Distinct CDH11+   circulating fibroblasts and immune cells co-expressing chemokine receptors in chronic inflammatory arthritis patients

doi:10.21203/rs.3.rs-4803279/v1

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Distinct CDH11⁺ circulating fibroblasts and immune cells co-expressing chemokine receptors in chronic inflammatory arthritis patients

https://doi.org/10.21203/rs.3.rs-4803279/v1

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The mechanisms underlying the progression of chronic inflammatory arthritis, affecting over 1% of adults, remain largely unclear. Using single-cell mass cytometry on peripheral blood of patients with active rheumatoid and psoriatic arthritis, we identified various cells co-expressing mesenchymal markers, including the homotypic adhesion molecule cadherin-11 (CDH11), and chemokine receptors. Circulating fibroblasts (podoplanin⁺CD45⁻CD3⁻CD19⁻CD4⁻CD8⁻CD56⁻CD66b⁻CD294⁻) co-expressing CDH11 and CCR7 were found exclusively in patients and not in paired bone marrow samples, suggesting their origin from inflamed joints. Increased fibrocytes (CD34⁺HLA-DR⁺CD45⁺CD3⁻CD19⁻CD4⁻CD8⁻CD56⁻CD66b⁻CD294⁻) co-expressing CDH11 and CCR7 were also found in patients, being more prevalent in bone marrow than blood, supporting their bone marrow origin. Among various leukocyte subsets, CDH11⁺CD90⁺neutrophils co-expressing CCR6 were markedly increased in patients. Paired measurements three months post-antirheumatic treatment revealed persistently increased circulating CDH11⁺fibroblasts, CDH11⁺fibrocytes and CDH11⁺CD90⁺CCR6⁺neutrophils, regardless of clinical responses. Moreover, CDH11⁺neutrophils were identified by confocal microscopy in close proximity to synovial fibroblasts in knee-surgery-obtained rheumatoid synovium. Combining our findings with previous data showing circulating pre-inflammatory mesenchymal cells to precede clinical arthritis flares, we suggest a drug-independent process orchestrated by chemokines that may contribute to ‘arthritis spreading’, wherein synovial fibroblasts and fibrocytes migrate into distant synovium, either alone or by forming complexes with CD90⁺CDH11⁺ neutrophils, through CDH11-mediated binding.

Health sciences/Rheumatology/Rheumatic diseases/Rheumatoid arthritis

Health sciences/Pathogenesis/Inflammation/Chronic inflammation

Health sciences/Rheumatology/Rheumatic diseases/Spondyloarthritis/Psoriatic arthritis

Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are the main chronic immune-mediated diseases causing peripheral joint inflammation, currently affecting more than 1% of the adult population^1,2. Both conditions share common pathogenetic mechanisms, but the role of adaptive immunity seems less important in patients with PsA and seronegative RA, who lack anti-citrulline autoantibodies and rheumatoid factors^3–6. Despite significant advances in the management of RA and PsA following the introduction of anti-TNF biologic therapies in 1998^7–10, the pathogenetic mechanisms driving the initiation and perpetuation of joint inflammation remain largely unclear^4,11,12. Efforts to understand the dynamic cellular interactions involved in these mechanisms include studying circulating synovial fibroblasts in the peripheral blood within the concept of cell migration. The pathogenic behavior of migrating synovial fibroblasts was first introduced in 2003 by Aidinis et al.¹³, and was further supported by other studies^14,15. Using experimental arthritis models, it was shown that trafficking of synovial fibroblasts through circulation results in arthritis spreading to unaffected joints. More recent evidence in patients with rheumatoid arthritis (RA) suggests the presence of circulating pre-inflammatory mesenchymal (PRIME) cells prior to disease flare, possessing features of inflammatory synovial fibroblasts¹⁶. Building upon these findings, a model wherein PRIME cells undergo activation by B cells in the weeks preceding a clinical flare, and then migrate from the periphery into the synovium, has been proposed¹⁶.

Our previous investigations have shown elevated cadherin-11 (CDH11) mRNA transcripts, as well as rare non-hematopoietic cells expressing CDH11 in the blood of RA patients, that correlate with the concomitant presence of active polyarthritis¹⁷. CDH11 is a cell-cell adhesion protein, traditionally expressed by synovial fibroblasts, contributing to synovial hyperplasia and joint inflammation in arthritis^18,19, but its expression has also been detected on rare circulating and tissue-resident leukocytes^17,20–23. These findings support a dynamic crosstalk between mesenchymal and hematopoietic origin cells, adding another layer of complexity to the pathogenetic processes in chronic inflammatory arthritis.

