CD142+ synovial fibroblast drives meniscus destruction in rheumatoid arthritis

doi:10.21203/rs.3.rs-4823074/v1

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CD142+ synovial fibroblast drives meniscus destruction in rheumatoid arthritis

https://doi.org/10.21203/rs.3.rs-4823074/v1

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Rheumatoid arthritis (RA) induced destruction of knee joints is a common cause of total knee arthroplasty (TKA). Although previous evidence suggests that bone and cartilage damage is the main pathogenesis of RA joint destruction, the meniscus, a special structure in the knee joint, has been ignored. Here, we identified CD142 + synovial fibroblasts as a novel SF sub-cluster located in the sublining layer in normal and osteoarthritis knee synovium, which is elevated and migrates to the lining layer (LL) in RA knee synovium. Intra-articular injection of CD142 + SF can quickly and drastically damage the meniscus but has a slight effect on cartilage. RNA sequencing revealed that ABCC4 was highly expressed in CD142 + SF, and the pharmacological blockade of ABCC4 by MK571 attenuated CD142 + SF-induced meniscal degradation. Long-term follow-up of the RA cohort indicated that enriched CD142 + SF in the LL was a risk factor for severe joint destruction and eventually underwent TKA. Our results demonstrate that CD142 + SF can be used as an indicator to assess prognosis and a therapeutic target to inhibit meniscal damage, thereby alleviating RA knee joint destruction.

Biological sciences/Immunology/Immunological disorders

Health sciences/Diseases/Immunological disorders

synovial fibroblast

CD142

meniscus

rheumatoid arthritis

The global prevalence of rheumatoid arthritis (RA) is approximately 0.5–1%, affecting approximately 18 million people¹. In China, the morbidity rate is 0.42%, with over 5 million diagnosed cases². RA primarily involves small joints, such as the metacarpophalangeal and interphalangeal joints; however, some patients present with large joint involvement, such as the knee joint, where severe cases require total knee arthroplasty (TKA). The proportion of TKA for RA is 3–9% in the United States^3,4 and approximately 6% in China⁵. The autoimmune response is the representative pathophysiology of RA⁶. Immune cells stimulate synovial fibroblasts (SF), which then undergo aggressive inflammatory and invasive phenotypes, damaging the cartilage and bone, and leading to joint dysfunction⁷. Recent single-cell RNA sequencing (scRNA-seq) studies revealed that FAPα + THY1 + SF located in the synovial sublining layer (SL) resulted in severe and persistent inflammatory arthritis, whereas the FAPα + THY1- SF located in the synovial lining layer selectively mediate bone and cartilage damage⁸. NOTCH3 signaling drives THY1 + sublining SF differentiation and exaggerates inflammation in RA⁹ cells.

Unlike other joints composed of bone, cartilage, and ligaments, the knee joint has a special structure, the meniscus¹⁰. The meniscus plays a vital role in knee-joint stability, shock absorption, and load distribution¹¹. In osteoarthritis (OA), meniscus degeneration is an important pathogenesis for OA development^12,13, and destabilization of the medial meniscus (DMM) is the most widely used model for traumatic OA in mice¹⁴. In RA, both direct observation during TKA surgery¹⁵ and magnetic resonance imaging (MRI) evaluation ¹⁶ show severe meniscal damage. However, the role of the meniscus in knee destruction in patients with RA remains unclear.

To determine the specific SF contributing to meniscal degradation in RA, we analysed the cellular interactions of the synovium, cartilage, and meniscus using scRNA-seq. We demonstrated that CD142 + SF was elevated in both human and mouse RA, and their intra-articular injection invaded and severely impaired the meniscus with only a minimal effect on cartilage destruction. Inhibiting of CD142 + SF invasion by MK571 attenuates meniscus damage. These results may offer insights into reducing RA knee damage by alleviating CD142 + SF-induced meniscal destruction.

Severe meniscus degradation in knee RA

We assessed meniscal degradation in human RA using radiology and histology. We compared the radiographs and MRI scans of knee joints of patients with OA (Kellgren–Lawrence grade 3–4) who required arthroplasty and those of patients with knee RA who also required arthroplasty. The radiograph at the weight-bearing position showed a significant reduction in both medial and lateral knee joint space in patients with RA compared with those having OA (Extended Data Fig. 1a–c). In addition to extensive synovial hyperplasia and cartilage destruction, the entire meniscus (lateral meniscus anterior horn, lateral meniscus body, lateral meniscus posterior horn, medial meniscus anterior horn, medial meniscus body, and medial meniscus posterior horn) was also drastically damaged compared with that in patients with OA (Extended Data Fig. 1a, d, e).

