Synovial fibroblasts integrate inflammatory and neuroendocrine stimuli to drive rheumatoid arthritis

Abstract

Rheumatoid arthritis is a chronic inflammatory disease that is characterized by pannus tissue consisting of synovial fibroblasts (SF), macrophages and lymphocytes. The inflammatory milieu in the joint activates resident SF and transforms them in a tumor-like phenotype. These changes manifest in resistance to Fas-induced apoptosis and production of cytokines, chemokines and matrix metalloproteinases. By alterations in DNA methylation, SF retain their transformed phenotype even in the absence of pro-inflammatory stimuli and are able to spread arthritis to unaffected joints. Furthermore, SF integrate neuroendocrine input to modulate inflammation since they possess receptors for several neurotransmitters (e.g., dopamine, norepinephrine and glutamate). Until now, no specific therapy targeting SF is available; however, reprogramming them to a regulatory phenotype might limit joint destruction and cartilage degradation.

Keywords:

• catecholamines
• cytokines
• sympathetic nervous system
• synovial fibroblasts
• TNF

Synovial fibroblasts (SF) are resident cells of the intimal lining layer of synovial tissue. They produce a large array of matrix proteins (e.g., collagens and fibronectin) thereby maintaining joint integrity. In addition, they also function as innate immune cells as they can attract neutrophils and respond to Toll-like receptor stimulation with production of pro-inflammatory cytokines [1]: While SF do not encounter immune cells in healthy joints, extensive SF-macrophage (type A synoviocytes) and SF-lymphocyte interactions occur during the course of rheumatoid arthritis (RA) with pro-inflammatory cytokines and cell–cell contacts being responsible for SF transformation. Macrophage/monocyte-derived cytokines drive the expression of cytokines and adhesion molecules in SF [2]. In addition, production of TNF and IL-1β by synovial B-lymphocytes is one major factor for SF conversion into aggressive RA-like SF [3]. Although B-cell cytokine production is reduced when co-cultured with SF, B-lymphocyte survival by secretion of B-lymphocyte activating factor and engagement of VCAM-1 is augmented by SF [3]. T-lymphocytes induce IL-6 and IL-8 production in SF upon contact, which is mediated by membrane-bound TNF. TNF also mediates resistance of rheumatoid arthritis SF (RASF) to Fas-induced apoptosis, since it up-regulates soluble Fas that binds Fas ligand and prevents its pro-apoptotic action [4]. In addition, binding of integrin α5β1 on RASF to fibronectin, which is abundant in RA synovial tissue, reduces the ability of Fas ligand to induce apoptosis [5]. SF activation is supported by synovial hypoxia, which is responsible for the up-regulation of a wide array of pro-inflammatory genes such as leptin, COX2 and transient receptor potential ankyrin.

After acquiring an activated phenotype, SF increase production of matrix degrading enzymes such as matrix metalloproteinase (MMP) and cathepsins thereby tipping the scale towards matrix and cartilage degradation. Calcium and potassium play an important role in this process since modulation of non-selective cation channels such as transient receptor potential vanilloid 1 and 2 or calcium-activated potassium channel KCa3.1 influence SF invasiveness and cytokine production [6].

RASF not only invade into cartilage but can also spread arthritis to unaffected joints, as demonstrated in the implanted severe combined immunodeficiency mouse model [7]. Here, not only cartilage co-implanted with RASF was degraded but also contralateral implanted human cartilage without adjacent RASF. RASF migration to distant sites and transmigration through endothelial barriers were not prevented by anti-TNF therapy but were reduced using VCAM-1 antibodies [7]. RASF maintained their migrative and invasive phenotype even when injected 14 days before cartilage implantation [7]. One prerequisite for this is the ability of RASF to grow and survive under anchorage-free conditions. While lung or skin fibroblasts cannot grow without attachment, SF thrive because of autocrine/paracrine stimulation with growth factors like PDGF [8].