In addition to circulating synovial fibroblasts, other lines of evidence suggest a potential involvement of circulating fibrocytes in RA progression, as well^24–27. Fibrocytes are bone marrow-derived progenitor cells that co-express hematopoietic stem cell antigens and can produce extracellular matrix components^26,28. Circulating fibrocytes have the potential to aggravate synovitis in RA through diverse mechanisms, including the secretion of pro-inflammatory cytokines and growth factors, antigen presentation via major histocompatibility complex class II (MHCII), as well as their direct interaction with endothelial cells, promoting angiogenesis²⁹. Moreover, evidence supports that circulating fibrocytes can migrate into the synovium where they may serve as precursors to fibroblast-like synoviocytes, further perpetuating joint inflammation^30–32. Studies on circulating fibroblasts and fibrocytes have not been performed in patients with PsA, in contrast to RA^16,24,25,33. On the other hand, within the inflamed joint, fibroblast-like synoviocytes have been indeed shown to interact with infiltrating leukocytes to regulate inflammation and tissue damage in the joints of patients with RA and PsA^34,35.

Mass cytometry (CyTOF) has emerged as an ideal technology for identifying and characterizing rare cells in the blood, i.e. 1–2 cells per ml, due to its ability to simultaneously analyze multiple parameters at the single-cell level with minimal background noise and enhanced sensitivity^36–38. Thus, by exploiting the high-dimensional power and sensitivity of single-cell proteomics, we aimed to identify and characterize fibroblasts, fibrocytes and leukocytes expressing CDH11 and/or other mesenchymal markers in the peripheral blood of patients with active RA and PsA, and compare their levels with controls, as well as with paired samples three months after antirheumatic treatment. Moreover, we sought to investigate whether CDH11-expressing synovium-infiltrating leukocytes co-localize with synovial fibroblasts in patients with RA.

Circulating fibroblasts expressing CDH11, CD90, HLA-DR and CCR7 characterize RA and PsA

Circulating fibroblasts, i.e. non-hematopoietic cells expressing podoplanin (PDPN⁺CD45⁻CD3⁻CD19⁻CD4⁻CD8⁻CD56⁻CD66b⁻CD294⁻)^16,22 (Fig. 1a), were identified in both active RA and PsA patients by mass cytometry. Specifically, a total of 26 circulating fibroblasts were detected in the peripheral blood samples of 7/11 RA patients (67%) and a total of 24 fibroblasts detected in 10/12 PsA patients (83%), with an estimated median frequency of 7 cells/ml (Supplementary Table 6). In contrast to patients with RA and PsA, a total of only 4 fibroblasts were detected in blood samples from 4/9 control individuals (44%) (p = 0.018, Fig. 1b). Moreover, two or more fibroblasts per sample were observed in 55% and 50% of active RA and PsA patients, respectively, contrasting with only one fibroblast per control sample (p = 0.012; Fig. 1c).

We also investigated the expression of CDH11, CD34, CD90 and Notch3 on the identified PDPN-expressing fibroblasts at baseline. These four proteins characterize mesenchymal cells in general, and their expression has been associated with higher cell motility, adhesion, migration and invasion^{18,19,39–41}. Notably, CDH11 and CD90 were expressed only by patient-derived fibroblasts, with higher frequency in RA than PsA patients (Fig. 1d, Supplementary Fig. 19a, b). Moreover, expression of HLA-DR, denoting antigen-presenting properties, was exclusively noted on patients’ fibroblasts (Fig. 1d). By exploring all potential combinations involving CDH11, CD34, CD90, and Notch3, a total of 16 distinct fibroblastic subsets were investigated (FB1-16; Supplementary Table 3). Out of these 16 phenotypes, 7 were identified in RA (FB1,2,3,7,8,12,16) and 6 in PsA (FB1,2,5,7,12,16) versus only 2 in controls (FB1,9; Fig. 2a).

Next, we examined the presence of a panel of chemokine receptors (CCR4, CCR6, CCR7, CXCR3 and CXCR5), known to orchestrate migration of cells in peripheral tissues, on the identified fibroblasts. Notably, CCR7 was only observed in patient-derived fibroblasts, suggesting their potential trafficking towards lymphoid organs or the inflamed joint (Fig. 1e, Supplementary Fig. 19c). Moreover, circulating fibroblasts primarily expressed CCR6 (Fig. 1e), with CCR6⁺ fibroblast counts being elevated in patients compared to controls (p = 0.047; Supplementary Fig. 19d).

Following three months of antirheumatic treatment, a total of 38 circulating fibroblasts were detected in 10/22 patients (46%), at comparable frequencies and having similar characteristics to those identified at baseline. Among the re-evaluated patients, there were 11/22 who achieved clinical remission/low disease activity (5 RA and 6 PsA patients), while the remaining 11 patients had persistently active disease, albeit less active than baseline. In these repeated measurements, 11 and 27 circulating fibroblasts were detected in the peripheral blood of 4/11 patients in remission and 6/11 patients with persistent active disease, respectively (Supplementary Table 6, Supplementary Fig. 20). The levels of CDH11⁺ fibroblasts remained elevated in the peripheral blood of both RA and PsA patients, regardless of clinical responses (Fig. 1f). Fibroblast phenotypic composition in terms of mesenchymal marker expression demonstrated inter-patient and intra-patient variability between the two time-points (Fig. 2b, c). Of note, fibroblasts co-expressing all examined mesenchymal markers (FB16) were detected only in active patients (Fig. 2b, c), indicating potential relevance to disease activity.

