This study enrolled n = 15 patients diagnosed with oligoarticular juvenile idiopathic arthritis (oJIA) based on the criteria set by the International League of Associations for Rheumatology (ILAR). The participants were recruited from the Department of Pediatric Rheumatology at Skåne University Hospital in Sweden between 2016 and 2023, whilst undergoing therapeutic joint aspiration. Patients were included upon written informed consent from the patients and/or their legal guardians/next of kin. Ethical approval for the study was granted by the Regional Ethical Review Board for southern Sweden (2016/128).

Synovial fibroblasts (S-Fib) were isolated from the synovial fluid (SF) of seven patients, while SF for priming experiments was pooled in equal proportions from a minimum of 8 patients with oJIA. Some patients were used only for S-Fib isolation, some for isolation of SF for pooling and some for both. The process of isolating cells and SF is detailed below. Clinical characteristics of the patients involved in the study are described in Table 1. Notably, patients were either newly diagnosed or in inactive disease without receiving disease-modifying anti-rheumatic drugs (DMARDs) or oral/intra-articular steroids for the past six months preceding inclusion, with the exception of one patient who had been administered methotrexate three months before sampling. All patients contributing with S-Fib had been treated with non-steroidal anti-inflammatory drugs (NSAIDs), but no other drugs. In addition, half of the patients donating SF for priming experiments received NSAIDs, while the other half was untreated at sampling.

Healthy S-Fib were purchased from Cell Applications and maintained in fibroblast-like synoviocytes (FLS) growth medium (Cell Applications). For patient S-Fib isolation, synovial fluid (SF) underwent centrifugation at 500 g for 10 min. The resulting cell pellet was washed twice with PBS and then placed in FLS medium in T-25 flasks (Sarstedt). A second centrifugation of the SF was performed at 800 g for 10 min, and the supernatant was frozen at −80°C for future use. The initially seeded cells were washed with fresh medium 24–48 h later to discard non-adherent cells. Subsequently, the cells were passaged approximately three times until S-Fib-like cells became the predominant cell type, as previously reported (17–19). The identity of the S-Fib was confirmed by flow cytometry for the expression of fibroblast markers, such as Cadherin-11 and CD90. The cells were then frozen at −80°C until needed, with usage limited to passage 10.

For co-culture experiments and priming, S-Fib cells were detached, and 2,000 cells/well were seeded in 96-well plates (Falcon) in FLS growth medium. The S-Fib were allowed to rest for 24 h, followed by either priming or no priming using FLS growth medium supplemented with 20% of pooled SF for an additional 48 h.

Granulocytes were isolated from heparinized whole blood from adult healthy controls through density centrifugation (lymphoprep) at 620 g for 20 min with low break. The upper layer were discarded and the lower fraction containing granulocytes (mainly neutrophils) and red blood cells (RBCs) was used. RBCs were sedimented using 1.5% dextran T500 (Pharmacosmos) in saline solution for 20 min, RT. The cell rich supernatant was collected, centrifuged and remaining RBCs were lysed by 20–25 s incubation with sterile H₂O, followed by restoration of isotonicity with 1:1 ratio of 1.8% saline. The cells were subsequently centrifuged, resuspended in RPMI-1640 with 10% normal human serum (NHS), counted (XN-350, Sysmex) and adjusted to 1 × 10⁶/ml. S-Fib (cultured as described above) were washed twice with RPMI-1640 medium before the addition of 10⁵ neutrophils. Neutrophils alone served as control. The cells were cultured for 24 h and analyzed as described below.

To generate S-Fib conditioned supernatants, S-Fib cells were initially seeded at a density of 10⁴ cells/well in a 24-well Falcon plate using FLS growth medium. After 72 h of cultivation, 20% of pooled SF was either added or omitted, and the S-Fib were subsequently cultured for 48 h (priming). Following this, the cells underwent two washes with RPMI-1640 medium, and serum-free RPMI-1640 was added, with the cells being cultured for an additional 24 h before harvesting the supernatants.

