Fibroblast-like synoviocytes (FLS) play a critical role in inflammation that contributes to disease progression in juvenile idiopathic arthritis (JIA). In rheumatoid arthritis (RA), FLS express tumor necrosis factor alpha (TNFα). TNFα signaling has been shown to be upstream of transforming growth factor beta (TGFβ) signaling. Overexpression of TNFα and TGFβ, as well as related proteins, can cause increased inflammation in RA. In this study, we examine the effects of inhibiting TNFα signaling with adalimumab on FLS isolated from synovial fluid of patients with JIA.
Methods
Synovial fluid samples were selected from 41 patients in our repository. Of these samples, 23 were diagnosed with persistent oligoarticular JIA and 18 were diagnosed with extended oligoarticular JIA. All samples were taken prior to patients extending to a polyarticular course, or what we termed extended-to-be (ETB), and were on no medications or nonsteroidal anti-inflammatory drugs (NSAIDs) only at the time of arthrocentesis. For cell studies, FLS were isolated from synovial fluid and treated with adalimumab for 24 h. Protein antibody arrays were performed by RayBiotech, Inc. according to their protocols.
Results
In persistent FLS, TNFα (fold change [FC] = 1.2 p = 0.001), TGFβ (FC = 1.5 p = 0.001), lymphotoxin alpha (LTα) (FC = 4.3 p = 0.015), soluble tumor necrosis factor receptor 1 (sTNFRI) (FC = 5.1 p = 0.008), and soluble tumor necrosis factor receptor 2 (sTNFRII) (FC = 3.8 p = 0.025) were significantly elevated in adalimumab treated cells compared to untreated cells. In ETB FLS, TNFα was significantly elevated (FC = 1.04 p = 0.023) while TGFβ (FC = 1.03 p = 0.037) was significantly decreased in adalimumab-treated cells compared to untreated cells.
Conclusions
This data suggests that, after only 24 h, adalimumab is effective at decreasing inflammation that occurs downstream of initial TNFα signaling in extended-to-be fibroblast-like synoviocytes.
Key Summary Point
Why carry out this study?
Fibroblast-like synoviocytes (FLS) have been shown to contribute to disease progression in rheumatoid arthritis (RA).
FLS are thought to play a similar role in disease progression in juvenile idiopathic arthritis (JIA), but little is known about how they contribute to the various disease subtypes.
Determining the pathogenesis of JIA through FLS could lead to clinicians intervening earlier in the disease course with advanced therapeutics that target the joint to prevent disease from evolving to more severe disease subtypes.
Tumor necrosis factor alpha (TNFα) and transforming growth factor beta (TGFβ) contribute to inflammation in RA and adalimumab; a TNFα inhibitor is an advanced therapy used in the treatment of JIA.
We hypothesize that inhibiting TNFα in FLS isolated from synovial fluid of patients who eventually extend to a more severe disease course could prevent increased inflammation and disease progression.
What was learned from the study?
After only 24 h, adalimumab is effective at decreasing inflammation that occurs downstream of initial TNFα signaling in extended-to-be fibroblast-like synoviocytes.
While TNFα itself did not show significant changes in expression when FLS from the extended to be samples were treated with adalimumab, TGFβ expression, downstream of TNFα, was significantly reduced in extended-to-be (ETB) FLS.
Longer exposure to adalimumab could affect TNFα expression in FLS.
Introduction
While the pathogenesis of juvenile idiopathic arthritis (JIA) is not completely understood, fibroblast-like synoviocytes (FLS) are thought to play a critical role in JIA progression and may contribute to increased inflammation associated with disease progression [1, 2]. In this study, we delve deeper into understanding FLS as effector cells for disease progression and the impact of advanced therapeutics on this cell type.
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The current classification scheme for JIA includes seven subtypes of disease based on onset [3, 4]. In this study we focused on the persistent oligoarticular subtype (persistent), which is the most benign, and extended oligoarticular subtype, in which there is a greater chance of disability when joint involvement evolves from a persistent oligoarticular to a polyarticular disease course. Specifically, we examined FLS and synovial fluid from patients with a persistent disease course and compared them to patients prior to extension to a polyarticular course, termed extended-to-be (ETB), to observe differences in inflammatory profiles before these patients evolved to a more severe disease course. Specifically, our objective was to show that disease originates within the FLS and that targeting cells directly with advanced therapeutics can have a direct impact on disease progression, particularly in the patients that eventually extend to a more severe disease course. Furthermore, our data suggests that attenuating inflammation within the FLS can lower the overall inflammatory response typically promoted by FLS.
