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. 2021 Feb 17:11:613975.
doi: 10.3389/fimmu.2020.613975. eCollection 2020.

GM-CSF Expression and Macrophage Polarization in Joints of Undifferentiated Arthritis Patients Evolving to Rheumatoid Arthritis or Psoriatic Arthritis

Sara Fuentelsaz-Romero  1 , Andrea Cuervo  2 , Lizbeth Estrada-Capetillo  1 , Raquel Celis  2 , Raquel García-Campos  1 , Julio Ramírez  2 , Sergi Sastre  3 , Rafael Samaniego  4 , Amaya Puig-Kröger  1 , Juan D Cañete  2
Affiliations

GM-CSF Expression and Macrophage Polarization in Joints of Undifferentiated Arthritis Patients Evolving to Rheumatoid Arthritis or Psoriatic Arthritis

Sara Fuentelsaz-Romero et al. Front Immunol. .

Abstract

Background and aims: GM-CSF-dependent macrophage polarization has been demonstrated in rheumatoid arthritis (RA). Our aim was to seek diagnostic/prognostic biomarkers for undifferentiated arthritis (UA) by analyzing GM-CSF expression and source, macrophage polarization and density in joints of patients with UA evolving to RA or PsA compared with established RA or PsA, respectively.

Methods: Synovial tissue (ST) from patients with UA evolving to RA (UA>RA, n=8), PsA (UA>PsA, n=9), persistent UA (UA, n=16), established RA (n=12) and PsA (n=10), and healthy controls (n=6), were analyzed. Cell source and quantitative expression of GM-CSF and proteins associated with pro-inflammatory (GM-CSF-driven) and anti-inflammatory (M-CSF-driven) macrophage polarization (activin A, TNFα, MMP12, and CD209, respectively) were assessed in ST CD163+ macrophages by multicolor immunofluorescence. GM-CSF and activin A levels were also quantified in paired synovial fluid samples. CD163+ macrophage density was determined in all groups by immunofluorescence.

Results: Synovial stromal cells (FAP+ CD90+ fibroblast, CD90+ endothelial cells) and CD163+ sublining macrophages were the sources of GM-CSF. ST CD163+ macrophages from all groups expressed pro-inflammatory polarization markers (activin A, TNFα, and MMP12). Expression of the M-CSF-dependent anti-inflammatory marker CD209 identified two macrophage subsets (CD163+ CD209high and CD163+ CD209low/-). CD209+ macrophages were more abundant in ST from healthy controls and PsA patients, although both macrophage subtypes showed similar levels of pro-inflammatory markers in all groups. In paired synovial fluid samples, activin A was detected in all patients, with higher levels in UA>RA and RA, while GM-CSF was infrequently detected. ST CD163+ macrophage density was comparable between UA>RA and UA>PsA patients, but significantly higher than in persistent UA.

Conclusions: GM-CSF is highly expressed by sublining CD90+ FAP+ synovial fibroblasts, CD90+ activated endothelium and CD163+ macrophages in different types of arthritis. The polarization state of ST macrophages was similar in all UA and established arthritis groups, with a predominance of pro-inflammatory GM-CSF-associated markers. CD163+ macrophage density was significantly higher in the UA phases of RA and PsA compared with persistent UA. Taken together, our findings support the idea that GM-CSF is a strong driver of macrophage polarization and a potential therapeutic target not only in RA but also in PsA and all types of UA.

