Background: Human peripheral blood contains rare hematopoietic and non-hematopoietic cells, including circulating tumor cells which have been investigated for their prognostic value in various malignancies. Additionally, evidence suggests that rare circulating fibroblast-like cells expressing the mesenchymal marker cadherin-11 (CDH11)[1], as well as pre-inflammatory mesenchymal cells[2], are found in the peripheral blood of rheumatoid arthritis (RA) patients.

Objectives: To detect and characterize circulating fibroblasts, identified as non-hematopoietic cells expressing podoplanin (PDPN), and fibrocytes, identified as hematopoietic antigen-presenting cells expressing CD34, in patients with RA and psoriatic arthritis (PsA), by exploiting the high sensitivity and multidimensionality of mass cytometry.

Methods: Peripheral blood samples of 270 μl from patients with active seropositive RA (n=9), active PsA (n=9) and healthy controls (n=9) were stained using a panel of metal-conjugated antibodies against 35 proteins, including 5 chemokine receptors (CCR4, CCR6, CCR7, CXCR3 and CXCR5) and 5 mesenchymal markers (CDH11, PDPN, CD90, CD34, and Notch3), and were analyzed by a 3 ^rd generation Helios mass cytometer. Patients were re-evaluated after three months of anti-rheumatic treatment.

Results: Compared to only 4 circulating fibroblasts (CD45 ^- CD3 ^- CD19 ^- CD4 ^- CD8 ^- CD56 ^- CD25 ^- CD66b ^- CD294 ^- PDPN ⁺ ) detected in 4/9 HC, 24 and 14 fibroblasts were detected in 5/9 RA and 7/9 PsA patients, respectively (median frequency of 7 cells/ml, p =0.009 vs HC). On the other hand, circulating fibrocytes (CD45 ⁺ CD3 ^- CD19 ^- CD4 ^- CD8 ^- CD56 ^- CD25 ^- CD66b ^- CD294 ^- CD34 ⁺ HLA-DR ⁺ ) were increased in RA (n=203) and PsA (n=283) patients compared to HC (n=138, p =0.003), with a median frequency of 70 cells/ml. Single-cell analysis of CDH11, CD90, CD34, and Notch3 expression on circulating fibroblasts revealed that CDH11 and CD90 were uniquely expressed in patients, with higher frequency in RA vs PsA. In addition, expression of CCR7 was exclusively observed in patient-derived fibroblasts, suggesting their potential migration to tissues expressing CCR7 ligands, such as secondary lymphoid organs and synovium. Regarding fibrocytes, significantly increased CDH11 ⁺ and CCR7 ⁺ fibrocyte counts were observed in both RA and PsA patients compared to HC. Additional analyses revealed a marked heterogeneity in expression pattern combinations of mesenchymal markers in both fibroblasts and fibrocytes, especially in RA patients. Following three months of treatment, all findings pertaining to fibroblasts were comparable to baseline, whereas increased fibrocyte frequencies were observed regardless of disease activity changes. However, in patients who achieved clinical remission, percentages of CDH11 ⁺ and PDPN ⁺ fibrocytes were significantly decreased, indicating a possible role of these cells in active disease.

Conclusion: Increased numbers of circulating fibroblasts and fibrocytes, having migration potential and properties of CDH11-mediated homotypic adhesion to synovial fibroblasts, are present in the blood of RA, as well as of PsA patients. Further studies into their possible pathogenetic role are warranted.

REFERENCES: [1] Sfikakis PP, et al. Cadherin-11 mRNA transcripts are frequently found in rheumatoid arthritis peripheral blood and correlate with established polyarthritis. Clin Immunol . 2014 Nov;155(1):33-41.

[2] Orange DE, et al . RNA Identification of PRIME Cells Predicting Rheumatoid Arthritis Flares. N Engl J Med . 2020 Jul 16;383(3):218-228.

Acknowledgements: We acknowledge the support of this work by the project “The Greek Research Infrastructure for Personalised Medicine (pMedGR)” (MIS 5002802) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund).

Disclosure of Interests: Maria Kyriakidi: None declared, Eleni-Kyriaki Vetsika: None declared, Nikolaos I. Vlachogiannis: None declared, Kleio Verrou: None declared, Maria G. Tektonidou ACTELION, PFIZER, GENESIS, MSD, UCB, BOEHRINGER INGELHEIM, ENORASIS, FARMASERV-LILLY, GILEAD, ABBVIE, NOVARTIS, ROCHE, FARAN, AMGEN, JANNSEN, ACTELION, PFIZER, GENESIS, MSD, UCB, BOEHRINGER INGELHEIM, ENORASIS, FARMASERV-LILLY, GILEAD, ABBVIE, NOVARTIS, ROCHE, FARAN, AMGEN, JANNSEN, George E. Fragoulis PFIZER, GENESIS, MSD, UCB, BOEHRINGER INGELHEIM, ENORASIS, FARMASERV-LILLY, ABBVIE, NOVARTIS, ROCHE, FARAN, AMGEN, JANNSEN,, Petros P. Sfikakis ACTELION, PFIZER, GENESIS, MSD, UCB, BOEHRINGER INGELHEIM, ENORASIS, FARMASERV-LILLY, GILEAD, ABBVIE, NOVARTIS, ROCHE, FARAN, AMGEN, JANNSEN, ACTELION, PFIZER, GENESIS, MSD, UCB, BOEHRINGER INGELHEIM, ENORASIS, FARMASERV-LILLY, GILEAD, ABBVIE, NOVARTIS, ROCHE, FARAN, AMGEN, JANNSEN.