In rheumatoid arthritis (RA), studies have shown that FLS express tumor necrosis factor alpha (TNFα) and secretion of TNFα contributes to the increased inflammatory response associated with active disease [2, 5‐8]. Furthermore, studies have demonstrated links between transforming growth factor beta (TGFβ) signaling and TNFα expression in various chronic inflammatory diseases and that anti-TNFα biologic medications cause changes in the TGFβ signaling pathway [9‐11]. On the basis of these studies, we examined the impact of adalimumab, an anti-TNFα biologic disease-modifying anti-rheumatic drug (bDMARD), on cytokine expression in FLS. Specifically, we measured global inflammatory profiles in FLS isolated from synovial fluid of patients prior to them extending to a polyarticular disease course and observed differences in expression of TNFα and TNFα-related proteins. We treated cells with adalimumab to determine if this advanced therapy had a direct impact on FLS. The emergence of biologics that target specific cytokines has led to dramatic improvements in the treatment of JIA without widespread risks to the patients [12]. Distinguishing between persistent and ETB is critical to the study of JIA because patients with these subtypes have the same clinical presentation at diagnosis, making it difficult for clinicians to determine which patients will extend to a more severe disease course. Understanding how ETB JIA differs from persistent JIA could allow for intervention with advanced therapies earlier in the disease course and possibly prevent extension to a more severe disease course. Specifically, treating the FLS directly with advanced medications could prohibit promotion of the inflammatory response in more severe subtypes and potentially prevent patients from extending to a polyarticular disease course.
Methods
Selection of Samples
Samples were selected using the same criteria from our previous work [13]. Briefly, synovial fluid samples were selected from a repository approved by our Nemours Foundation BioMedical Research Institutional Review Board (IRB#84709-30). Written informed consent was obtained from patients who had clinically indicated arthrocentesis and were offered participation in the repository. This study was performed in accordance with the Helsinki Declaration of 1964, and its later amendments. In our database, 320 patients with oligoarticular JIA were identified. Patients with or diagnoses of psoriatic, inflammatory bowel disease-related, Lyme, syndromic arthritis, and polyarticular JIA were excluded, leaving 83 individuals who underwent one or more arthrocentesis. Out of the 83 patients identified, 41 patients with oligoarticular JIA who underwent an intra-articular arthrocentesis while the patient was on no medications or nonsteroidal anti-inflammatory drugs (NSAIDs) only, and before having received any disease-modifying anti-rheumatic drugs (DMARDs) or biologic disease-modifying anti-rheumatic drugs (bDMARDs), were selected. All patients were followed between 1990 and 2021.
Of the 41 patients who met inclusion criteria and adhere to International League for Associations in Rheumatology (ILAR) classification criteria, 23 patients were in the persistent oligoarticular JIA group and 18 in the extended oligoarticular group. Fifteen of the 23 patients in the persistent group received no medication or received NSAIDs only during their disease course, whereas eight eventually received DMARDs/bDMARDs. From this group, for the cell culture experiments, we selected three persistent oligoarticular synovial fluid samples from which to isolate FLS. All samples had no prior steroid injections, were only on NSAIDs or no medications at time fluid was taken, never received DMARDs/bDMARDs, and were followed for at least 2 years to ensure they remained persistent and did not extend to a more severe disease course. In the extended oligoarticular JIA group, 17 of the 18 patients eventually received DMARDs/bDMARDs, while one did not and remained on no medications. From this group, for the cell culture experiments, we selected three ETB synovial fluid samples from which to isolate FLS. All samples had no prior steroid injections, were only on NSAIDs or no medications at time fluid was taken, had not received DMARDs/bDMARDs at time of injection, and extended after the first 6 months of disease. All selected synovial fluid samples analyzed in this study were obtained before patients received advanced medications, including DMARDs or bDMARDs. At the time of the arthrocentesis, patients were taking either NSAIDs or no medications. If the initial synovial fluid sample could not be located, a sample obtained at least 3 months after the initial arthrocentesis was selected. Paired samples were two samples collected at least 3 months apart from the same patient. For paired synovial fluid samples, all patients were either on NSAIDs or no medications at time of injection, were collected at least 3 months apart, and had received no DMARDs or bDMARDs. All synovial fluid samples were taken from knee joints.