Finally, to gain insights into the potential origin of circulating fibroblasts, we further analyzed paired samples of peripheral blood and bone marrow in 2 PsA patients. Rare fibroblasts characterized by CD34 expression were indeed present in the bone marrow, however, none expressed CDH11. These findings suggest that circulating fibroblasts expressing CDH11 and exclusively found in patients with chronic arthritis are more likely to derive from inflamed joints than the bone marrow.

Circulating CDH11⁺ and CCR7⁺ fibrocytes are increased in chronic inflammatory arthritis

Circulating fibrocytes (CD34⁺HLA-DR⁺CD45⁺CD3⁻CD19⁻CD4⁻CD8⁻CD56⁻CD66b⁻CD294⁻)^25,42 (Fig. 3a) were detected in all examined blood samples from patients and controls. A total of 253 fibrocytes were detected in RA (range, 6–86) and 322 in PsA patients (range, 7–89), with an estimated median frequency of 70 cells/ml, versus a total of 138 (range, 2–52) fibrocytes in controls (Fig. 3b, Supplementary Table 6). Certain fibrocytes expressed CDH11, PDPN, Notch3, or CD90, with higher percentages detected in RA, followed by PsA patients, compared to controls (Supplementary Fig. 21a). Of them, a significant elevation of CDH11⁺ fibrocyte counts was found in patients compared to controls (p = 0.004), as shown in Fig. 3c. Through the examination of all potential combinations involving CDH11, PDPN, CD90, and Notch3 in our fibrocyte pool, a total of 16 subsets were formed (FC1-16; Supplementary Table 4). Out of these distinct phenotypes, 13 and 11 were identified in RA and PsA patients, respectively, versus only 5 (FC1-5) in controls (Fig. 4a). No co-expression of the examined mesenchymal markers (FC6-16) was detected in controls’ fibrocytes.

Regarding chemokine receptor expression on fibrocytes, we found that these cells predominantly expressed the chemokine receptor CCR6, possibly facilitating their recruitment into sites of inflammation (Supplementary Fig. 21b), and to a lesser extent CCR7 and CCR4. Interestingly, patients exhibited increased counts of CCR7⁺ fibrocytes compared to controls (p = 0.026; Fig. 3d).

Upon patients’ re-evaluation after three months of antirheumatic treatment, fibrocytes were increased compared to baseline, either in patients who achieved remission (p = 0.008) or in patients with persisting active disease (p = 0.019), regardless of RA or PsA (Supplementary Fig. 22). As in the case of CDH11⁺ fibroblasts, CDH11⁺ fibrocyte counts remained elevated three months post-treatment, regardless of the clinical status of the patients (Fig. 3e). Moreover, the phenotypic composition of fibrocytes in terms of mesenchymal marker combinations displayed inter-patient and intra-patient variability between the two time-points (Fig. 4b, c).

Finally, highly elevated fibrocyte counts were evident in bone marrow aspirates when compared to the matched blood samples in two patients with PsA. Indeed, fibrocyte counts were increased by 50 to 100 times compared to the peripheral blood (Fig. 3f), indicating that circulating fibrocytes originate primarily from bone marrow.

Neutrophils co-expressing CDH11, CD90 and CCR6 are increased in chronic inflammatory arthritis

To expand on our previous observations on CDH11 expression on circulating hematopoietic CD45⁺cells¹⁷, and given that CD90 may promote leukocyte extravasation^41,43, we sought to investigate the expression of these mesenchymal cell markers on circulating leukocytes. Hematopoietic cells co-expressing CD90 and CDH11, which mediates cell-to-cell binding, were indeed identified by mass cytometry (Fig. 5a, Supplementary Fig. 23), comprising approximately 1% of total CD45⁺ cells in the peripheral blood, with comparable proportions across RA, PsA and controls. Further analysis revealed that co-expression of CDH11 and CD90 was present in all 12 tested major leukocyte subpopulations (neutrophils, eosinophils, basophils, monocytes, natural killer cells, dendritic cells, innate lymphoid cells, mucosal-associated invariant T cells/invariant natural killer cells, γδ T cells, CD4⁺ T cells, CD8⁺ T cells and B cells), with the highest counts being detected in cells of innate immunity, particularly eosinophils, followed by neutrophils and monocytes (Fig. 5b). No significant differences were noted between patients and controls in all CDH11⁺CD90⁺leukocyte subpopulations except one. Specifically, CDH11⁺CD90⁺ neutrophils were found to be highly enriched in patients compared to controls (p = 0.006; Fig. 5c).