Neuroendocrine factors can alter SF function and this might be particularly important, as RA is characterized by an increase in sensory nerve fibers and a decrease in sympathetic fibers in joints and spleen [9]. This alters neurotransmitter/neuropeptide composition with direct effects on SF behavior. Furthermore, B- and T-lymphocyte responses are altered upon exposure to neuroendocrine stimuli, which also reflects on SF properties. Norepinephrine can stimulate IFN-γ production in T-cells during early experimental arthritis and this cytokine stimulates B-lymphocyte activating factor production in SF [10]. On the other hand, β-adrenergic stimulation leads to the generation of anti-inflammatory B-cells that produce IL-10 and this cytokine inhibits cartilage destruction by RASF [11]. Pro-inflammatory neuropeptides, such as substance P and calcitonin-gene related peptide, increase SF cytokine production by up-regulating transient receptor potential vanilloid 1 [12]. The excitatory neurotransmitter glutamate promotes inflammation by increasing MMP-2 and IL-6 expression, while dopamine, opioids and norepinephrine can decrease IL-6 and IL-8 production [9,13]. SF actively shape sympathetic and sensory innervation of the joints, since they are a major source of nerve growth factor that supports growth and sprouting of sensory and sympathetic nerve fibers and also enhances SF proliferation [14]. Under pro-inflammatory conditions, however, SF (together with macrophages) produce semaphorin 3C, a nerve repellent factor specific for sympathetic nerves [15]. Repulsion of sympathetic fibers leads to a disequilibrium between sensory and sympathetic innervation observed in RA joints [9]. Furthermore, RASF express axonal guidance receptor Roundabout-3 (ROBO3), which is modulated by its ligand, Slit3. Analog to neuronal cells, RASF migration and invasion is controlled by Roundabout-3 (ROBO3) and ligation with Slit3 abrogates these processes [16]. Although expression levels of Slit3 have not been investigated in RA, studies in other cell types revealed glucocorticoids and Toll-like receptors to be involved in Slit3 regulation [17].

Due to DNA modifications under pro-inflammatory conditions, SF retain their aggressive features even after initiation of therapy, which might explain continuing (albeit reduced) joint destruction. Histone deacetylase 5 expression is negatively regulated by pro-inflammatory cytokines, which is associated with increases in pro-inflammatory type I interferon signaling [18]. In addition, DNA methyltransferases are down-regulated by TNF and IL-1β, leading to DNA hypomethylation, which results in increased expression of respective genes. When treated with the hypomethylation drug 5-azacytidine, healthy SF acquire an irreversible, RA-like phenotype [19].

Current RA therapy often involves a daily regimen of glucocorticoids and this also impacts on function of SF. Glucocorticoids dampen pro-inflammatory signaling pathways and cytokine production in SF, but prolonged treatment also unveils detrimental effects. In SF from osteoarthritic patients (OASF), prednisolone induced the production of leptin, which has been implicated in supporting pro-inflammatory signaling by activating the transcription factor STAT3 and initiating IL-6 production [20].

An optimal therapeutic approach in RA might need to combine traditional anti-rheumatic drugs with a specific SF targeted therapy to dampen or even reverse their activated phenotype. In vivo, this reversion might occur by transforming a portion of SF into catecholamine-producing cells, which act anti-inflammatory, but factors pinpointing this transformation have not been identified yet. Tyrosine hydroxylase-positive catecholamine-producing SF appear in RA synovial tissue after loss of sympathetic nerve fibers. In vitro, brain-derived neurotrophic factor is an important factor for mesenychmal stem cell differentiation into catecholaminergic cells and brain-derived neurotrophic factor production by osteoarthritic patients (OASF) in vivo is correlated with the density of sympathetic nerve fibers in the joint, suggesting that ‘sensing’ the synovial sympathetic tone is required for catecholaminergic differentiation [21]. A possible therapeutic intervention would be ex vivo generation of catecholamine-producing, anti-inflammatory RASF isolated from synovial fluid or tissue [22]. Since RASFs have the ability to migrate to distant joints, these anti-inflammatory RASFs can be administered to the donor and might accumulate in affected joints. There, reprogrammed ‘regulatory’ RASF might dampen inflammation and joint destruction by the generation of dopamine, norepinephrine and other sympathetic neurotransmitters.

Other therapeutic avenues targeting RASF might be available because of the pro-inflammatory environment in the synovium. TNF up-regulates and sensitizes several receptors that are quiescent under basal conditions. Transient receptor potential channels are up-regulated by pro-inflammatory cytokines and these channels are required for calcium homeostasis and cell activation. Elevated intracellular calcium is necessary for the generation of pro-inflammatory mediators and blocking/desensitizing respective channels might prevent SF activation. A recent study identified transient receptor potential vanilloid 2 as a possible target in SF since its activation decreased MMP-2, MMP-3 and SF invasion [6].

In conclusion, SF have a strong pro-inflammatory and cartilage-degrading phenotype in RA. Their re-programming towards a regulatory phenotype by hormones/neurotransmitters or epigenetic means can be a future therapeutic approach. It is necessary to find these long-lasting therapies because joint destruction can continue, although anti-inflammatory therapies have already blocked cells of hematopoietic origin.

Financial & competing interests disclosure

The authors were supported by the Deutsche Forschungsgemeinschaft (DFG Research unit FOR 696, LO 1686/2-1). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.