Meniscal degeneration in OA is accompanied by collagenous fibre disruption; Safranin O-positive areas appear in Safranin-O-Fast Green staining¹⁷. Compared with the OA meniscus, the RA meniscus showed a remarkable volume reduction and severe fibre structure destruction, and a large number of invaded areas emerged (Fig. 1a–c). Synovial hyperplasia is a common feature of RA, and various cell types, such as fibroblasts and immune cells in the synovium, play an important role in the destruction of the bone and cartilage of the knee joint^7,18. Therefore, we assessed the relationship between histopathological synovitis score (Krenn Score) and meniscal degradation. The Krenn score evaluation included the width of the synovial lining layer, cell density of the stroma, and density of inflammatory infiltration¹⁹. The Krenn score of the RA synovium was significantly higher than that of the OA synovium, with similar results obtained when the lining layer (LL) scores were compared independently (Fig. 1d–f). Spearman’s correlation analysis indicated that the LL score was positively associated with meniscal degradation and invasion area (Fig. 1g, h). Collectively, these data demonstrate that severe meniscal destruction in RA is associated with synovitis and synovial invasion.

CD142 + SFs elevate in RA

To investigate the mechanism of joint destruction by SF in RA, we performed scRNA-seq analysis of chondrocytes, meniscal cells, and synovial cells from patients with RA requiring TKA. Eight major cell types have been identified: meniscal cells, chondrocytes, SF, macrophages, smooth muscle cells, T cells, endothelial cells, and mast cells. SF were more closely distributed with meniscal cells than with chondrocytes in the uniform manifold approximation and projection (UMAP) (Fig. 2a, b, Extended Data Fig. 2a, b). Next, we re-analysed SF separately and identified three distinct cell subpopulations: the PRG4 + cluster (expressing PRG4 and HBEGF) from the LL, the DKK3 + cluster (expressing DKK3 and CHI3L2) from the SL ⁸, and the CD142 + cluster (expressing CD142 and CXCL12), a novel cell cluster neighbouring meniscal cells in UMAP (Fig. 2c, d). CellChat analysis showed that the interactions between CD142 + SF and meniscal cells were the most abundant, indicating a stronger relationship (Fig. 2e, Extended Data Fig. 2c). Collectively, these data suggested that CD142 + SF have the potential to invade the meniscus.

We examined the number of CD142 + SF in healthy OA and RA knee joints. Healthy synovium was obtained from knees that underwent thigh amputation or hemipelvectomy, whereas OA and RA synovium were acquired from knee arthroplasty cases. Cell flow cytometry showed that the number of CD142 + SF in RA synovium was significantly elevated compared to that in healthy and OA synovium (Fig. 2f, g). We also collected synovial fluid from patients with OA and RA during arthroplasty and obtained the deposits by centrifugation. The deposits were cut into histological sections, and immunohistochemical (IHC) staining revealed that the amount of CD142 + SF in RA synovial fluid deposits was also significantly higher than that in OA; however, the THY1 + and PRG4 + cells were not significantly changed (Fig. 2h, i, Extended Data Fig. 2d–f).

We evaluated the number of CD142 + SF in the mouse model. The surgical model of DMM in the C57BL/6 strain was used to study OA, whereas the model of collagen-induced arthritis (CIA) in the DBA/1 strain was used to induce autoimmune arthritis to study RA. CD142 + SF was slightly increased in OA mice and drastically increased in RA mice (Fig. 2j–m). THY1 + and PRG4 + cells were slightly elevated in the RA model, but no significant changes were observed in the OA model (Extended Data Fig. 2g–l).

CD142 + SFs migrate from SL to LL in RA

Recently, scRNA-seq revealed that fibroblasts in the LL have a strong effect on cell adhesion and invasion, whereas fibroblasts in the SL exhibit severe proinflammatory function⁸. Therefore, we examined the spatial distribution of CD142 + SF in the synovium. We used multiplex immunohistochemistry (mIHC) staining to label different cell subtypes: PRG4 to label LL, THY1 to label SL, and CD146 to label blood vessels. Consistent with the cell flow cytometry results, CD142 + SF levels were markedly increased in patients with RA. Notably, the spatial distribution was also altered. In healthy and OA synovium, CD142 + fibroblasts are mainly located in the SL, whereas they are massively distributed in the LL in RA. Additionally, we observed that CD142 + SF was closer to the superficial layer than PRG4 + fibroblasts (Fig. 3a–c, Extended Data Fig. 3a, b).

We calculated the distance from each CD142 + SF to the nearest CD146 + cell. No significant difference was observed between the healthy and OA synovium, whereas the distance was significantly elongated in RA, suggesting that CD142 + fibroblasts migrated from SL to LL during RA progression (Fig. 3d). To distinguish whether CD142 + fibroblasts are a new cell cluster or a population overlapping with previously identified subpopulations, we counted the number of CD142 single- or double-positive cells with PRG4 or THY1. In the healthy synovium, most CD142 + cells expressed THY1(52.2%) or PRG4 (28.9%), whereas in RA synovium, the majority of CD142 + cells (76.8%) did not overlap with THY1 or PRG4 (Fig. 3e, Extended Data Fig. 3c). Together, these results identified CD142 + fibroblasts as a novel cell cluster significantly elevated in the LL of RA synovium, having migrated from the SL to the LL.