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Cell Culture and Adalimumab Treatment
We selected primary cells from three subjects with persistent oligoarticular JIA and three subjects who eventually extended to a polyarticular disease course (termed extended-to-be, ETB). All samples had no prior steroid injections and were on NSAIDs or no medications at time synovial fluid was obtained. Synovial fluid was spun at 500 rpm for 5 min, and cell pellets were plated in T-25 culture flasks with 15% fetal bovine serum/Dulbecco’s modified Eagle’s medium. Initial primary cultures were passaged into T-75 flasks and, at confluence, passaged three more times, to ensure FLS were the predominant cell type in culture. FLS from persistent oligoarticular and ETB oligoarticular JIA were grown in medium containing 15% FBS. At sub-confluence, media were removed, and cells were incubated overnight in growth medium containing 0.2% serum. The serum-starved cells were then seeded on six-well plates for experiments. Cells were stimulated with either 10 µg/ml adalimumab [14] or normal media for 24 h. After 24 h, cell media supernatants were obtained from each condition. Power analysis was performed on the basis of synovial fluid data to determine the number of FLS samples needed to meet 0.80 power with an error rate of 5%. On the basis of this data, it was determined that for inflammatory proteins measured, two to four samples were needed to reach a power of 0.80 with a 5% error rate. As a result of the number of proteins analyzed, we chose the median of three samples per group.
Antibody Arrays and Proteomic Analysis
Data analysis was performed using the same techniques from our previous work [13]. Briefly, protein concentrations were calculated using a Bradford assay. RayBiotech performed Human Inflammation Array C3 (AAH-INF-3) according to the manufacturer’s protocols. The array membranes were processed similarly to a western blot (chemiluminescent readout). Densitometry and background correction was performed. Averages were calculated between technical duplicates for each sample. Semiquantitative changes in protein expression were determined and compared between JIA subtypes. Scaling was performed by converting intensity measurements or relative protein amounts, to the log10 value. Statistical analysis was performed using Excel. For full data calculations please reference our previous work [13]. Briefly, we calculated average log10 values and standard deviation for the combined biological replicates in each group. Using a Welsh’s t test, we determined significant differences between two comparisons as having a p value of less than 0.05.
Results
FLS Isolated from Synovial Fluid of Patients Prior to Disease Extension to a More Severe Disease Course Overexpress Pro-inflammatory Cytokines Related to TNFα Signaling
FLS are thought to play a critical role in disease pathogenesis in JIA [2, 15]. Using protein antibody arrays to measure relative changes in protein expression of 40 different cytokines, we tested FLS isolated from synovial fluid of six patients. FLS from patients that remained persistent throughout disease course were compared to FLS from patients that eventually extended to a more severe disease course (ETB). Of the 40 cytokines, six proteins were elevated in ETB FLS compared to persistent FLS (Fig. 1). Transforming growth factor beta 1 (TGFβ1) had a 1.1-fold increase in ETB FLS compared to persistent FLS (p = 0.022) (Fig. 1). TNFα family members, lymphotoxin alpha (LTα) (fold change [FC] = 3.5 p = 0.014), soluble TNF receptor 1 (sTNFRI) (FC = 4.4 p = 0.014), and soluble TNF receptor II (sTNFRII) (FC = 3.4 p = 0.036) were significantly elevated in ETB FLS compared to persistent FLS (Fig. 1). Platelet-derived growth factor (PDGF), which can stimulate TNFα synthesis and has been shown to be elevated in RA FLS when TGFβ1 expression is increased [16, 17], had a 3.5-fold increase in ETB FLS compared to persistent FLS (p = 0.035) (Fig. 1). Lastly, interleukin-3 (IL-3), a promoter of inflammatory cell proliferation, was elevated in ETB FLS compared to persistent FLS (FC = 1.1 p = 0.008) (Fig. 1). This data suggests that FLS from ETB, prior to extension, secrete higher levels of cytokines that induce TNFα signaling when compared to those patients who remain persistent throughout disease course.