For the migration assay, a 24-well plate with 5 µm pore-sized transwell inserts (Corning) was used. Briefly, HMEC endothelial cells (ATCC) were seeded in the wells and allowed to reach confluence in MCDB-131 medium supplemented with 10% fetal bovine serum, 10 ng/ml hEGF, non-essential amino acids, L-glutamine (1 mM), sodium pyruvate, penicillin, and streptomycin (PenStrep). Prepared inserts were placed in a new 24-well plate containing S-Fib supernatants pooled from four donors. A negative control with RPMI-1640 medium alone was included. Neutrophils, isolated as previously outlined and adjusted to 2 × 10⁶/ml in serum-free RPMI-1640, were added (100 µl) to the top of the inserts. The plate was then transferred to an incubator (37°C, 5% CO₂), allowing migration to occur for 80 min. Subsequently, the inserts were discarded, and cells at the bottom of the wells were harvested by gentle pipetting in PBS/1 mM EDTA. Neutrophils were incubated with a 1:200 dilution of anti-CD66b (clone: G10F5, Alexa Fluor 647, BD) for 20 min at RT. Finally, the cells were washed with PBS, resuspended in 100 µl, and subjected to analysis using flow cytometry (CytoFLEX). The number of CD66b⁺ neutrophils/µl was used for subsequent analysis.

Following co-culture with S-Fib as described above, neutrophils were detached through gentle pipetting and one wash of the wells using PBS/1 mM EDTA, and centrifuged. Next, they were stained with anti-CD66b (1:50, clone: G10F5, FITC, BD) and anti-CD11b (1:200, clone: ICRF44, Alexa fluor 700, BD). The cells were incubated for 25 min, RT. Next, the cells were washed with PBS and resuspended in Annexin V binding buffer (BD biosciences) and stained with 1:200 Annexin V (FITC, BD) for 15 min, followed by analysis using flow cytometry (CytoFLEX).

In some experiments, supernatants were collected from S-Fib cultured for 24 h in 96-well plates (Falcon) in RMI-1640 with 10% NHS. These were then used at 1:1 ratio with fresh medium to stimulate freshly isolated neutrophils for 24 h, followed by analysis of apoptosis (as described above).

Supernatants were collected following S-Fib-neutrophil co-culture and stored at −80°C until use. An elastase activity assay (EnzChek) was performed according to the manufacturer's instructions. The plate was read at 485/535 nm and analysed using the Wallac 1420 software (version 3.0, PerkinElmer Life Sciences) and Microsoft Excel.

Following co-culture between S-Fib and neutrophils, as described above, the neutrophils were detached using gentle pipetting and the wells were washed once with PBS/1 mM EDTA. The cells were subsequently washed once with PBS and resuspended in 100 µl RPMI-1640 supplemented with 5% NHS. The cells were transferred to a black 96-well plate (Thermo Fisher) and placed in a 37°C pre-heated plate reader (VICTOR³, 1420 Multilabel Counter, PerkinElmer Life Sciences) for 10 min. Next, 10 µM H₂DCFDA was added to each well, and reactive oxygen species (ROS) production was induced by stimulation using 10 ng/ml PMA. Unstimulated cells and unstained cells served as controls. The plate was analysed at different time points up to 1 h of incubation. The plate was read at 485/535 nm and analysed using the Wallac 1420 software (version 3.0, PerkinElmer Life Sciences) and Microsoft Excel.

Following co-culture between S-Fib and neutrophils, supernatants were collected and frozen in −80°C until use for MPO analysis. The samples were analyzed using the LSBio Human MPO ELISA kit (LifeSpan BioSciences) according to the manufacturer's instructions. Supernatants were diluted 1:30. The plate was read at 450 nm and was analyzed using the Wallac 1420 software (version 3.0, PerkinElmer Life Sciences) and Microsoft Excel.