CD142 + SFs invade meniscus

To examine the role of CD142 + SF in meniscal destruction, we performed histological staining of the synovial invasion area in human RA and CIA mouse menisci. IHC staining of the invaded area showed strong CD142 expression in both human RA menisci and CIA mouse menisci, whereas only a small number of cells expressed PRG4 and THY1(Fig. 4a–c). We established a knee joint injection mouse model for SF invasion. CD142 + and CD142- SFs were sorted by flow cytometry and then cultured in a plate. The cells were labelled with fluorescence before each injection. The knee joint of DBA/1 mice was injected CD142 + or CD142- SFs at 8, 9, 10, and 11 weeks of age, and these mice were sacrificed at 12 or 16 weeks of age for histological assessment (Fig. 4d).

Frozen sections from 16-week-old mice showed significant invasion of CD142 + SF, particularly in the contact area of the meniscus and synovium, whereas the invasion of CD142- SF was attenuated. Cartilage invasion was weaker than that of the meniscus, with only one or two layers of cells invading the cartilage surface (Fig. 4e). Paraffin sections from both groups showed severe synovitis; however, meniscal synovial invasion and destruction were more severe in the CD142 + group. The meniscus volume was smaller, and the meniscal white zone (without blood supply) was completely disrupted and replaced by synovial tissue (Fig. 4f–i). Notably, cartilage damage was mild in both groups. At 12 weeks, both groups showed minimal cartilage damage with no significant difference in OARSI scores, while OARSI scores in the CD142 + group were higher than those in the CD142- group at 16 weeks (Fig. 4j). This suggests secondary cartilage damage resulting from meniscus degradation rather than direct synovial inflammation or cartilage invasion.

ABCC4 blockade attenuated CD142 + SFs invasion

We examined the invasive and migratory abilities of CD142 + SFs. Compared with CD142- SF, CD142 + SF exhibited higher invasion and migration abilities (Extended Data Fig. 4a, b). The mRNA expression of cell migration- and invasion-related genes (SNAI1, SNAI2, and TWIST1) and matrix metalloproteinase genes (MMP3, MMP9, and MMP13) were also significantly upregulated, whereas the migration suppressor gene CDH1 (encoding E-cadherin)²⁰ was significantly downregulated (Extended Data Fig. 4c, d). Co-culture of human OA meniscal cells with CD142 + or CD142- SF showed that COL1A1 mRNA expression in human meniscal cells co-cultured with CD142 + SF was significantly decreased (Fig. 4k).

We then identified potential targets of CD142 + SF invasion using RNA-seq to compare gene expression between CD142 + and CD142- SF. Gene Ontology (GO) analysis revealed enrichment of cell adhesion-related terms in CD142 + SF. ABCC4, also known as multidrug resistance protein 4, a member of the C subfamily of ABC transporters, was upregulated (3.19-fold increased, P < 0.01) in CD142 + SF and was associated with cell migration and adhesion (Fig. 5a–c)²¹. IHC staining confirmed increased ABCC4 expression in the synovial LL and synovial fluid deposits of patients with RA (Fig. 5d–g).

We further assessed whether pharmacological blockade of ABCC4 using MK571, a selective ABCC4 blocker²², could inhibit CD142 + SF invasion (Fig. 5h, i). CD142 + SF cells treated with MK571 resulted in reduced migration and invasion, accompanied by decreased mRNA expression of SNAI1, SNAI2, TWIST1, MMP3, MMP9, and MMP13, and increased CDH1 expression (Fig. 5j, k). Additionally, MK571 alleviated the effect of COL1A1 mRNA inhibition in human OA meniscal cells co-cultured with CD142 + SF (Fig. 5l).

MK571 attenuates CD142 + SFs invasion induced meniscus degradation

We investigated whether MK571 could inhibit CD142 + SF invasion and attenuate meniscal degradation in vivo. The knee joints of DBA/1 mice were injected with CD142 + SF separately or in combination with MK571 at 8, 9, 10, and 11 weeks of age, and sacrificed at 12 or 16 weeks for histological assessment, as previously described (Fig. 6a). Frozen sections from 16-week mice showed that MK571 treatment protected the meniscus from CD142 + SF invasion (Fig. 6b). Safranin-O-Fast Green staining of paraffin sections revealed that MK571 treatment significantly reduced the meniscal invasion area, lowered the Krenn score at both 12 and 16 weeks, and decreased the OARSI score at 16 weeks (Fig. 6c–g). Collectively, these results demonstrate that MK571 attenuates meniscal damage by inhibiting CD142 + SF invasion, thereby protecting against secondary damage to the articular cartilage.

CD142 + SFs migrating to LL is the risk factor for knee dysfunction in RA

To investigate the role of CD142 + SF in the progression of knee dysfunction in RA, we followed up with patients with RA having long-term knee symptoms. Seventeen patients with RA having knee symptoms were included in this study, and all patients underwent synovial biopsy at an early stage of knee pain or swelling. The follow-up duration was from 2013 to 2024, and the patients were categorised into two groups based on whether they eventually underwent TKA. We defined synovial biopsy as the starting point for this study, with the time of TKA surgery as the endpoint of follow-up for the TKA group (seven knees, mean follow-up time 82.4 months, 64–108 months), and May 2024 as the endpoint for the non-TKA group (10 knees, mean follow-up time 92.3 months, 60–120 months). Detailed demographic data are presented in Supplementary Data Table 1.