Fig. 1
Protein antibody array on FLS isolated from synovial fluid from patients with persistent and ETB JIA revealed elevation of proteins related to TNFα and TGFβ. We measured 40 cytokines in 3 samples from persistent JIA and 3 samples from ETB JIA. All patients were steroid naïve and on either no medication or only NSAIDs at the time of the arthrocentesis. All synovial fluid samples from the ETB group were taken prior to their disease extending to a more severe disease course. TGFβ1 had a 1.1-fold increase in ETB FLS compared to persistent FLS (p = 0.022). LTα (FC = 3.5 p = 0.014), sTNFRI (FC = 4.4 p = 0.014), sTNFRII (FC = 3.4 p = 0.036) were significantly elevated in ETB FLS compared to persistent FLS. PDGF had a 3.5-fold increase in ETB FLS compared to persistent FLS (p = 0.035). Interleukin-3 (IL-3) was elevated in ETB FLS compared to persistent FLS (FC = 1.1 p = 0.008). FLS fibroblast-like synoviocytes, JIA juvenile idiopathic arthritis, ETB extended-to-be, TNFα tumor necrosis factor alpha, TGFβ1 transforming growth factor beta 1, NSAIDs nonsteroidal anti-inflammatory drugs, FC fold change, LTα lymphotoxin alpha, sTNFRI soluble TNF receptor 1, sTNFRII soluble TNF receptor 2, PDGF platelet-derived growth factor, IL interleukin
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There is an Elevated Inflammatory Response in Synovial Fluid from Patients with ETB JIA When Compared to Synovial Fluid from Patients with Persistent JIA
Since FLS isolated from synovial fluid of patients with ETB JIA secrete factors that induce TNFα signaling, we examined the overall effects on inflammatory cytokines in the milieu of synovial fluid. Using protein antibody arrays to measure relative changes in protein expression of 40 different cytokines, we tested 41 samples of synovial fluid from patients with JIA. We divided these synovial fluid samples into two groups: 23 samples from those that remained persistent throughout their disease course and 18 ETB samples. Of the 40 cytokines measured, 22 were significantly elevated in the ETB group when compared to persistent, suggesting that those patients who eventually extend to a more severe disease course have an elevated inflammatory response and dysregulation of their inflammatory network when compared to persistent samples (Fig. 2). TGFβ1, an activator of T helper cells, was increased 1.3-fold in ETB compared to persistent (p = 0.008) (Fig. 2). Similarly, TNFα, a major pro-inflammatory protein, was increased 1.3-fold in ETB compared to persistent (p = 0.026) (Fig. 2). LTα, a member of the TNF family, was also elevated 1.3-fold in ETB compared to persistent (p = 0.037) (Fig. 1). Interleukin-6 (IL-6), a notable marker of disease activity in JIA and RA, was elevated 1.2-fold in ETB compared to persistent (p = 0.024) (Fig. 2). Additionally, there were 14 pro-inflammatory cytokines that were significantly elevated between one- and threefold in ETB compared to persistent (p < 0.05) (Fig. 2). In response to pro-inflammatory protein levels, anti-inflammatory proteins interleukin-10 (IL-10) (FC = 2.3 p = 0.000) and interleukin-11 (IL-11) (FC = 1.7 p = 0.002) were elevated in ETB compared to persistent (Fig. 2). Notably, tissue inhibitor of metalloproteinases-2 (TIMP-2), was significantly less in ETB compared to persistent (p = 0.001), suggesting a lack of inhibition of metalloproteinases (Fig. 2). Taken together with findings in FLS, this data suggests that ETB JIA have a more robust inflammatory response than persistent JIA and that this could possibly be driven by FLS.
Fig. 2
Protein antibody array on synovial fluid from patients with persistent and ETB JIA reveal an increase in inflammatory proteins in ETB samples compared to persistent samples. We compared 23 samples from persistent JIA to 18 samples from ETB JIA. All patients were steroid naïve and on either no medication or only NSAIDs at the time of the arthrocentesis. All synovial fluid samples from the ETB group were taken prior to their disease extending to a more severe disease course. TGFβ1 was increased 1.3-fold in ETB compared to persistent (p = 0.008). TNFα was increased 1.3-fold in ETB compared to persistent (p = 0.026). LTα was also elevated 1.3-fold in ETB compared to persistent (p = 0.037). IL-6 was elevated 1.2-fold in ETB compared to persistent (p = 0.024). There were 14 pro-inflammatory cytokines that were significantly elevated between one- and threefold in ETB compared to persistent (p < 0.05). Anti-inflammatory proteins IL-10 (FC = 2.3 p = 0.000) and IL-11 (FC = 1.7 p = 0.002) were elevated in ETB compared to persistent. TIMP-2 was significantly less in ETB compared to persistent (p = 0.001). ETB extended-to-be, JIA juvenile idiopathic arthritis, NSAIDs nonsteroidal anti-inflammatory drugs, TGFβ1 transforming growth factor beta 1, TNFα tumor necrosis factor alpha, LTα lymphotoxin alpha, IL interleukin, FC fold change, TIMP-2 tissue inhibitor of metalloproteinases-2, MIP-1-alpha macrophage inflammatory protein 1 alpha, MIP-1-beta macrophage inflammatory protein 1 beta, M-CSF macrophage colony-stimulating factor, CCL chemokine ligand, IFN-gamma interferon-γ, GM-CSF granulocyte-macrophage colony-stimulating factor, G-CSF granulocyte colony-stimulating factor, MIG monokine induced by interferon-γ
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Of All Patients Who Eventually Required a DMARD/bDMARD, Synovial Fluid Samples from Patients with ETB JIA had an Elevated Inflammatory Response When Compared to Synovial Fluid Samples from Patients with Persistent JIA