The analysis of flow cytometry data was performed using the CytExpert software (version 2.3) or Kaluza (version 2.1, both from Beckman Coulter). Presentation of data includes the mean with standard deviation unless specified otherwise. Comparison of fold change (FC) data was performed by one-sample t-test and the hypothetical mean of 1. Comparison of data among three groups was performed through repeated measures-one-way ANOVA, followed by Tukey's multiple comparisons test. GraphPad Prism 9 and Microsoft Excel were used for data analysis.

We hypothesized that the inflammatory environment could be important for the phenotype of synovial fibroblasts (S-Fib) in oligoarticular juvenile idiopathic arthritis (oJIA). Thus, we primed the S-Fib with 20% of pooled synovial fluid (SF) from oJIA. Subsequently, we collected supernatants to investigate if these cells could be responsible for neutrophil recruitment. In a transwell system (Figure 1A), supernatants from cultured oJIA S-Fib were able to induce migration of healthy neutrophils (n = 5, p = 0.0491), which was further pronounced using supernatants from SF-primed S-Fib (n = 5, p = 0.0063, Figure 1B). Therefore, these findings could be compatible with that S-Fib are able to recruit neutrophils from the circulation, especially following exposure to the arthritic environment of the joint.

As S-Fib drive neutrophil migration, we aimed to explore if they promote activation. Healthy neutrophils were thus co-cultured with S-Fib or SF-primed S-Fib from oJIA patients (Figure 1C). As a negative control, we also included healthy S-Fib in these experiments. Co-culture between SF-primed oJIA S-Fib and neutrophils (n = 5) prevented neutrophil apoptosis (Figures 1D,E, p = 0.0094), as measured by the percentage of annexin V positive neutrophils. A similar trend, although not significant, was observed in co-cultures with unprimed S-Fib (p = 0.2678). Furthermore, at the surface marker level, there were no significant changes in expression of either CD66b or CD11b on neutrophils (n = 5) following co-culture, although there was a trend of higher expression in neutrophils co-cultured with SF-primed S-Fib (Figures 1F,G). The effect of healthy S-Fib om neutrophils followed a similar trend but did not significantly affect apoptosis (Figure 1H) or the expression of activation markers (Figure 1I). Thus, co-culture with primed oJIA S-Fib results in prolonged viability, but without inducing expression of activation markers, in healthy neutrophils. Finally, the effect of healthy S-Fib on neutrophils was minor.

As co-culture with oJIA S-Fib prolonged neutrophil viability, we set out to investigate if this effect was due to soluble factors. Supernatants were collected from S-Fib cultures and used to stimulate neutrophils for 24 h. Compared to medium alone, S-Fib supernatants did not prolong neutrophil viability (n = 4), regardless of whether the supernatants originated from unprimed S-Fib (p = 0.6939) or primed S-Fib (p = 0.9102) (Figure 1J). Thus, direct cell-cell interactions are likely important in prolonging neutrophil viability.

Co-culture between healthy S-Fib and neutrophils did not influence the neutrophil viability or phenotype, whilst oJIA S-Fib prolonged neutrophil viability but had little effect on activation markers. Thus, as oJIA S-Fib did display some influence on neutrophils, we set out to investigate further whether neutrophils are functionally affected following co-culture with oJIA S-Fib. First, we stimulated neutrophils with PMA following co-culture and measured reactive oxygen species (ROS) production (Figures 2A,B). There was no statistical difference between the groups, although co-cultured neutrophils tended to have decreased ROS production compared to neutrophils alone (n = 4). A similar observation was made analyzing elastase activity in supernatants (Figure 2C), were there was no statistical difference, but a trend towards lower activity in supernatants from co-culture with SF-primed S-Fib. Finally, we analyzed MPO levels in supernatants (n = 4), and again, we found no difference between the conditions (Figure 2D). Hence, the effect of co-culture between S-Fib and neutrophils are minor at the functional level, with a trend towards lower activity in co-cultures with SF-primed S-Fib, potentially reflecting the increased viability in this setting.