The New Knee Society Score System (KSS Score)²³ was used to evaluate knee function. The TKA group showed significantly lower KSS scores than the non-TKA group, including worse knee alignment, stability, and range of motion (Fig. 7a, b). We analysed the histological sections from synovial biopsies using mIHC staining. Although the number of CD142 + SF cells in the SL and the distance of each CD142 + SF from the nearest CD146 + cells did not differ, the CD142 + SF in the LL was significantly higher in the TKA group than in the non-TKA group (Fig. 7c–f), suggesting that increased CD142 + SF in the synovial lining layer is a risk factor for severe knee deformity in patients with RA who require TKA.

Recently, many studies have investigated the phenotype of SF in RA and clarified their spatial distribution and corresponding functions^24,25. Synovial B cells, T cells, and macrophages promote SF proliferation, invasion, inflammatory mediator production, and matrix metalloproteinase secretion, eventually leading to joint destruction^6,26. However, most studies have focused on bone and cartilage damage and ignored a specific anatomical part of the knee joint, the meniscus. In our study, we identified CD142 + SF as a novel SF sub-cluster, which is located in the SL in normal and OA synovium but migrated to the LL in the RA synovium. Intra-articular injection of CD142 + SF can quickly and drastically lead to meniscus damage, but has a slight effect on cartilage. ABCC4, which is highly expressed in CD142 + SF, could be targeted to block CD142 + SF-induced meniscal degradation. Using the synovia from synovial biopsy in our long-term follow-up RA cohort, we found that CD142 + SF were highly expressed in the LL of patients who eventually underwent TKA due to severe joint destruction.

Croft et al. reported two distinct SF subsets, FAPα + THY1 + SF located in the sublining for persistent inflammation, and FAPα + THY1 − SF located in the LL for bone and cartilage damage⁸. However, our CD142 + SF did not belong to the preceding two cell populations but are classified as FAPα-THY1 + SF and located variably. These differentiated results may be due to the different synovial sources, as our synovium was obtained from TKA, whereas other studies used synovial biopsies of different joints. Zhang et al. recently used scRNA-seq and surface protein histology of synovial tissues from 79 donors to classify them into six groups¹⁸. According to Zhang's criteria, the synovium used for scRNA-seq in our study was fibroblast-abundant, the type with the poorest response to different treatments. Furthermore, in a separate fibroblast cluster analysis conducted by Zhang et al., CD142 + SF coincided with CXCL12 + SFRP1 + SF. These data indicate that CD142 + SF are mainly present in RA synovial tissue with poor prognosis, which is also consistent with the synovial biopsy results from our long-term follow-up RA cohort.

Previous studies have evaluated meniscal damage in RA using morphology¹⁵, histology²⁷, and radiology¹⁶, which showed that both the cartilage and meniscus were severely damaged; however, it is unclear which was damaged first. This may be because we could not obtain the data at the early stage of knee RA, and radiological evaluation requires expensive MRI, whereas histological evaluation of the cartilage and meniscus was derived from arthroplasty for end-stage knee RA. Our study found that CD142 + SF intra-articular injection first invaded and damaged the meniscus, whereas the effect on the cartilage was slight, suggesting that knee joint dysfunction in RA might originate from meniscal destruction, and cartilage erosion might partly result from the weakened protective effect after meniscal damage. In our clinical study, we noted that patients with RA having mild knee damage support this hypothesis. For example, during knee arthroplasty in a 62-year-old woman with RA, we found that the lateral meniscus was completely defective, and only a small part of the medial meniscus remained. However, the articular cartilage was mainly worn in the weight-bearing area, and the cartilage is still present in the non-weight-bearing areas (Extended Data Fig. 5). The meniscus body is connected to the synovium, and the anatomical distance is shorter than the distance between the cartilage and synovium, which may explain why synovial tissue is more likely to invade the meniscus.

In conclusion, our study identified a new RA fibroblast subpopulation that proliferates and migrates from the sublining to the LL in RA. The invasion of CD142 + SF-induced meniscal degradation may be the major cause of knee damage in RA. High CD142 + SF expression in the LL of patients with early RA is a risk factor for severe knee destruction, ultimately requiring TKA (Fig. 7g). CD142 + SF could be used as an indicator for assessing prognosis and as a therapeutic target for the destruction of the RA knee joint.

Human subjects research

All patients included in this study signed a written informed consent form for the use of abandoned specimens (knee meniscus, cartilage, synovium, and synovial fluid) and publication of any potentially identifiable images or data, as approved by the Ethics Committee of XXX Hospital (2009-06, 2020 − 126). We included patients with RA who met the 1987 American Rheumatism Association criteria²⁸ and the 2010 ACR/EULAR criteria²⁹.

