accompanied by pro-inflammatory markers. This phenotype extended to FCN1hi lung macrophages in SSc patients with interstitial lung disease, indicating a systemic pro-inflammatory and phagocytic profile. hMDMs from SSc, RA and PsA patients demonstrated enhanced phagocytic activity in vitro. Elevated FcγRI and FcγRII levels were identified as key drivers of increased phagocytic activity and subsequent IL-6-driven inflammation. Nintedanib showed reduction in FcγRI expression, suggesting its potential therapeutic benefit in attenuating the phagocytic process.

Conclusion: This study highlights FcγR-expressing macrophages as drivers of phagocytosis and inflammatory responses in SSc. Dysregulated activation of these macrophages could lead to persistent inflammation and fibrosis in rheumatic diseases, highlighting new potential therapeutic approaches.

Keywords: SSc; fc gamma receptors; inflammatory pathways; macrophage activation; phagocytosis; rheumatic autoimmune inflammatory diseases; single-cell transcriptomics.

Graphical abstract

Figure 1.

Skin and lung tissue macrophages from SSc patients display a pro-phagocytic signature. (A) Schematic representation of skin and lung tissue transcriptomic analysis. (B) Log₁₀-adjusted P values (q-value) vs log2 fold changes of DEG comparing dcSSc patients vs HC in FCGR3A^hi skin macrophages. (C) Upregulated pathways comparing dcSSc patients vs HC in FCGR3A^hi skin macrophages. (D) Dot plot of a selection of macrophage polarization or phagocytosis-associated genes is shown comparing HC vs dcSSc patients in CCR1^hi, FCN1^hi and FCGR3A^hi skin macrophages. (E) Heatmap of pathway scores is shown comparing the different CCR1^hi, FCN1^hi and FCGR3A^hi skin macrophages from dcSSc patients. (F) Heatmap of DEG from the pseudobulk analysis comparing early dcSSc patients (disease duration <5 years) and HC in the total skin macrophage population (only DEG with log2FC ≥ 0.5 and P value < 0.05 are shown). (G) Log₁₀-adjusted P values (q-value) versus log2 fold changes of DEG comparing SSc-ILD patients vs HC in FCN1^hi lung macrophages. (H) Upregulated pathways comparing SSc-ILD patients vs HC in FCN1^hi lung macrophages (FDR). (I) Dot plot of a selection of macrophage polarization or phagocytosis-associated genes is visualized comparing HC vs SSc-ILD patients in FABP4^hi, FCN1^hi and SPP1^hi lung macrophages. (J), Heatmap of pathway scores is shown comparing the different FABP4^hi, FCN1^hi and SPP1^hi lung macrophages from SSc-ILD patients. Illustration created with BioRender.com

Figure 2.

SSc hMDMs display upregulated phagocytosis in all subtypes alongside an alternatively-activated phenotype. (A) Experimental workflow of in vitro phagocytosis assessment in M0 hMDMs. CD14⁺ monocytes were isolated from PBMCs from HC and SSc patients and differentiated into hMDMs with rh M-CSF (50 ng/ml) and left unpolarized as M0 hMDMs. (B) M0 HC (n = 36) and SSc (n = 57) hMDMs were incubated for 1 h with pHrodo bioparticles and the uptake was assessed by flow cytometry. A representative histogram of phagocytic activity (measured as MFI) and corresponding analysis of M0 HC (grey histogram) and SSc (purple histogram) hMDMs. (C) MFI fold change results of phagocytic activity of hMDMs from SSc patients were further divided into the different subtypes and compared. (D) and (E) Macrophage phagocytosis was correlated with disease duration from with patients without current treatments (n = 28) (D), or all patients (n = 44) (E). F–J, Differentiated HC (n = 10) and SSc (n = 14) hMDMs were either left unpolarized M0, stimulated with LPS (10 ng/ml) to M(LPS) or IL-4 (10 ng/ml) to M(IL-4). hMDMs were stained with fluorescently labelled surface antibodies and analysed by flow cytometry. (F) Representative gating into PD-L2 and CD206 markers already gated into CD40⁺CD86⁺CD163⁺ cells. (G) Percentages of living CD40⁺CD86⁺CD163⁺PD-L1⁺CD206⁺ cells comparing HC and SSc patients in M0, M(LPS), and M(IL-4) hMDMs. (H–J) Quantifications of MFI (fold changes) of CD206 (H), CD163 (I) and CD86 (J) comparing HC and SSc hMDMs. Data are shown as mean (s.d.) or median ± interquartile range. Significance was determined using unpaired non-parametric Mann–Whitney U test, unpaired two-tailed parametric t test or Kruskal-Wallis test with Dunn’s multiple comparison. Correlations were assessed by Pearson coefficient analysis. Illustration created with BioRender.com

Figure 3.