The expression of chemokine receptors on the surface of CDH11⁺CD90⁺ neutrophils was subsequently investigated (Supplementary Fig. 24). Among the examined receptors, CDH11⁺CD90⁺CCR6⁺ neutrophil counts were significantly higher in patients compared to controls (p = 0.041; Fig. 5d, Supplementary Fig. 25). All examined chemokine receptors were preferentially overexpressed on CD90⁺CDH11⁺ neutrophils as compared to CDH11⁻CD90⁻, CDH11⁻CD90⁺ and CDH11⁺CD90⁻ neutrophils (Supplementary Fig. 26). As Notch3 is a receptor whose expression on immune cells has been associated with enhanced extravasation⁴⁴, we also examined its presence on CDH11⁺CD90⁺ neutrophils. Although our analysis revealed no significant differences in CDH11⁺CD90⁺Notch3⁺ neutrophil counts between patients and controls, investigation of chemokine receptor expression showed CDH11⁺CD90⁺Notch3⁺ neutrophils co-expressing CCR6⁺ or CCR7⁺ were enriched in patients versus controls (p = 0.027, and p = 0.019, respectively; Supplementary Fig. 27).

In repeated measurements upon patients’ re-evaluation following antirheumatic treatment, no significant differences were observed concerning CDH11⁺CD90⁺ leukocytes, despite changes in disease activity. Specifically, total CDH11⁺CD90⁺ neutrophil counts and CDH11⁺CD90⁺CCR6⁺ neutrophil counts remained elevated in both patients with persisting active disease and patients who achieved clinical remission/low disease activity (Fig. 5e, Supplementary Fig. 28).

Finally, we searched for individual correlations between CDH11⁺CD90⁺ leukocyte subpopulations and fibroblasts and fibrocytes expressing CDH11 in patients with RA and PsA at baseline. No correlation was observed between circulating CDH11⁺ fibroblasts and CDH11⁺CD90⁺ leukocytes. Notably however, we found that individual numbers of circulating fibrocytes expressing CDH11 correlated positively with corresponding numbers of CDH11⁺CD90⁺ neutrophils (r_s = 0.498; p = 0.015), as well as with monocytes, natural killer cells, innate lymphoid cells, dendritic cells and γδ T cells co-expressing CD90 and CDH11 (Supplementary Table 7); no correlations were found with CD4⁺ T, CD8⁺ T or B cells.

Synovium-infiltrating neutrophils may interact with synovial fibroblasts via CDH11

To investigate whether circulating leukocytes with mesenchymal phenotypic features could be found within the synovial tissue and potentially interact with synovial fibroblasts, we conducted confocal microscopy analysis on synovial tissue biopsies obtained during knee arthroplasty from four patients with RA and three with osteoarthritis (OA). Using immunofluorescence staining for CD45, CDH11, PDPN, and CD90 on paraffin sections, the presence of CDH11⁺ leukocytes in close proximity to CDH11⁺ synovial fibroblasts was observed in RA (Fig. 6a), indicating a potential intercellular communication between these cells through homotypic binding. Moreover, co-expression of CD90 and CDH11 was detected on leukocytes and more specifically on neutrophils (Ly6G⁺) in RA, being closely located to CDH11⁺ fibroblasts (Fig. 6b, c). Of note, none of these observations were evident in OA synovium. These findings suggest a neutrophil-fibroblast crosstalk mediated by CDH11, as well as a potential migratory pathway for the described cells from the periphery into the RA tissue, highlighting their possible relevance in the pathogenesis of inflammatory arthritis.

Using mass cytometry, we show herein that rare circulating fibroblasts and fibrocytes, expressing CDH11 and chemokine receptors, possibly originating from inflamed joints and bone marrow, respectively, are increased in patients with inflammatory arthritis. Single-cell proteomics by mass cytometry is an ideal technology for identifying and characterizing rare cells in whole blood samples^36,37. Its minimal background noise and improved sensitivity make it particularly well-suited for the detection and characterization of low-abundance cellular events, as, for example, circulating tumor cells in cancer patients, detected in frequencies ranging from 1 to 100 cells per ml of blood^38,45. Circulating fibroblasts and fibrocytes in our patients had a median cell count of 7 and 70 per ml of peripheral blood, respectively, whereas their presence has been previously studied in RA¹⁶, but not in PsA patients.

By investigating the expression of markers that are known to characterize synovial fibroblasts, such as CDH11, CD90, CD34, and Notch3, we unveiled the heterogeneity of circulating fibroblasts, akin to their synovial counterparts^22,46,47. This heterogeneity was more evident in the circulating fibroblast pool of RA compared to PsA, suggesting potential differences in fibroblast involvement between the two types of arthritis, as also observed in synovium studies³⁵. On the other hand, fibrocytes have been previously studied in RA by flow-cytometry, but not in PsA, being elevated compared to controls at baseline, as well as during re-evaluation after three and six months²⁵.