On the basis of our data that FLS isolated from synovial fluid of patients with ETB JIA have increased secretion of TNFα-related cytokines and may contribute to an elevated inflammatory response in synovial fluid from patients with ETB, we tested a subset of 25 samples of synovial fluid from patients with persistent and ETB JIA who eventually required either a DMARD or bDMARD. Using protein antibody arrays, we measured relative changes in protein expression of 40 different cytokines between eight samples from those that remained persistent throughout their disease course and 17 ETB samples. TNF family member LTα (p = 0.040) was elevated 1.5-fold in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD (Fig. 3a). TGFβ1 was elevated 1.4-fold in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD (p = 0.003) (Fig. 3a). In addition to these TNFα-related cytokines, there were 18 pro-inflammatory cytokines (FC between 1.5-fold and 9.0-fold) that were significantly elevated in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD (p < 0.05) (Fig. 3a). In response to this increase in inflammatory cytokines, IL-10 (FC = 8.5 p = 0.000) and IL-11 (FC = 4.2 p = 0.000), anti-inflammatory proteins were significantly elevated in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD (Fig. 3a). The elevation of LTα and TGFβ suggests that patients with ETB JIA who eventually require a DMARD/bDMARD may benefit from TNFα inhibitors earlier in the disease course.
Fig. 3
Protein antibody array on synovial fluid from patients with persistent and ETB JIA who eventually required a DMARD or bDMARD reveal that prior to receiving advanced therapies, patients who eventually extend to a more severe disease course have elevated inflammatory protein expression. We measured protein expression of 40 cytokines between 8 samples from those that remained persistent throughout their disease course and 17 ETB samples (a). All patients had not yet received a DMARD or bDMARD, were steroid naïve, and on either no medication or NSAIDs only at the time of arthrocentesis and all synovial fluid from the ETB group were taken prior to their disease extending to a more severe disease course (a). LTα (p = 0.040) was elevated 1.5-fold in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD. TGFβ1 was elevated 1.4-fold in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD (p = 0.003). There were 18 pro-inflammatory cytokines (FC between 1.5-fold and 9.0-fold) that were significantly elevated in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD (p < 0.05). IL-10 (FC = 8.5 p = 0.000) and IL-11 (FC = 4.2 p = 0.000) were significantly elevated in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD. We tested serial synovial fluid samples from the 25 persistent and ETB samples (b). At the time of arthrocentesis, none of the patients had received a DMARD or bDMARD, had only one previous steroid injection, and were on no medication or NSAIDs only (b). TNFα (p = 0.003) and LTα (p = 0.007) were both elevated 1.8-fold in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD. TGFβ1 was significantly elevated in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD (FC = 2.1-fold p = 0.001). In addition to these promoters of inflammation, 20 other cytokines were elevated in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD after a single intra-articular corticosteroid injection (FC range 1.1–5.1 p < 0.050) (c). ETB extended-to-be, JIA juvenile idiopathic arthritis, DMARD disease-modifying anti-rheumatic drug, bDMARD biologic disease-modifying anti-rheumatic drug, LTα lymphotoxin alpha, NSAIDs nonsteroidal anti-inflammatory drugs, IL interleukin, TGFβ1 transforming growth factor beta 1, FC fold change, M-CSF macrophage colony-stimulating factor, MIP-1 macrophage inflammatory protein 1, TIMP-2 tissue inhibitor of metalloproteinases-2, G-CSF granulocyte colony-stimulating factor, CCL chemokine ligand, IFN-gamma interferon-γ, GM-CSF granulocyte-macrophage colony-stimulating factor, MIG monokine induced by interferon-γ
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Of All Patients Who Eventually Required a DMARD/bDMARD, TNFα Remains Elevated in ETB After Intra-articular Corticosteroid Injection Compared to Persistent After Intra-articular Corticosteroid Injection
To determine if intra-articular corticosteroids change the expression of inflammatory promoter TNFα, we tested serial synovial fluid samples from the 25 persistent and ETB samples analyzed above using protein antibody arrays. At the time of arthrocentesis, none of the patients had received a DMARD or bDMARD, had only one previous steroid injection, and were on no medication or NSAIDs only. In the synovial fluid samples of the subsequent arthrocentesis, TNF family members TNFα (p = 0.003) and LTα (p = 0.007) were both elevated 1.8-fold in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD (Fig. 3b). TGFβ1 was significantly elevated in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD (FC = 2.1-fold p = 0.001) (Fig. 3b). In addition to these TNFα-related promoters of inflammation, after a single intra-articular corticosteroid injection, 20 other cytokines were elevated in ETB who eventually required a DMARD/bDMARD compared to persistent who eventually required a DMARD/bDMARD (FC range 1.1–5.1 p < 0.050) (Fig. 3c). This data suggests that while intra-articular corticosteroids may initially lower inflammatory response in those patients who eventually extend to a more severe disease course, intra-articular corticosteroids did not alter overall inflammatory response in the long term.