For patients with RA who underwent systemic treatment without remission of severe knee pain or knee deformity (knee varus or knee valgus), we performed knee arthroplasty and obtained meniscus, cartilage, synovium, and synovial fluid samples. For patients with OA who met Kellgren-Lawrence Ⅲ or Ⅳ grading³⁰ and had severe knee pain which could not be relieved by NASIDs, we performed knee arthroplasty and obtained knee tissue samples. For the control knee synovium samples, we obtained healthy knees by thigh amputation or hemipelvectomy. Radiological data from patients with OA and RA were used for radiological analysis, and samples from normal, OA, and RA knees were used for histological analysis, single-cell RNA sequencing, cell cytometry, cell culture, in vitro and in vivo experiments, and transcriptomic RNA sequencing.

The cohort was established in 2013 for long-term follow-up in RA cohort studies. All patients with RA underwent knee Parker-Pearson fine needle biopsy at the onset of knee symptoms (knee pain and swelling) to obtain knee synovium tissue samples. After the biopsy, all patients underwent systematic antirheumatoid treatment. By the end of May 2024, 17 patients completed the follow-up period. We categorised the patients into non-TKA and TKA groups based on whether they underwent TKA. For the TKA group, the follow-up endpoint was set at the time of surgery; for the non-TKA group, the follow-up endpoint was set at May 2024. Knee joint function was assessed using the KSS score²³ for both groups.

Radiological analysis of human knee data

An X-ray in the anteroposterior view in the standing posture was used to measure the medial joint space and the lateral joint space by selecting the minimum segment between the medial and lateral compartments ³¹. All measurements were based on a real-world plotting scale. MRI with a T2-weighted sequence was used for meniscal measurement. In the coronal sequence, the image plan in which the medial tibial spine volume was maximal was selected for measuring the thickness and width of the meniscal body segment, whereas in the sagittal sequence, the image plan on the midpoint of the medial/lateral compartment was selected for measuring the thickness and width of the medial/lateral meniscal anterior and posterior horn³².

Histological morphology analysis and scoring of human synovium and meniscus

For histological sections, fresh meniscus, cartilage, and synovial tissues obtained from knee arthroplasty surgery (for RA and OA subjects) were fixed in 4% paraformaldehyde and embedded in paraffin. Human knee synovial fluid was centrifuged at 2000 rpm for 5 min, and the deposit was fixed in 4% paraformaldehyde and embedded in paraffin. All paraffin-embedded specimens were dissected into 5 µm sections and stored at room temperature. Sections of the human synovium and meniscus were stained with haematoxylin and eosin (HE), and Safranin-O-Fast Green. The lining (cell) layer of the synovium was identified on HE staining as the membrane facing the joint cavity, characterised by ellipsoidal cells arranged in layers³³. Synovial sections were scored using the total Krenn synovitis score (ranging from 0–9 points) and the enlargement of the synovial lining cell layer component of the Krenn synovitis score (ranging from 0–3 points)¹⁹.

Meniscal sections were scored using the Pauli degeneration score¹⁷ (ranging from 0–18 points). Synovium invasion area on the meniscus was defined as the area containing limited clustered or widely infiltrated cells, usually accompanied by new angiogenesis. The area was calculated as the ratio of the invasion area to the total meniscal area using ImageJ software (version 1.54).

Immunohistochemistry and mIHC

For immunohistochemistry (IHC), antigen retrieval was performed at pH 9.0 using Tris-EDTA for 20 min by microwave heating. Sections were incubated with primary antibodies of CD142 (Bioss, 1:200), PRG4 (Abcam, 1:200), THY1 (Proteintech, 1:200), and ABCC4 (Proteintech, 1:200) at 4℃ overnight, followed by incubation with anti-rabbit horseradish peroxidase (HRP) secondary antibody (Servicebio, 1:200) at room temperature for 50 min. HRP staining was performed using DAB Peroxidase HRP Substrate (Servicebio).

For mIHC, antigen retrieval was performed at pH 9.0 using Tris-EDTA for 20 min by microwave heating. The main steps of mIHC are as follows: sections were incubated with primary antibody at 4℃ overnight, and then with anti-rabbit HRP secondary antibody at room temperature for 50 min, followed by tyramide signal amplification (TSA) staining at room temperature for 10 min. Antigen retrieval was performed again for the next primary antibody incubation. Primary antibodies against PRG4 (Abcam, 1:1000), CD146 (Proteintech, 1:1000), CD142 (Cell Signaling Technology, 1:600), and THY1 (Abcam, 1:600) were incubated. IF440-TSA (anti-CD146, Servicebio, 1:500), iF488-TSA (anti-CD142, Servicebio, 1:500), iF555-TSA (anti-PRG4, Servicebio, 1:500) and iF647-TSA (anti-THY1, Servicebio, 1:500) were used for TSA staining. DAPI staining (Servicebio) was performed last to label nuclei.