In both RA and PsA patients, circulating neutrophils co-expressing CD90 and CDH11, were increased compared to controls, with their levels remaining elevated after a three-month anti-rheumatic standard treatment, regardless of disease activity. CD90, by binding to integrins, including αVβ3, has been shown to be involved in the adhesion of leukocytes to activated human endothelial cells (EC), facilitating their extravasation into different tissues^43,48,49. Although certain studies have reported CDH11 and CD90 expression on immunocytes^17,21,43,50, this is the first proteomic study to investigate their expression at the single-cell level in the peripheral blood. In our study, the presence of leukocytes co-expressing CDH11 and CD90 was also detected in RA synovial biopsies, and not in OA, further supporting their potential migration into the synovium. These cells were located closely to CDH11-expressing synovial fibroblasts, indicating their possible interaction through CDH11-mediated homotypic binding. In the context of RA, CD90 expression has been observed on CD14⁺ cells in perivascular areas of the synovium, possibly indicative of the migration of these cells from the peripheral blood to the synovial tissue⁵¹. Moreover, mass cytometry data from synovial tissues has also revealed the expression of CDH11 and CD90 by certain monocytes, T and B cells²². Interestingly, the levels of CDH11⁺CD90⁺ neutrophils that co-expressed Notch3 and CCR6 were also elevated in our patients, which may further support the migratory potential of this subset, as there is evidence suggesting the involvement of Notch3 in the process of immunocyte transmigration into the inflamed tissue, by mediating leukocyte adhesion to the endothelium⁴⁴.

Circulating fibroblasts, fibrocytes and CDH11⁺CD90⁺ neutrophils mainly expressed CCR6, among the examined chemokine receptors, CCR4, CCR7, CXCR3 and CXCR5. This may be indicative of their increased capacity to receive inflammatory signals from the joint, where its ligand, CCL20, is upregulated during arthritis⁵². Moreover, increased counts of fibroblasts and fibrocytes expressing CCR7 were observed in patients compared to controls. Although, CCR7 is traditionally involved in the migration of cells towards lymphoid organs, its ligands CCL19 and CCL21 have been found to be highly produced in the synovium of both RA and PsA⁵³. Thus, CCR6 and CCR7 expression may facilitate the guided movement of these cells from the bloodstream towards distant joints.

The concept of circulating synovial fibroblasts was initially proposed as a hypothesis to explain inflammatory monoarthritis/oligoarthritis progression to polyarthritis, with studies showing the ability of injected pathogenic fibroblasts to a joint to cause arthritis and then migrate to unaffected joints, where they undergo proliferation and promote a pro-inflammatory microenvironment¹³. Given that the mesenchymal CDH11, which is essential for the development of the synovium⁵⁴, has been detected in RA patients’ peripheral blood¹⁷ and is also a marker gene for circulating PRIME cells¹⁶, it can be hypothesized that specific fibroblast subpopulations are activated and migrate into the bloodstream toward distant joints, thus spreading arthritis.

Building upon the findings from the aforementioned studies^13,15–17 and based on the results presented herein, we propose a model where fibroblasts and fibrocytes, expressing CDH11, originating from the joints and bone marrow, respectively, enter the bloodstream and migrate into distant synovia, either alone or by forming complexes with CDH11⁺CD90⁺ neutrophils (Fig. 7). Such migration processes may operate independently of adaptive immunity-mediated pathogenetic mechanisms, which is in line with our mass cytometry findings. According to our proposed model, migration of fibroblasts and fibrocytes along with neutrophils may be facilitated by binding of CD90 to integrins of endothelial cells during extravasation, with these cells being attracted towards the joint by synovium-derived chemokines. Homotypic binding via CDH11 may enable the formation of cell clusters with enhanced migratory potential that could possibly insert the joint. As in the case of circulating tumor cell clusters, where non–tumor cells, such as platelets and neutrophils, can interact with tumor cells to enhance their survival and metastasis^55,56, we could assume that the formation of cell aggregates between circulating fibroblasts/fibrocytes and neutrophils may protect these rare circulating cells against harmful shear stress and mechanical forces formed in the bloodstream. Moreover, PDPN on fibroblasts may bind to C-type lectin‐like receptor 2, which is mainly expressed by platelets, potentially resulting in platelet activation⁵⁷. Platelets are known to favor tumor dissemination and metastasis by protecting tumor cells from mechanical forces during circulation, enabling them to evade immune surveillance, and mediating adherence to the vascular endothelium, thus favoring extravasation^57,58. Therefore, circulating fibroblasts may bind to not only neutrophils, but also platelets, via PDPN, further facilitating their trafficking through the circulation.

Current biologic therapies for RA and PsA have significantly improved not only the quality of life but also the prognosis of these patients⁵⁹. Nonetheless, attaining and sustaining long-term remission remains a considerable challenge. One of the reasons for the suboptimal management of these patients could be the insufficient targeting of critical players, such as cells with mesenchymal characteristics. We showed herein that the levels of circulating CDH11-expressing fibroblasts, fibrocytes and neutrophils remain elevated regardless of the disease activity of patients, underscoring that our proposed model is drug-independent. Our results suggest the potential involvement of these circulating cells, as well as the possible formation of fibroblast/fibrocyte-neutrophil aggregates, in the pathogenesis of chronic joint inflammation, therefore supporting the endeavors for the development of anti-mesenchymal therapies, targeting for example CDH11^60,61.