Heightened FcyR expression reveals initial steps in the phagocytic process as drivers of enhanced macrophage phagocytosis observed in SSc. (A) Inhibition efficiency of cytochalasin D in M(0) SSc hMDMs [representative histogram and calculated fold changes (MFI) to untreated are shown, n = 13]. Data are presented as mean (s.d.). Significance was determined using one-way ANOVA test with Dunnett’s multiple comparison. (B) Phagocytic activity of untreated and after inhibition with 1 and 5 μM cytochalasin D of M(0) HC (n = 10) and SSc (n = 13) hMDMs are shown (Fold changes MFI to HC untreated). Data are presented as mean (s.d.). Two-way ANOVA test with Bonferroni’s multiple comparison was performed. (C) Inhibition efficiency of cytochalasin D in M(0) HC and SSc hMDMs is shown (fold changes MFI to HC or SSc untreated, respectively). Data are presented as mean (s.d.). Two-way ANOVA test with Bonferroni’s multiple comparison was performed. (D) Gene expression of ARPC1B, ARPC2, ARPC3 and ARPC5 from HC (n = 9–12) and SSc (n = 15–20) M(0) hMDMs were measured by RT-qPCR. Data are shown as mean (s.d.) or median ± interquartile range. Significance was determined using unpaired non-parametric Mann-Whitney U test or unpaired two-tailed parametric t-test. (E) Inhibition efficiency of CK-666 in pooled M(0) HC and SSc hMDMs [representative histograms and calculated fold changes (MFI) to untreated are shown, n = 5]. Data are presented as mean (s.d.) Significance was determined using one-way ANOVA test with Dunnett’s multiple comparison. (F–K) Expression of surface FcγRI (CD64) (F), FcγRII (CD32) (G) and FcγRIII (CD16) (H) of M(0) hMDM from HC (n = 14–16) and SSc patients (n = 15–22) were assessed by flow cytometry. (I–K) M(0) hMDM phagocytosis results from HC and SSc patients were correlated with the expression of FcγRI (n = 14) (I), FcγRII (n = 14) (J) and FcγRIII (n = 9) (K). Data are shown as mean (s.d.) or median ± interquartile range. Significance was determined using unpaired non-parametric Mann-Whitney U test or unpaired two-tailed parametric t-test. Correlations were assessed by Pearson coefficient analysis

Figure 4.

Subsequent pro-inflammatory cascades are induced following phagocytosis. (A) Heatmap of pathway scores is shown comparing FCGR3A^hi skin macrophages from HC and dcSSc patients. (B) Heatmap of pathway scores is shown comparing FCN1^hi lung macrophages from HC and SSc-ILD patients. (C) Representative Western Blot of NF-κB pathway assessment at timepoints 0’, 15’, 30’ and 60’ (in min). (D) Quantification of p-IKKαβ/IKKβ, p-NF-κB/NF-κB and p-IκBa/IκBa ratios are shown for SSc hMDMs (n = 8–9). (E) Quantification of total NF-κB expression at timepoint 0’ comparing HC (n = 12) vs SSc (n = 14) M(0) hMDMs. Data are shown as mean (s.d.). Significance was assessed with an unpaired two-tailed parametric t-test. (F) 5 h after incubation with pHrodo bioparticles gene expression was assessed by RT-qPCR. IL6, IL8 and CCL22 expression were measured in M(0) hMDMs from HC (n = 5) and SSc (n = 6–7) patients. Data are shown as mean (s.d.). One sample t-test was used

Figure 5.

Tyrosine kinase inhibitor nintedanib diminished phagocytic activity of M0 hMDMs, paralleled by a decrease in FcγRI expression. (A–F) SSc hMDMs were treated with 0.1 μM nintedanib or left untreated simultaneously with their polarization into M(0) or M(LPS) for a total of 24 h. (A and B), hMDMs were then incubated for 1 h with pHrodo bioparticles and uptake was assessed by flow cytometry. Representative histogram and quantification of MFI (fold changes) for M(0) (A) or M(LPS) (B). Significance was assessed using a paired t-test. Expression of surface FcγRI (CD64) (C), FcγRII (CD32) (D), FcγRIII (CD16) (E) and CD18 (F) of M(0) and M(LPS) hMDM from SSc patients (n = 5–6) were assessed by flow cytometry. Significance was assessed using a paired t-test

Figure 6.

Enhanced macrophage phagocytosis in RA and PsA cohorts. (A–I) CD14⁺ monocytes were isolated from PBMCs from HC (n = 8–10), RA patients (n = 10), PsA patients (n = 11) and axSpA patients (n = 9). CD14⁺ monocytes were then differentiated into hMDMs using rh M-CSF (50 ng/ml). M(0) hMDMs were incubated for 1 h with pHrodo bioparticles and uptake was assessed by flow cytometry. A representative histogram of phagocytic activity (measured as MFI) of M(0) hMDMs and corresponding analysis of M(0) HC and RA (A), PsA (D), and axSpA (G) patient hMDMs is shown. Macrophage phagocytosis was correlated with CRP and ESR from RA patients (n = 9) (B), PsA patients (n = 10) (E), and axSpA patients (n = 8) (H). Effects of total number of previous immunosuppressive treatments were assessed from RA patients [≤4, (n = 6), >4, (n = 3)] (C), PsA patients (≤3, (n = 7), >3, (n = 4)] (F) and axSpa patients [<3, (n = 6), ≥3, (n = 3)] (I). Macrophage phagocytosis was correlated with number of previous immunosuppressive treatment from RA patients (n = 9) (C), PsA patients (n = 11) (F) and axSpA patients (n = 9) (I). Data are shown as mean (s.d.). Significance was determined using unpaired two-tailed parametric t-test