Adalimumab is Effective at Targeting Downstream TNFα Signaling and Overall Inflammatory Response in FLS Isolated from Synovial Fluid of Patients with ETB JIA
We showed that FLS isolated from synovial fluid of patients with ETB JIA have increased TNFα expression and that this may contribute to the elevated inflammatory response seen in synovial fluid from patients with ETB JIA that eventually required a DMARD/bDMARD. Therefore, using protein antibody arrays we examined the effects of a short course (24 h) of adalimumab, a TNFα inhibitor, on inflammatory cytokines in FLS isolated from synovial fluid of six patients. In the persistent FLS treated with adalimumab for 24 h, 17 of the 40 inflammatory proteins remained significantly elevated when compared to untreated cells (Fig. 4). Specifically, TNFα (FC = 1.2 p = 0.001) and TGFβ (FC = 1.5 p = 0.001) were significantly elevated in persistent FLS treated with adalimumab compared to untreated persistent FLS (Fig. 4). LTα (FC = 4.3 p = 0.015), sTNFRI (FC = 5.1 p = 0.008), and sTNFRII (FC = 3.8 p = 0.025) were significantly overexpressed in persistent FLS treated with adalimumab compared to untreated persistent FLS (Fig. 4). The remaining 12 markers of inflammation fold change ranged from 1.1 to 4.4 increase in persistent FLS treated with adalimumab compared to untreated FLS (p < 0.05) (Fig. 4). Interestingly, this data suggests that one dose of adalimumab for 24 h is not effective at inhibiting an inflammatory response in persistent FLS, the least severe disease course of JIA.
Fig. 4
Adalimumab, a TNFα inhibitor, did not decrease expression of pro-inflammatory cytokines in FLS isolated from synovial fluid of patients with persistent JIA after 24 h of treatment. All patients were steroid naïve and on no medication or only NSAIDs at the time of the arthrocentesis. All FLS isolated from synovial fluid from the ETB group were taken prior to their disease extending to a more severe disease course. TNFα (FC = 1.2 p = 0.001), TGFβ (FC = 1.5 p = 0.001), LTα (FC = 4.3 p = 0.015), sTNFRI (FC = 5.1 p = 0.008), and sTNFRII (FC = 3.8 p = 0.025) were significantly elevated in persistent FLS treated with adalimumab compared to untreated persistent FLS. The remaining 12 markers of inflammation fold change ranged from 1.1 to 4.4 increase in persistent FLS treated with adalimumab compared to untreated FLS (p < 0.05). TNFα tumor necrosis factor alpha, FLS fibroblast-like synoviocytes, JIA juvenile idiopathic arthritis, ETB extended-to-be, NSAIDs nonsteroidal anti-inflammatory drugs, TGFβ transforming growth factor beta, LTα lymphotoxin alpha, FC fold change, sTNFRI soluble TNF receptor 1, sTNFRII soluble TNF receptor 2, IL interleukin, MIP-1 macrophage inflammatory protein 1, PDGF platelet-derived growth factor
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Adalimumab is Effective at Decreasing Inflammation that Occurs Downstream of Initial TNFα Signaling After 24 h in FLS Isolated from Synovial Fluid of Patients Who Eventually Extend to a More Severe Disease Course
In the ETB FLS treated with adalimumab eight of the 40 inflammatory markers had significant changes in protein expression compared to the untreated ETB FLS. Interestingly, seven proteins decreased after exposure to adalimumab for 24 h and only TNFα remained significantly elevated in treated ETB FLS compared to untreated ETB FLS (FC = 1.04 p = 0.023) (Fig. 5). TGFβ (FC = 1.03 p = 0.037) was significantly decreased in treated ETB FLS compared to untreated ETB FLS (Fig. 5). Additionally, granulocyte colony-stimulating factor (G-CSF), interferon gamma (IFNγ), IL-10, macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein 1 alpha (MIP-1-alpha), and macrophage inflammatory protein 1 delta (MIP-1-delta) were all significantly decreased in treated ETB FLS compared to untreated ETB FLS (FC range from 1 to 3.6 p < 0.05) (Fig. 5). This data suggests that although adalimumab did not lower TNFα expression in ETB FLS, after 24 h, adalimumab is effective at decreasing inflammation that occurs downstream of initial TNFα signaling after a short time. Differing responses to adalimumab between persistent and ETB FLS are another distinguishable characteristic between these two JIA subtypes.