The major limitation of our study is that we have not explored potential functional implications associated with the examined cell populations or assessed their migratory capacity through, for instance, migratory assays. Another limitation of our study is the fact that cell recovery during acquisition in mass cytometry experiments is always less than 100%, which, combined with the cell-cell adhesion properties of CDH11, which may exclude a subset of CDH11-expressing cells from the single-cell analysis, suggests that the actual counts of the described cells might be indeed much higher than the observed values.

To conclude, our results form the basis for future experiments to extend our understanding of the pathogenetic role of circulating cells with mesenchymal phenotype, forming cell-cell complexes, and the dynamic cellular interactions leading to arthritis “metastasis”. Based on previous studies^13,15–17 and our findings, arthritis may be spread by a process shared by RA and PsA which operates independently of adaptive immunity and current drugs, thus, holding promise for novel therapeutic interventions.

Patient enrollment

A total of 23 consecutive patients with moderately or highly active chronic inflammatory arthritis who attended the outpatient rheumatology clinics (Supplementary Table 1) were recruited. There were 11 patients with active RA (RA 2010 criteria), of whom 9 were seropositive (positive for RF and/or ACPA) and 2 were seronegative (negative for RF and ACPA), and 12 patients with active PsA (CASPAR criteria). Moderate to high disease activity was defined as DAS28-ESR > 3.2 for RA (active RA) and as DAPSA > 14 and/or ASDAS > 1.2 (for those having axial disease confirmed by X-rays or magnetic resonance) for PsA (active PsA). Exclusion criteria included prior treatment with the b cell-depleting rituximab or chemotherapy, those with a history of solid or hematological neoplasms, as well as individuals who had been vaccinated within the preceding two weeks and those having clinical evidence of concurrent infection. A total of 9 healthy age and sex-matched individuals served as controls.

All patients, but one with PsA, were re-evaluated three months after the initiation or modification of antirheumatic treatment regimens for active disease, following standard clinical practice in accordance with national and international guidelines. Specifically, 8 patients received conventional synthetic disease-modifying antirheumatic drugs (csDMARDs; RA, n = 3; PsA, n = 5), and 14 patients received biologic/targeted-synthetic DMARDs (b/tsDMARDs; RA, n = 8; PsA, n = 6).

Mass cytometry was performed on peripheral blood samples at baseline and at the three-month timepoint. In addition, matched bone marrow samples were collected and analyzed from two PsA patients, of whom one with inactive PsA and hypergammaglobulinemia, and one with active PsA and monoclonal gammopathy of undetermined significance (MGUS).

Confocal microscopy was employed in synovial tissues derived from three additional patients with RA undergoing knee joint arthroplasty. Three patients with osteoarthritis (OA) served as controls.

The study complied with the Ethical Principles for Medical Research Involving Human Subjects according to the World Medical Association Declaration of Helsinki and the Oviedo Convention. Approval was obtained from the local Ethics and Scientific Committees of the University Hospitals of the National and Kapodistrian University of Athens (No. 314/2021). Written informed consent was obtained from all participating individuals.

Mass cytometry

Whole blood and bone marrow staining

Peripheral blood was obtained by venipuncture and stored in ethylenediaminetetraacetic acid (EDTA) tubes for subsequent analysis by high-dimensional mass cytometry. To prevent potential contamination from skin-derived fibroblasts in our analysis, the initial 5 ml of blood collected were discarded. No other intervention was performed, and clinical data were collected from the patients’ files, as recorded during their regular visits to the Rheumatology Unit.

One ml of whole blood was incubated with heparin (at a final concentration of 100 U/ml) for 20 min at room temperature (RT). Next, 270 µl of heparin-blocked blood were incubated into the tube containing the dry antibody pellet of the Standard BioTools Maxpar Direct Immune Profiling Assay (MDIPA, Catalogue number: 201334) supplemented by the addition of antibodies against CDH11, PDPN, CD90 (or Thy-1), CD34 and Notch3, henceforth referred to as mesenchymal markers (Supplementary Table 2) for 30 min at RT. Then, 250 µl of lysing solution (1:10 dilution of BD FACS Lysing Solution, BD Biosciences, San Jose, CA, USA, in Maxpar Water) were added for red blood cell lysis, followed by incubation for 10 min at RT in the dark. Following the addition and incubation of 3 ml of Maxpar Water for 10 min at RT in the dark, the tube was centrifuged at 300 x g for 5 min and the supernatant was carefully aspirated. Cells were washed 3 times using 3 ml of Maxpar Cell Staining Buffer (CSB) and centrifuged at 300 x g for 5 min.

Bone marrow samples were resuspended and incubated in 10x volume of lysing solution for 10 min at RT. Following centrifugation at 350 x g for 5 min at 4°C, the supernatant was carefully aspirated, and then lysis was repeated. The pellet was then resuspended in 1 ml of cold Maxpar Phosphate Buffer Solution (PBS) and filtered through a 70 µm cell strainer. Cells were washed using 4 ml cold PBS and centrifuged at 350 x g for 5 min at 4°C. The pellet was then resuspended in 1 ml of CSB and cells were counted. A maximum of 5 million cells were aliquoted and centrifuged at 350 x g for 5 min at 4°C. After carefully aspirating the supernatant, the pellet was resuspended in 50 µl CSB. FcR-blocking was achieved by adding 5 µl of Human TruStain FcX (Biolegend, San Diego, CA, USA). After incubating for 10 min at RT, 215 µl of CSB were added, for a final volume of 270 µl. Then, the 270 µl were transferred into the tube containing the dry antibody pellet of MDIPA, supplemented by the addition of antibodies against CDH11, PDPN, CD90, CD34, and Notch3 for 30 min at RT. Cells were, then, washed twice by adding 3 ml CSB and were centrifuged at 300 x g for 5 min.

