Synovial fibroblasts from children with oligoarticular juvenile idiopathic arthritis induce migration and prolong viability of neutrophils

Abstract

Introduction: Little is known of the processes that trigger neutrophil activation in the joint of patients with oligoarticular juvenile idiopathic arthritis (oJIA), and if synovial fibroblasts (S-Fib) play an important role in the activation. Therefore, we aimed to investigate whether S-Fib derived from oJIA patients drive neutrophil activation.

Methods: Synovial fluid (SF) was collected from patients with oJIA. S-Fib were isolated from the SF of n = 7 patients through passaging. Subsequently, the S-Fib were primed or not with 20% of pooled SF. Supernatants were used to study migration of neutrophils in a transwell system. Additionally, the influence of S-Fib on neutrophils were studied in co-cultures. Phenotype and viability were assessed by flow cytometry. Neutrophil function was tested through the production of reactive oxygen species (ROS), and supernatants were tested for myeloperoxidase (MPO) release and elastase activity.

Results: Supernatants of S-Fib induced neutrophil migration (n = 5, p = 0.0491), which was further pronounced using supernatants from SF-primed S-Fib (p = 0.0063). Additionally, co-culture between SF-primed S-Fib and neutrophils resulted in prolonged viability (n = 5, p = 0.0094), with little effect on activation markers, e.g., CD11b. Conversely, co-culture did not induce functional alterations (n = 4), such as production of ROS (p > 0.1570), release of MPO (p > 0.4934) or elastase activity (p > 0.0904). Finally, supernatant stimulation did not replicate the results of prolonged viability (p = 0.9102), suggesting a role of cell-contact.

Conclusion: S-Fib from patients with oJIA induce migration of neutrophils via soluble mediators and, in addition, S-Fib prolong neutrophil viability in a cell-contact dependent manner. These mechanisms could be important for accumulation of neutrophils during arthritis.

Keywords: fibroblasts; inflammation; juvenile idiopathic arthritis; neutrophils; rheumatology.

Figure 1

Synovial fibroblasts drive neutrophil recruitment and prolong their viability, with limited effect on activation markers. (A) Schematic illustration of how supernatants from four donors of oJIA synovial fibroblasts (S-Fib), with or without prior priming for 48hrs with a pool of synovial fluid (SF), were used to induce migration of healthy neutrophils in a transwell system seeded with endothelial cells (HMEC). (B) Neutrophils in the lower chamber were identified based on CD66b expression and counted using flow cytometry. (C) Overview of how neutrophils were co-cultured with S-Fib for 24 h, with or without prior priming of the S-Fib. They were subsequently analyzed by flow cytometry for apoptosis and surface marker expression (D) representative histograms of the degree of apoptosis following flow cytometry using annexin V staining. (E) level of apoptosis as percentage annexin V positive cells in n = 5 neutrophil donors. (F) the expression of two surface markers in neutrophils following co-culture as representative histograms following flow cytometry analysis. (G) the expression as MFI values in n = 5 neutrophil donors. In parallel, healthy S-Fib (n = 3) were also used as a negative control to study (H) the degree of apoptosis in n = 5 neutrophil donors and (I) the expression of activation markers. (J) Supernatants from S-Fib cultures were used to activate neutrophils at a 1:1 ratio for 24 h. Viability was measured using Annexin V, and data represents the fold change (FC) to the control (neutrophils cultured in medium alone). Each data point represents a unique neutrophil donor, which in turn is the average of co-culture with 3–5 different donors of S-Fib. Data is presented as mean ± standard deviation. Statistics was performed using repeated measures-one-way ANOVA with Tukey's multiple comparisons test, except for (J), which was performed using one-way t-test and the hypothetical mean of 1. S-Fib, synovial fibroblasts; oJIA, oligoarticular juvenile idiopathic arthritis; SF, synovial fluid; MFI, median fluorescence intensity. Figures 1A,B were created using BioRender.com.

Figure 2

The effects of synovial fibroblast co-culture on neutrophil function are minor. Neutrophils were investigated for functional alterations following co-culture for 24 h with synovial fibroblasts (S-Fib). (A) Reactive oxygen species (ROS) was measured in n = 4 neutrophil donors following H₂DCFDA staining and PMA activation, over different time points, or (B) at 60 min. Data is presented as fold change/ratio vs. unstimulated control. (C) Elastase activity or (D) MPO levels were measured in supernatants following co-culture (n = 4). Each data point represents a unique neutrophil donor, which in turn corresponds to the average of co-culture with n = 5 donors of S-Fib. Data were analyzed using repeated measures-one-way ANOVA with Tukey's multiple comparisons test. S-Fib, synovial fibroblasts; ROS, reactive oxygen species; PMA, phorbol 12-myristate 13-acetate; OD, optical density, MPO, myeloperoxidase.