Fig. 5
Effects of a short course (24 h) of adalimumab, a TNFα inhibitor, on inflammatory cytokines in FLS isolated from synovial fluid of patients with ETB JIA reveal a decrease in pro-inflammatory cytokines downstream of TNFα. In the ETB FLS treated with adalimumab, TNFα remained significantly elevated compared to untreated ETB FLS (FC = 1.04 p = 0.023). TGFβ (FC = 1.03 p = 0.037) was significantly decreased in treated ETB FLS compared to untreated ETB FLS. G-CSF, IFNγ, IL-10, M-CSF, MIP-1-alpha, and MIP-1-delta were all significantly decreased in treated ETB FLS compared to untreated ETB FLS (FC range from 1 to 3.6 p < 0.05). TNFα tumor necrosis factor alpha, FLS fibroblast-like synoviocytes, JIA juvenile idiopathic arthritis, ETB extended-to-be, FC fold change, TGFβ1 transforming growth factor beta 1, G-CSF granulocyte colony-stimulating factor, IFNγ interferon-γ, IL interleukin, M-CSF macrophage colony-stimulating factor, MIP-1 macrophage inflammatory protein 1
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Discussion
Our data suggests that when well-characterized markers of inflammation were measured, FLS isolated from synovial fluid of patients with ETB JIA had increased expression of inflammatory markers, namely TNFα-related cytokines, when compared to FLS isolated from synovial fluid of patients with persistent JIA, suggesting that FLS may contribute to the overall inflammation associated with disease pathogenesis. When these cells were treated with TNFα inhibitor, adalimumab, expression of pro-inflammatory proteins downstream of TNFα signaling were significantly reduced in ETB FLS treated with adalimumab.
FLS are the major component of the synovial membrane and contribute to inflammation by producing pro-inflammatory cytokines and participating in cell-to-cell communication to recruit inflammatory cells to affected joints [18, 19]. FLS are thought to play a key role in disease pathogenesis in JIA [2] which prompted us to examine the inflammatory profiles of FLS isolated from synovial fluid of patients with persistent and ETB JIA and study the effects of adalimumab on this cell type. ETB FLS have increased expression of inflammatory promoters TGFβ and LTα and both TNFα receptors, suggesting that targeting the TNFα signaling pathway earlier in the disease course could be effective at delaying or preventing disease extension.
It has been shown that cell death can induce inflammation in autoimmune diseases and chronic inflammatory diseases [20]. Specifically, binding of TNFα to TNFR1 exacerbates inflammation by inducing cell death [21]. This is thought to be achieved by overcoming cell death checkpoints that allow for the activation of downstream inflammatory pathways MAPK and NF-κB [22, 23]. TGFβ signaling acts as an intermediary activator of both MAPK and NF-κB activation, downstream of TNFα binding to TNFRI [22, 23]. Specifically, TNFα induces TGFβ expression in lung fibroblasts and, conversely, TGFβ1 caused increased expression of TNFα in FLS isolated from patients with RA, and subsequent activation of NF-κB signaling, suggesting TGFβ is downstream of TNFα and can contribute directly to the inflammatory response of FLS [10, 24].
Secretion of TNFα-related cytokines by FLS prompted us to examine inflammatory cytokine profiles in synovial fluid of patients with JIA. Inflammatory cytokine expression is significantly elevated in synovial fluid from patients who eventually extend to a more severe disease course when compared to those patients who remain persistent. In RA, patients with chronic inflammation have elevated IL-1, IL-6, and TNFα [25] as well as elevated GM-CSF, IL-10, and TGFβ [26]. Similarly, we observed increased expression of these cytokines in synovial fluid from patients with ETB JIA when compared to patients with a persistent disease course.
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When we tested a subset of synovial fluid from patients who eventually required either a DMARD/bDMARD but prior to receiving an intra-articular corticosteroid injection or DMARD/bDMARD treatment in persistent and ETB JIA, we observed significant elevation of inflammatory cytokines related to TNFα signaling in patients with ETB JIA when compared to persistent. When we measured cytokine protein expression after patients received an intra-articular corticosteroid, we still observed significant increases in TNFα and cytokines downstream of TNFα. Elevation of these cytokines in synovial fluid from patients with ETB JIA who eventually require a DMARD/bDMARD could indicate the need for introduction of DMARD/bDMARD earlier in the disease course. Specifically, a TNFα inhibitor introduced earlier in the disease course may effectively delay extension to a polyarticular course in these patients. While there currently exists no clinical indicators for patients who will eventually extend to a more severe disease course, global protein expression of cytokines could be used to indicate the need for intervention with advanced medications earlier in the disease course.