Next, cells from both whole blood and bone marrow samples were fixed and permeabilized by adding 1 ml of 1.6% freshly prepared formaldehyde solution in Maxpar PBS. After incubating for 10 min at RT, the tube was centrifuged at 800 x g for 5 min and the supernatant was carefully aspirated. Finally, DNA staining was performed by incubating the cell pellet with 1 ml of 125 nM Cell-ID Intercalator-Ir solution in Maxpar Fix and Perm Buffer and incubating at 4°C overnight. All antibodies and buffers were purchased from Standard BioTools Inc., San Francisco, CA, USA, unless otherwise stated.

Data acquisition and processing

Sample acquisition was conducted using a 3rd generation Helios mass cytometer. Prior to acquisition, the instrument was first tuned with Tuning Solution and a bead sensitivity test was performed using EQ Four Element Calibration Beads. Samples were washed twice with 1 ml of Maxpar CSB and twice with 1 ml of Cell Acquisition Solution (CAS). Subsequently, cells were resuspended in CAS containing 0.1X EQ Four Element Calibration Beads, to a maximum cell concentration of 1 x 10⁶ cells/ml. Finally, cells were filtered through a 35-mm nylon mesh cell strainer (BD Biosciences, San Jose, CA, USA). Using the CyTOF Software version 7.0.8493, an average of 500,000 events were acquired per file, at a flow rate of 300–500 cells/sec. The generated FCS files were normalized using the CyTOF software and then Cytobank (Beckman Coulter Life Sciences, Indianapolis, IN, USA) was used for further processing and analysis.

The gating strategy employed for the initial pre-processing stages is shown in Supplementary Fig. 1. The phenotype of circulating fibrocytes and fibroblasts was based on previous studies^16,22,25,42. Specifically, fibroblasts were defined as PDPN⁺CD45⁻ cells strictly gated in lineage-negative lymphoid and granulocytic cells (CD3⁻CD19⁻CD4⁻CD8⁻CD56⁻CD66b⁻CD294⁻). Based on all the possible combinations of the mesenchymal markers CDH11, CD34, CD90 and Notch3, a total of 16 fibroblast phenotypes were formed (Supplementary Table 3). Similarly, fibrocytes were defined as CD34⁺HLA-DR⁺CD45⁺ cells strictly gated in lineage-negative lymphoid and granulocytic cells (CD3⁻CD19⁻CD4⁻CD8⁻CD56⁻CD66b⁻CD294⁻). Again, based on all the possible combinations of CDH11, PDPN, CD90 and Notch3, a total of 16 fibrocyte phenotypes were formed (Supplementary Table 4). The presence of the chemokine receptors C-C chemokine receptor (CCR) 6, CCR7, C-X-C chemokine receptor (CXCR) 3 and CXCR5 was investigated in both fibroblasts and fibrocytes. In addition, 12 leukocyte subpopulations were gated and investigated for the co-expression of CD90 and CDH11. The phenotypes of the identified leukocyte subpopulations are listed in Supplementary Table 5. The detailed gating strategy for all cell populations is shown in Supplementary Fig. 2–18.

To visualize the high-dimensional data in two dimensions, the dimensionality reduction algorithm tSNE-CUDA (GPU-accelerated t-distributed stochastic neighbor embedding) was used through Cytobank, based on the expression of the 30 surface markers of the MDIPA panel. For this analysis, 15,000 randomly selected cells from each sample were analyzed, totaling 810,000 cells.

Immunofluorescence staining and imaging analysis of synovial tissue

Synovial tissue was processed and cryopreserved as follows: each synovial tissue specimen was dissected into 1 to 5 mm fragments, then submerged in Cryostor CS10 (Sigma-Aldrich, St. Louis, MO, USA), then stored at − 80°C prior to transferring to long-term storage in liquid-nitrogen. Imaging of the RA tissue was carried out in the BSRC Al. Fleming Imaging facility on FFPE synovial tissue sections.