Keywords: GM-CSF; macrophages; psoriatic arthritis; rheumatoid arthritis; synovial tissue; undifferentiated arthritis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
GM-CSF expression in stromal cells from synovial tissue of undifferentiated arthritis patients. (A) Representative immunofluorescence image of persistent undifferentiated arthritis (UA) synovial tissue (ST) determined by confocal microscopy using anti-CD90, anti-FAP (white), and anti-CD163 (red)-specific antibodies; nuclei were counterstained with DAPI. Lining and sublining layers are shown. The region of interest in the sublining layer (yellow square) is located 50 µm from the lining layer. (B) Immunofluorescence analysis of normal synovium (NS), persistent undifferentiated arthritis (UA), undifferentiated arthritis evolving to RA (UA>RA), rheumatoid arthritis (RA), undifferentiated arthritis evolving to psoriatic arthritis (UA>PsA) and psoriatic arthritis (PsA) ST determined by confocal microscopy using anti-GM-CSF (green), anti-CD90, anti-FAP or anti-CD163 (red)-specific antibodies; nuclei were counterstained with DAPI. Arrowheads indicate CD90+FAP+ fibroblast and white arrows indicate CD163+ macrophages. Samples were devoid of staining when incubated with isotype-matched irrelevant antibodies as negative controls. The experiment was carried out in independent samples from each type (NS, n=6; UA, n=16; UA>RA, n=8; RA, n=12; UA>PsA, n=9; and PsA, n=10) and representative experiments are shown. CD163+ macrophages are indicated by white arrows. Scale bars, 50 µm. Determination of levels of GM-CSF (C) and activin A (D) in the synovial fluid of patients with UA (n=12), UA>RA (n=9), RA (n=9), UA-PsA (n=7), and PsA (n=9), as determined by ELISA.
Figure 2
Figure 2
Expression of polarization markers by RA macrophages and in-vitro generated monocyte-derived macrophages. (A) Gene set enrichment analysis (GSEA) on the ranked list of genes obtained from the comparison of the transcriptome of RA macrophages (RA mac) versus monocyte-derived macrophages (M-MØ) (GSE10500) using the set of genes with the highest GM-CSF-induced upregulation in monocyte-derived macrophages differentiated with GM-CSF (GSE68061). (B) INHBA, MMP12, CD209, and TNFA mRNA expression levels determined by qRT-PCR on monocytes differentiated with GM-CSF (GM-MØ) and M-CSF (M-MØ). Mean ± SEM of four independent donors are shown (* p < 0.05, ** p < 0.01, Student t test).
Figure 3
Figure 3
Expression of macrophage-pro-inflammatory polarization markers by CD163+ macrophages from undifferentiated arthritis patients. (A) Immunofluorescence analysis of synovial tissues as determined by confocal microscopy using anti-activin A, TNFα-, MMP12-specific antibodies; nuclei were counterstained with DAPI. Samples were devoid of staining when incubated with isotype-matched irrelevant antibodies as negative controls. The experiment was carried out in independent samples from each type (NS, n=6; UA, n=16; UA>RA, n=8; RA, n=12; UA>PsA, n=9; and PsA, n=10) and representative experiments are shown. Scale bars, 50 µm. (B) Summary dot plot showing mean MFI values of activin A, TNF-α and MMP12 expression in CD163+ macrophages from NS, UA, UA>RA, RA, UA>PsA, and PsA synovial tissues samples. Mean ± SEM are shown (* p < 0.05, ** p < 0.01, ***p < 0.001, Mann-Whitney test).
Figure 4
Figure 4
CD163+ CD209 macrophage subsets in synovial tissue. (A) CD163 (green) and CD209 (red) staining of NS, UA, UA-RA, RA, UA-PsA, and PsA synovial tissues. Scale bar, as shown. (B) Summary dot plot showing mean MFI values of CD209 expression in CD163+ macrophages from synovial tissue samples. The experiment was carried out in independent samples from each type (NS, n=6; UA, n=16; UA>RA, n=8; RA, n=12; UA>PsA, n=9; and PsA, n=10) and representative experiments are shown. Mean ± SEM are shown (* p < 0.05, Mann-Whitney test). (C) Percentage of CD209-expressing macrophages in NS, UA, UA>RA, RA, UA>PsA, and PsA synovial tissues. The MFI values of eight to 12 tissues samples of each group were used for classification as “low/neg” (white) or “high” (> 34 a.u., black) CD209 expression.
Figure 5
Figure 5
Macrophage density in normal, undifferentiated arthritis, rheumatoid arthritis and psoriatic arthritis synovial tissue. (A) Quantification of CD163+ macrophages from normal (NS, n=6), undifferentiated arthritis (UA, n=16), undifferentiated arthritis evolving to RA (UA>RA, n=8), undifferentiated arthritis becoming psoriatic arthritis (UA>PsA, n=9), rheumatoid arthritis (RA, n=12), and psoriatic arthritis (PsA, n=10) synovial tissues. The total number of macrophages was normalized based on selected tissue area (mm2). Mean ± SEM is shown. Significant differences are indicated (** p < 0.01, *** p < 0.001, Mann-Whitney test). (B) Immunofluorescence analysis of NS, UA, UA>RA, RA, UA>PsA, and PsA synovial tissues as determined by confocal microscopy using anti-CD163 specific antibody (green); nuclei were counterstained with DAPI (blue). The experiment was carried out in independent samples from each type (six normal synovial tissue controls and eight to 16 remaining synovial tissues) and representative experiments are shown. Scale bars, 100 µm.
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