Quantitative and spatial analysis of mIHC

Human synovial sections stained with mIHC were scanned using Akoya whole-slide multispectral imaging in fluorophore Spectral DAPI (excitation: 368; emission: 461), Opal Polaris 480 (excitation: 450; emission: 500), Opal 520 (excitation: 494; emission: 525), Opal 570 (excitation: 550; emission: 570), and Opal 690 (excitation: 6676; emission: 694). Three fields (923 µm × 692 µm) in each section were chosen for analysis, and their data were summed as one sample. Akoya InForm (version 1.6) loaded the chosen fields to output phenoptic data, followed by tissue segmentation, cell segmentation, and phenotyping. For tissue segmentation, the lining and SLs were segmented using haematoxylin and corresponding HE staining. For cell segmentation, the minimum nuclear size was set at 10, the cytoplasm thickness was set at 5, and the membrane search distance was set at 10. For cell phenotyping, 20 cells for each fluorophore were manually chosen for classifier training, and the classifier was used for field phenotyping to label each segmented cell. The R package PhenoptrReports (version 0.3.3) processed the phenoptics data and calculated all phenotyping cells in the LL and SL of each section and the nearest distance between each pair of phenotyping cells. The results from three fields were summed and averaged for each sample.

Human meniscus, cartilage, and synovium tissue processing for cell suspensions and cell culturing

Fresh meniscus, cartilage, and synovial tissues were obtained from knee arthroplasty (for RA and OA subjects) and thigh amputation or hemipelvectomy (for control subjects). For flow cytometry, cell culturing and RNA sequencing, fresh synovium tissues were dissected in 2 mm pieces and then digested in collagenaseⅠsolution (2 mg/mL, Gibco; +10% FBS, Gibco; +1% penicillin-streptomycin, Gibco) for 2 h in 37℃, after which solution was filtered by 70 nm cell strainer (BIOFIL) and centrifuged at 1000 rpm for 5 minutes to obtain cells and made into single cell suspensions. Meniscal and cartilage tissues were dissected in 2 mm pieces and then digested in collagenase P solution (2 mg/mL for meniscus and 0.25 mg/mL for cartilage, Roche; +10% FBS, Gibco; +1% penicillin-streptomycin, Gibco) for 6–8 h in 37℃ for cell suspensions.

For human primary meniscal cells and SF, we cultured them in Dulbecco’s modified Eagle’s medium/Hams F12 (DMEM/F-12, Gibco) with 10% FBS and 1% penicillin-streptomycin in an incubator at 37℃ and 5% CO₂.

Flow cytometry and synovial fibroblast collection

For the flow cytometric analysis of human synovial cells, red blood cell lysis buffer (Solarbio) was added to the cell suspensions for 10 min at room temperature, and the suspensions were centrifuged, washed, and resuspended. After removing red blood cells, synovial cells were stained with antibodies against CD31 (BV421, BioLegend, CA, USA), CD45 (APC, BioLegend), CD142 (PE, BioLegend), and 1% BSA in PBS for 20 min at room temperature. 7-AminoactinomycinD (7-AAD, BioLegend) was added to cell suspensions and cells were passed through a 100 µm filter. Data were acquired using BD FACSverse and analysed using FlowJo (version 10.8). Based on the above protocol, we sorted CD31-/CD45- living fibroblasts from primary human synovial cells and collected CD142 + and CD142- fibroblasts using BDJAzz for further in vivo and in vitro experiments.

Generation and analysis of single-cell RNA sequencing data

Fresh meniscus, cartilage, and synovial tissues obtained from the same RA knee by TKA were made into cell suspensions, in which meniscal cells, chondrocytes, and synovial cells were captured with 10x Genomics based on the NovaSeq platform. Fastp was applied with a default parameter for filtering the adaptor sequence and removing low-quality reads³⁴. Umi tools were used for single-cell transcriptome analysis to identify the cell barcode whitelist, extract the cell barcode UMIs, and calculate the cell expression counts based on the filtered clean FASTQ data³⁵. Downstream analysis was performed using the Seurat R package (version 4.0.2) as follows: cells with > 20% mitochondrial reads, < 200 genes, or < 200 UMI were excluded from the analysis. We merged cells from the meniscus, cartilage, and synovium, and the per-cell counts were normalised and scaled. The fastMNN function (k = 5, d = 50, approximate = TRUE) in the R package scran (version 1.12.1) was used to apply the mutual nearest-neighbour method to correct for batch effects among samples. The first ten principal components were retained for the UMAP projection. GraphCluster and K-means were used for cell clustering (resolution set at 0.8 for the full analysis), and the Wilcoxon rank-sum test was used for marker gene analysis (min.pct = 0.1, logfc. threshold = 0.25). We identified the meniscal cells as COL1A1+/CEMIP+, chondrocytes as ACAN+/COL2A1+, SF as PRG4+/COL1A1+, macrophages as CD14+/LYZ+, smooth muscle cells as MYL9+/NOTCH3+, T cells as CD3D+/CD8A+, endothelial cells as MCAM+(CD146+)/PECAM1+, and mast cells as CD79A+/ TPSB2+. SF were re-analysed to identify subclusters in the fastMNN-based UMAP zoom with a resolution set at 1.8, and marker genes were calculated (min.pct = 0.1, logfc. threshold = 0.25).