RA synovial tissues were first fixed in 4% PFA for at least 12 h followed by xylene and ethanol processing and embedded in paraffin. 5um sections were cut using the manual microtome LeicaRM2125RTS onto histology slides. Prior to staining, sections were rehydrated, and antigen retrieved in 10 mM Sodium Citrate buffer (pH 6.0) for 20 min at 96°C and further 20 min at RT. Slides were rehydrated with PBS before blocking with 10% normal donkey serum. Primary antibodies were then applied in PBS-10% serum, THY1/CD90 (sheep polyclonal, AF2067, Bio-Techne), CD45 (clone D3F8Q, Cell Signaling), PDPN (NZ-1.3, eBioscience, PE), CDH11 (MA1-06306, Thermo Fisher Scientific) and Ly6G (ab122501, Abcam). Appropriate isotype controls were used on a separate section. Secondary antibodies included Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Invitrogen- A-21206), Donkey anti-Sheep IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (Invitrogen, A-21448), Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (Invitrogen, A-31571), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (Invitrogen, A-11032). Primary antibodies were titrated and used at a concentration that ranged from 1:100 to 1:1000, depending on the antibody, whereas secondary antibodies were all used at 1:400 dilution. Slides were mounted in prolong diamond (Thermo Fisher Scientific, Waltham, MA, USA) and stored at 4°C before imaging.

Images were obtained using a Leica TCS SP8X White Light Laser confocal system in linear deconvolution or sequential mode and were analyzed using ImageJ (FIJI, publicly available NCBI software).

Statistical analysis

Given the exploratory nature of the study, proposing a statistical hypothesis to determine the appropriate sample size was not possible. Statistical analysis was performed using GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, CA, USA). Normal distribution was assessed using the D'Agostino-Pearson normality test. Fisher’s exact test was employed for comparing categorical variables. Comparisons of numerical values between the two groups were performed using an unpaired t-test or Mann-Whitney U test, as appropriate. Comparisons of numerical values between multiple groups were conducted using One-Way ANOVA or a Kruskal–Wallis test, followed by the two-stage step-up method of Benjamini, Krieger and Yekutieli for post-hoc analysis. Paired comparisons were performed using the Wilcoxon test. Correlation coefficients between variables were calculated according to Spearman's rank correlation coefficient and were performed using IBM SPSS Statistics 28.0.0.0 (IBM, Armonk, NY, USA). The level of significance was established at a p-value < 0.05 for two-sided tests.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Author information

These authors contributed equally: Maria Kyriakidi, Eleni-Kyriaki Vetsika

Competing interests

The authors declare no competing interests.

Author contributions

E.-K.V. and P.P.S. conceived the project and designed the experiments. Μ.Κ. and E.-K.V. performed mass cytometry experiments. M.S performed immunofluorescence staining. E.-K.V., M.K, M.S., A.M. M.G.T. and G.E.F. participated in data and samples collection. M.K., E.-K.V., K.-M.V, M.S. and P.P.S. analysed and interpreted the data. M.K, E.-K.V., M.S. and PPS wrote the manuscript. M.K, E.-K.V. N.I.V. and PPS reviewed and edited the manuscript. P.P.S. supervised the project. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

The authors express their gratitude to the patients and healthy volunteers who participated in the study. We would like to thank Prof.George Kollias and Prof. Christopher Buckley for their valuable advice, helpful suggestions and discussions. This work was supported by the Hellenic Rheumatology Society and Professional Association of Rheumatologists (Protocol Numbers: 2021/1081 and 2021/1084), and 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).

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Distinct CDH11⁺ circulating fibroblasts and immune cells co-expressing chemokine receptors in chronic inflammatory arthritis patients

Status:

Version 1

Abstract

Figures

Introduction

Results

Circulating fibroblasts expressing CDH11, CD90, HLA-DR and CCR7 characterize RA and PsA

Circulating CDH11⁺ and CCR7⁺ fibrocytes are increased in chronic inflammatory arthritis

Neutrophils co-expressing CDH11, CD90 and CCR6 are increased in chronic inflammatory arthritis

Synovium-infiltrating neutrophils may interact with synovial fibroblasts via CDH11

Discussion

Methods

Patient enrollment

Mass cytometry

Immunofluorescence staining and imaging analysis of synovial tissue

Statistical analysis

Declarations

Data availability

Author information

Competing interests

Author contributions

Acknowledgements

References

Additional Declarations

Supplementary Files

Status:

Version 1

Distinct CDH11+ circulating fibroblasts and immune cells co-expressing chemokine receptors in chronic inflammatory arthritis patients

Status:

Version 1

Abstract

Figures

Introduction

Results

Circulating fibroblasts expressing CDH11, CD90, HLA-DR and CCR7 characterize RA and PsA

Circulating CDH11+ and CCR7+ fibrocytes are increased in chronic inflammatory arthritis

Neutrophils co-expressing CDH11, CD90 and CCR6 are increased in chronic inflammatory arthritis

Synovium-infiltrating neutrophils may interact with synovial fibroblasts via CDH11

Discussion

Methods

Patient enrollment

Mass cytometry

Immunofluorescence staining and imaging analysis of synovial tissue

Statistical analysis

Declarations

Data availability

Author information

Competing interests

Author contributions

Acknowledgements

References

Additional Declarations

Supplementary Files

Status:

Version 1

Distinct CDH11⁺ circulating fibroblasts and immune cells co-expressing chemokine receptors in chronic inflammatory arthritis patients

Circulating CDH11⁺ and CCR7⁺ fibrocytes are increased in chronic inflammatory arthritis