Priming in response to pro-inflammatory cytokines is a feature of adult synovial but not dermal fibroblasts

Rheumatoid arthritis (RA), a chronic inflammatory disease of the joint, is one of the most common autoimmune diseases in the UK, affecting over 1% of the population [1]. In RA, high levels of pro-inflammatory cytokines are maintained within the joint, chemokine gradients continue to recruit leukocytes and the cells within the synovium are retained and are prevented from undergoing apoptosis. Further, the tissue-resident stromal cells become hyperplastic, leading to the growth of an invasive pannus, which narrows the joint space and degrades both articular cartilage and bone.

One of the predominant cell types of the synovial stroma is the fibroblast-like synoviocyte (FLS). It has become increasingly apparent that the FLS is crucial to normal and aberrant inflammatory responses in the joint [2‐4]. FLS can release large quantities of pro-inflammatory cytokines. IL-6, for example, is secreted at higher basal levels in FLS in RA than in osteoarthritis (OA) [5]. FLS in RA increase leukocyte adhesion to [6] and transmigration through [7] endothelial layers, and the high concentration of CXCL12 produced by FLS in RA restricts the egress of leukocytes from the joint [8]. Further, FLS release of granulocyte macrophage colony stimulating factor (GMCSF) keeps neutrophils alive for twice the normal span in vitro, and this growth factor is produced at higher levels by FLS in RA than in healthy controls [9‐11]. FLS in patients with RA have an increased rate of proliferation [12], are more resistant to apoptosis than FLS in their healthy counterparts [13], and also have increased invasive capabilities and matrix metalloproteinase (MMP) production [14]. Finally, the strongest evidence for the pathogenic role of FLS in RA comes from the experiments of Muller-Ladner et al., who showed that FLS in RA could be cultured in vitro and then implanted in mice with severe combined immunodeficiency (SCID) to induce RA-like disease in the absence of immune cells [15]. Thus, FLS in RA are now viewed as “imprinted aggressors” rather than the traditional “passive responders” [16].

It is well-known that inflammation does not passively end, but is actively switched off [17‐21]. It is therefore possible that chronic inflammatory diseases such as RA involve failure of mechanisms in the resolution of inflammation. The negative regulation of inflammation has been well-characterised in monocytes/macrophages (reviewed in [22]). Macrophages respond to a variety of inflammatory stimuli, both endogenous, such as TNFα and IL-1, and exogenous, such as lipopolysaccharide (LPS). Whilst these stimuli elicit strong inflammatory responses, they also induce anti-inflammatory pathways that curtail macrophage inflammation [19, 22, 23]. Negative regulation of inflammation occurs at a range of levels, both within and without the cell. Pro-inflammatory stimuli often elicit anti-inflammatory mechanisms to curtail their own action [22]. This is exemplified by the nuclear factor (NF)-κB-IκBα negative feedback loop. Once a stimulatory signal causes degradation of its inhibitor IκBα, NF-κB is free to translocate to the nucleus and facilitate transcription of pro-inflammatory mediator genes. Another gene also induced by NF-κB is NF-κB-inhibitor-α (Nfkbia), which encodes IκBα. Nascent IκBα shuttles NF-κB out of the nucleus to once again be held inactive, thereby curtailing the transcriptional response to stimulus [24, 25].

Despite the abundant literature on negative regulation of inflammation in macrophages, there is a comparative paucity of information on fibroblasts. Evidence from Lee et al. suggests that FLS in RA lack the inherent negative regulation seen in macrophages [26]. Rather, FLS continued to produce pro inflammatory mediators for the duration of incubation with TNFα, even after four days of stimulation. This paper also showed significantly lower expression of negative regulators such as A20-binding inhibitor of NFκB activation 3 (ABIN3), activating transcription factor 3 (ATF3), IL-1 receptor-associated kinase M (IRAK-M), and suppressor of cytokines signalling 3 (SOCS3) in FLS in RA compared to macrophages. The implication is that FLS are deficient in the negative feedback regulation of inflammation in RA. However, no comparison was made with FLS obtained from non-inflamed joints. A similar study showed that gingival fibroblasts (HGF) lack SOCS1, IRAKM and SH2 domain-containing inositol phosphatase 1 (SHIP1) proteins, which are all used in negative regulation of inflammation [27].

We sought to compare the inflammatory responses of FLS obtained from inflamed (RA) and non-inflamed joints, with the aim of elucidating which (if any) mechanisms are used by FLS to limit their inflammatory responses, and to test the hypothesis that such mechanism(s) would be aberrant or absent in FLS in RA. Experimental design has been based upon the model of endotoxin tolerance in macrophages, which has been extensively used to elucidate mechanisms of negative regulation of inflammatory responses in those cells [28‐31]. Exposure of macrophages to a single dose of LPS reprogrammes their responses to a second dose, such that expression of pro-inflammatory mediators is attenuated, whilst expression of anti-microbial products is spared [30, 31]. In a similar fashion, we exposed fibroblasts to two doses of TNFα, with an intervening rest period in the absence of the pro-inflammatory cytokine. We then compared the first and second responses. Contradictory to our initial hypothesis, we found FLS to augment their cytokine-induced IL-6 secretion when primed with either TNFα or IL-1α. We found this priming phenomenon to occur in FLS derived from both inflamed and non-inflamed joints, but not fibroblasts from adult skin. The augmented second response was gene-specific and transient. Sustained activation of NF-κB in response to the second stimulation may play a role in fibroblast priming.