Cell-to-cell interaction analysis

After identifying the three subclusters of SF, we performed a cell-to-cell interaction analysis using the R package CellChat (version 1.6.1). We created a CellChat object with clusters of meniscal cells and chondrocytes and three subclusters of SF. CellChat database for humans (CellChatDB.human) was loaded, and the “Secreted Signaling” category was chosen for analysis. Overexpressed genes and interactions were identified and projected onto the protein-protein interactions (PPI) of the CellChat object. We then computed the communication probability based on the algorithm’s truncated mean (cutoff = 20%) and filtered out the cell-to-cell communication if there were < 10 cells in certain cell groups, which were aggregated into the final cell-to-cell communication network. Three subclusters of SF were set as communication senders, while meniscal cells and chondrocytes were set as communication receivers, and the interaction numbers were output for a heatmap and bubble plot.

Transwell assays for SF

CD142 + and CD142- fibroblasts were used for in vitro experiments. For transwell assays, we used 8.0 µm PET membrane in a 24-well format (Corning) for migration assays and 8.0 µm Matrigel coated PET membrane in a 24-well format (Corning) for invasion assays. Note that MK571 (Selleck) was dissolved in DMSO, and when added to the experiment system of DMEM/F-12, the concentration of MK571 was 5 µM, and DMSO was lower than 0.1%. We added 1×10⁵ fibroblasts and 200 µL DMEM/F-12 (0% FBS, 1% penicillin-streptomycin, and 5µM MK571 for the specified group) in the upper chamber, and 600 µL DMEM/F-12 (10% FBS, 1% penicillin-streptomycin) in the nether chamber. Twenty-four well plates were placed in a 37℃ incubator for 24 h for migration assay and 48 h for invasion assay.

Co-culture of SF-meniscal cells

Primary human RA MCs were used in this study. We used 0.4µm PET membrane in 6-well format (Corning). We added 1×10⁵ CD142 + or CD142- fibroblasts, 1 ml DMEM/F-12 (10% FBS, 1% penicillin-streptomycin, and 5 µM MK571 for the certain group) in the upper chamber, 1×10⁵ meniscal cells, and 2 mL DMEM/F-12 (10% FBS, 1% penicillin-streptomycin) in the nether chamber. Six well plates were placed in a 37℃ incubator for 72 h; then, meniscal cells were washed with PBS and extracted to total RNA (details written below) for further analysis.

Mouse OA and RA models establishment

The animal study design was reviewed and approved by the Ethics Committee of XXX Hospital (2020-B0270). We created OA models by surgically destabilizing the medial meniscus (DMM) in C57BL/6 male mice at 8 weeks of age¹⁴. After 8 weeks (16 weeks of age), DMM mice were sacrificed to obtain knee samples.

We created an RA model with CIA in DBA/1 male mice³⁶. Mice were immunised with 200 µg chick typeⅡcollagen (CⅡ, Chondrex) emulsified 1:1 in complete Freund’s adjuvant (CFA, Sigma) at 8 weeks of age, and were boosted 3 weeks later with 200 µg CⅡemulsified 1:1 in Freund’s incomplete adjuvant (IFA, Chondrex). At 16 weeks of age, CIA mice were sacrificed to obtain knee samples.

Mouse invasive SF arthritis model establishment

Human RA CD142 + and CD142- SF were used to create an invasive synovial fibroblast arthritis model. Living CD142 + and CD142- fibroblasts were first labelled with autofluorescence using CellTrace CFSE dye (Thermo Fisher Scientific), which diffused into cells and bound covalently to intracellular amines, resulting in fluorescent staining (excitation: 488 nm; Ex/Em: 592/571 nm). Resuspended fibroblasts in PBS were then added to the CFSE dye at a working concentration of 5 µM and incubated for 20 min at room temperature, protected from light. Five times the original staining volume of the culture medium was added to the fibroblasts, incubated for 5 min, centrifuged, and resuspended in DMEM/F-12. For intra-articular injection, each knee joint was injected with 4µl cell suspensions (5×10⁴ fibroblasts in DMEM/F-12, without FBS and penicillin-streptomycin), while in MK571 group, each knee joint was injected 2µl cell suspensions (5×10⁴ fibroblasts in DMEM/F-12, without FBS and penicillin-streptomycin) plus 2 µL MK571 (10 µM).