Anti-TNF agent adalimumab is a neutralizing antibody that effectively binds to soluble and membrane-bound TNFα and prevents receptor/ligand binding to inhibit TNFα-driven inflammation [27, 28]. In patients treated with adalimumab, a response to treatment is usually seen in 131 ± 56 h [29]. Here, we treated FLS isolated from synovial fluid of patients with a persistent and ETB disease course for 24 h, to determine if a short course of treatment could have a direct effect on FLS in culture. In both persistent FLS and ETB FLS treated with adalimumab, TNFα remained elevated compared to respective untreated cells. In the persistent FLS, TGFβ1 was also still significantly overexpressed in treated cells compared to untreated cells. Berkhout et al. showed that circulating TNF actually increased in patients with RA treated with adalimumab and they saw an increase in healthy volunteers after one dose of adalimumab, implying that TNF ligand in circulation may not be associated with disease activity in patients with RA [30]. Interestingly, TGFβ expression levels significantly decreased in ETB FLS treated with adalimumab when compared to untreated ETB FLS, suggesting that after 24 h, adalimumab may have an effect downstream of TNFα on TGFβ signaling. In persistent FLS, sTNFRI and sTNFRII were significantly overexpressed in adalimumab-treated cells, suggesting that soluble receptor expression in response to elevated TNFα could bind available ligand and prevent membrane-bound receptor signaling. Interestingly, there were no significant differences in the soluble receptors in ETB FLS treated with adalimumab. Increased expression of sTNFRI could indicate increased cell death in persistent FLS compared to ETB FLS [20, 31]. Increased cell death has been linked to increased inflammation in chronic inflammatory diseases [22, 23, 31‐33].
Adalimumab may induce a faster response in more severe disease in JIA. Our findings show that TGFβ expression is lower in ETB FLS treated with adalimumab compared to untreated cells. Both soluble TNF receptors protein expression were not significantly different and IFNγ expression was significantly lower in ETB FLS treated with adalimumab compared to untreated cells, suggesting that ETB FLS are not secreting these downstream signaling proteins that activate other inflammatory cell types like macrophages and neutrophils [34].
While our data demonstrates increased inflammation in the ETB subtype of JIA when compared to persistent JIA in both FLS isolated from synovial fluid and synovial fluid samples, we recognize there are some limitations to this study. Cells were treated with adalimumab for 24 h. While in the more severe disease course we saw downregulation of TNFα downstream signaling, with prolonged exposure using a similar timeline to in vivo studies we may see more pronounced effects of adalimumab on FLS directly.
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Conclusion
We demonstrate that FLS isolated from synovial fluid of patients with JIA are capable of secreting pro-inflammatory cytokines and promoters of inflammation. Specifically, in patients who ultimately extend to a polyarticular course, FLS overexpress cytokines associated with TNFα signaling. We suggest that FLS may contribute to overall increased presence of pro-inflammatory cytokines in synovial fluid from patients who eventually extend to a more severe disease course. Finally, we establish that downstream TNFα-related proteins like TGFβ expression can be downregulated when FLS are treated with a short course of adalimumab, a TNFα inhibitor. These findings could potentially determine which patients will extend earlier in the disease course and allow for intervention sooner with advanced therapies.
Acknowledgements
Medical Writing, Editorial, and Other Assistance.
We would acknowledge Theresa Michel through Nemours Children’s Health editorial services who provided editorial assistance with this manuscript.
Declarations
Conflicts of Interest
Megan Simonds, Samuel Freer, Lina Al-Jaberi, and AnneMarie Brescia declare that they have no competing interests and no interests to disclose. All author affiliations at the time of the study are accurate on the title page; however, Samuel Freer is now affiliated with the University of Texas as a graduate student and Lina Al-Jaberi is now affiliated with Arkansas Children’s Hospital after completing her fellowship at Nemours Children’s Health.
Ethical Approval
Synovial fluid and fibroblast-like synoviocyte samples were obtained from the Nemours BioMedical Institutional Review Board-approved repository (IRB#84709-32). This study was performed in accordance with the Helsinki Declaration of 1964, and its later amendments. Patients who underwent clinically indicated arthrocentesis were offered inclusion into the repository and informed consent was obtained. There is no identifying information in this publication.
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc/4.0/.
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