LPS tolerance involves a range of epigenetic and signalling mechanisms that extensively reprogramme the responses of macrophages to re-stimulation, suppressing the induction of overtly pro-inflammatory genes whilst sparing those with anti-microbial properties [28‐31]. This may be viewed as a form of innate immune memory that guards against excessive inflammation without compromising defence against pathogens. The present study was devised to identify equivalent negative regulatory mechanisms by which synovial fibroblasts constrain their responses to repeated pro-inflammatory challenge. Surprisingly, we instead found that synovial fibroblasts were primed by a first exposure to either TNFα or IL-1α, such that they secreted significantly greater amounts of IL-6 when challenged a second time, after a 24-h rest period.

Fibroblasts derived from RA synovium retain an imprinted, invasive phenotype throughout prolonged culture in vitro [15, 16, 34]. Nevertheless, we found no evidence that the priming phenomenon was specific to RA-derived synovial fibroblasts. Similar priming responses were demonstrated by synovial fibroblasts from ostensibly healthy, non-inflamed joints of patients who did not have RA, and who did not later develop RA. A neonatal foreskin fibroblast line was primed by TNFα or IL-1α in a manner very similar to synovial fibroblasts, and was used in this study as a tool to explore mechanisms. In contrast, adult dermal fibroblasts (obtained from patients with OA and patients with RA at the time of joint surgery) showed no evidence of priming.

Synovial fibroblasts from joints affected by RA were previously shown to mount unremitting inflammatory responses, continuing to express IL-6 and other inflammatory mediators for at least four days in the presence of TNFα [26]. The same group also demonstrated priming by TNFα of synovial fibroblasts in RA, resulting in enhanced responses to later challenge with interferons [35]. In those studies no comparison was made with “normal” synovial fibroblasts. It is therefore possible that the properties described are characteristic of synovial fibroblasts as a whole, rather than acquired during the pathogenesis of RA. It is not a novel concept that fibroblasts differ in their patterns of gene expression and response to stimulation, according to the anatomical location from which they are obtained [11, 36‐38]. The important questions are why synovial fibroblasts should possess “inflammatory memory” [35], and whether such memory could play a role in the development of chronic joint disease.

One argument is that fibroblast responses to pro-inflammatory stimuli are dictated by their usual pattern of exposure to such stimuli [27]. In mucosal or dermal fibroblasts that are repeatedly exposed to pro-inflammatory insults, priming responses would be severely maladaptive. In contrast, fibroblasts from privileged sites such as the joint or the eye presumably seldom face sustained or repeated exposure to pro-inflammatory cytokines in healthy individuals, and may have evolved different responses to such danger signals. This hypothesis will be tested by investigating priming responses of fibroblasts from different anatomical locations of healthy and diseased individuals. Another possibility is suggested by the apparently gene-specific nature of the phenomenon, in which primed responses of IL-6 and CCL5 were enhanced, whereas that of IL-8 was not. Perhaps, as an inflammatory response evolves, differential priming allows the stroma to influence leukocyte recruitment and activation by modifying the profile of cytokines and chemokines expressed. This possibility is also under investigation.

Finally, the molecular basis of synovial fibroblast priming remains unknown. Increased expression of cytokine receptors is unlikely to provide an explanation, because of the gene-specific and signaling-pathway-specific effects described. Sustained activation of NF-κB has been implicated in the unremitting inflammatory response of synovial fibroblasts to prolonged TNFα exposure in RA [26]. Although we found NF-κB activity to decline after withdrawal of TNFα, the dynamics of activation in response to the second challenge were altered, with sustained nuclear localization of the RelA subunit and phosphorylation of its serine 536. It appears that the first exposure of FLS to TNFα induces a transient burst of IL6 gene expression, whereas the second exposure induces more sustained expression, dependent on prolonged activation of NF-κB. We speculate that priming influences the expression or function of negative feedback regulators required for the termination of the NF-κB activation signal.

We demonstrated here that inflammatory memory is a general property of synovial fibroblasts rather than a peculiarity of synovial fibroblasts in RA. We show that this phenomenon is transient, gene-specific rather than universal, and is accompanied by alterations of cytokine-induced signal transduction pathways. We hypothesize that the propensity of synovial fibroblasts to respond more strongly to re-stimulation contributes to the development of chronic joint inflammation.