DBA/1 male mice were anaesthetised using isoflurane. We made a 1 cm vertical incision above the ligamentum patellae to fully expose it, then used a microinjector at a range of 0–10 µL to fetch cell suspensions. The needle tip was parallel to the platform and pointed at the midpoint of the medial or lateral side of the ligamentum patellae. The knee joint was then penetrated, and cell suspensions were injected until visible swelling was observed on the joint without spillage. The tip of the needle was withdrawn, the penetration point was pressed for 2 min using a cotton ball, and the skin was sutured. Intra-articular injection was administered once a week at 8, 9, 10, and 11 weeks of age. Mice were sacrificed at the age of 12 weeks for paraffin-embedded slices and at 16 weeks for both frozen OCT and paraffin-embedded slices. OCT embedded slices were stained with DAPI and directly observed under a fluorescence microscope to detect the invasive fibroblasts. OCT and paraffin slices were prepared according to the protocol described above.

Histological analysis of mouse knee slices

The knee joints of mice were decalcified for 3 weeks with 10% EDTA solution (pH 7.2, Solarbio) and embedded in paraffin. Consecutive slices were made (paraffin in 5 µm thick, OCT in 10 µm thick) at the knee lateral compartment in the sagittal view. We used the degeneration score and invasion area to assess the destruction of the mouse meniscus, as discussed above. The Krenn score was calculated to assess synovitis in the mouse knees. To evaluate cartilage damage, the OARSI score was calculated, with a maximum of six scores³⁷. The scores of three slices (one slice after an interval of nine slices) were summed and averaged as one joint, whereas both the left and right joints were summed and averaged as an independent sample.

Quantitative reverse transcription PCR

RNA was isolated from the cell suspensions using an RNA isolation kit (EZ Bioscience) according to the manufacturer’s instructions. cDNA synthesis was performed for all samples (500 ng of RNA was transcribed) using the RT Premix cDNA synthesis kit (Accuracy Biology). Reverse transcription quantitative PCR (RT-PCR) was performed using the SYBR GreenPro Taq qPCR kit (Accuracy Biology) on a real-time PCR detection system (Roche LightCycler480Ⅱ). The gene expression levels were quantified using the 2 − ΔΔCt method. Each primer (forward, F; reverse, R) was designed based on the sequences available in the NCBI database (Supplementary Data Table 2).

Transcriptomic RNA sequencing data

Human RA primary CD142 + and CD142- fibroblasts with three independent samples sorted by flow cytometry from the RA knee synovium obtained by TKA surgery were used to extract RNA. We applied fastp with the default parameters to filter the adaptor sequence and remove low-quality reads. The clean reads were aligned to 9606 (Taxonomy ID) genomes (version: GRCh38) using Hisat2. HTseq was used to calculate the gene counts. Reads/fragments per kilobase million reads (RPKM/FPKM) were used to standardise expression data. We applied the DEseq2 algorithm to filter the differentially expressed genes and then filtered the fold change and FDR under the following criteria: i) log2FC > 0.585 or < -0.585; ii) FDR < 0.05. Volcano plots and heat maps were drawn using R based on the analysis of differentially expressed genes, and the colour was determined using the filtering criteria. GO analysis was performed to elucidate the biological implications of unique genes in the significant or representative profiles of the differentially expressed gene in the experiment³⁸. We downloaded from the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/), UniProt (http://www.uniprot.org/), and GO (http://www.geneontology.org/) databases. Fisher's exact test was used to identify significant GO categories, and FDR was used to correct p-values.

Statistical analysis

Statistical analyses were performed as described in each section using GraphPad Prism 9 software. Data are presented as mean ± s.d. from at least three independent experiments. Spearman’s correlation analysis was used to test the correlations between ordinal variables. Differences were considered significant at P < 0.05. Multiple testing corrections were applied where appropriate.

SUPPLEMENTARY DATA

Supplementary Data Table 1

Follow-up information of the RA cohort patients

Supplementary Data Table 2

Primers in this study for qPCR

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CD142+ synovial fibroblast drives meniscus destruction in rheumatoid arthritis

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Abstract

Figures

INTRODUCTION

RESULTS

Severe meniscus degradation in knee RA

CD142 + SFs elevate in RA

CD142 + SFs migrate from SL to LL in RA

CD142 + SFs invade meniscus

ABCC4 blockade attenuated CD142 + SFs invasion

MK571 attenuates CD142 + SFs invasion induced meniscus degradation

CD142 + SFs migrating to LL is the risk factor for knee dysfunction in RA

DISCUSSION

MATERIALS AND METHODS

Human subjects research

Radiological analysis of human knee data

Histological morphology analysis and scoring of human synovium and meniscus

Immunohistochemistry and mIHC

Quantitative and spatial analysis of mIHC

Human meniscus, cartilage, and synovium tissue processing for cell suspensions and cell culturing

Flow cytometry and synovial fibroblast collection

Generation and analysis of single-cell RNA sequencing data

Cell-to-cell interaction analysis

Transwell assays for SF

Co-culture of SF-meniscal cells

Mouse OA and RA models establishment

Mouse invasive SF arthritis model establishment

Histological analysis of mouse knee slices

Quantitative reverse transcription PCR

Transcriptomic RNA sequencing data

Statistical analysis

Declarations

SUPPLEMENTARY DATA

References

Additional Declarations

Supplementary Files

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