Endoplasmic reticulum stress cooperates with Toll-like receptor ligation in driving activation of rheumatoid arthritis fibroblast-like synoviocytes

Genetic studies have shed light on our understanding of the causes of autoimmune diseases by identifying shared and unique risk loci among these diseases. However, in rheumatoid arthritis (RA), only a fraction of disease susceptibility can be explained by genetic variation [1], and the temporal link between the break of self-tolerance and development of clinical disease remains elusive because circulating autoantibodies are detectable long before the onset of arthritis [2]. In RA, stromal cells in the joint, fibroblast-like synoviocytes (FLS), exhibit an imprinted and epigenetically maintained aggressive phenotype, predisposing them to participate in an inflammatory positive feedback loop in response to the cues from the synovial environment [3]. Identifying local tissue conditions able to initiate and perpetuate the ensuing inflammatory cycle is therefore of critical importance to understanding and intervening in the disease process.

Endoplasmic reticulum (ER) stress is a common cellular response to many of the conditions RA FLS encounter in the inflamed synovium [4], and it occurs when the amount of newly synthesized proteins in the ER exceeds the organelle’s capacity to ensure their proper folding. The resulting accumulation of misfolded proteins in the ER triggers a set of signals collectively referred to as the unfolded protein response (UPR), aimed at relieving the burden by slowing down the global translation rate while increasing production of a selected set of proteins, particularly ER chaperones [5]. The UPR depends upon the triggering of inositol-requiring enzyme 1α (IRE1α), protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6, sensors embedded in the ER membrane, by unfolded protein aggregates in the lumen. In response, IRE1α homodimerizes and causes the unconventional splicing of X-box binding protein 1 (XBP1) messenger RNA (mRNA). This causes a frameshift mutation in XBP1, making it a powerful transcription factor instrumental in restoring homeostasis. Additional transcription factors are activated by the two other sensors [6].

Although primarily a safeguard for protein folding homeostasis, ER stress is tightly associated with immunological processes via crosstalk occurring between the UPR and inflammatory signaling pathways. For example, the decrease in translation rate caused by PERK activity limits expression of proteins with a short half-life, such as nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), resulting in enhanced activation of the nuclear factor (NF)-κB pathway [7]. Autophosphorylated IRE1α interacts with the tumor necrosis factor receptor-associated factor 2 adaptor molecule, facilitating activation of NF-κB and mitogen-activated protein (MAP) kinase pathways [8]. Transcription factors involved in ER stress can directly drive expression of inflammatory gene products such as interleukin (IL)-6 and tumor necrosis factor (TNF) [9], and elements of the UPR are necessary for maturation of several immune cell populations [10, 11]. Consequently, ER stress has been linked to a number of human disease conditions, including autoimmunity, where it has been postulated to drive inflammatory activation, act as the source of or as an adjuvant for autoantigens, or contribute to pathology by modulating apoptotic pathways [12, 13].

Despite this, understanding of the relevance of ER stress to pathology in RA is largely incomplete. Analysis of publicly available datasets of microarrays performed on synovial tissue has identified genes related to ER stress and protein processing in the ER as those most significantly differentiating between RA and osteoarthritis (OA) synovia, whereas no such difference was observed between OA and normal synovia [14]. Prominent staining for ER stress markers was observed throughout RA synovial tissue, particularly in the lining layer, indicating that these differences were unlikely to reflect changes in numbers of minor cell populations. A similar enhancement of ER stress and ER stress signaling to the nucleus in synovial fluid macrophages has been observed, and the ER chaperone binding immunoglobulin protein (BiP) is an important regulator of synovial angiogenesis, synoviocyte proliferation and survival, and disease severity in animal models of RA [14]. In experimental arthritis, strong expression of ER stress markers is observed during disease development in both synovial macrophages and fibroblasts [15]. Whereas myeloid-specific targeting of UPR pathways resulted in decreased cytokine expression and ameliorated disease in K/BxN serum-induced arthritis [16], studies involving RA FLS focus predominantly on changes in cellular viability and their potential consequences for synovial hyperplasia [17]. Unlike other cell types, RA FLS are resistant to apoptosis induced by ER stress, likely due to enhanced rates of autophagy and proteasomal activity [18, 19]. However, little is known about how ER stress changes the potential of FLS to directly modulate synovial inflammation, and recent studies have indicated that splicing of XBP1 may be associated with the activation of RA FLS by Toll-like receptor (TLR) signaling, IL-1β, and TNF [20]. The aim of this study was therefore to examine if ER stress could regulate inflammatory gene expression in RA FLS.

ER stress plays an important role in both physiological and pathological immune responses, and RA and OA synovia are distinguished by a strong UPR signature [14]. Similarly, macrophages isolated from RA patient synovial fluid were demonstrated by several groups to bear signs of ER stress, as compared with both peripheral blood monocyte-derived macrophages or macrophages isolated from OA patient synovial fluid [14, 16]. In the present study, we demonstrate that also stromal cells, FLS, are readily responsive to ER stress induction, but by itself this has little significant effect on cellular activation, nor does it affect viability. Instead, ER stress primes stromal cells for enhanced cytokine and chemokine production in the presence of other agonistic signals. The magnitude and range of this synergistic response are in stark contrast with the negligible changes in cytokine expression induced by thapsigargin alone, and they are a result of effects on transcriptional rates, to some extent, and primarily on mRNA decay rates of inflammatory genes.

In regard to cell survival, RA FLS were previously described as more resistant to such challenge than OA FLS, with altered expression of CHOP and synoviolin postulated as possible mechanisms [17, 18]. Similarly, the ER chaperone BiP has been identified as an important survival factor for stressed synoviocytes, and its expression regulates joint destruction in animal models [14]. On the other hand, inflammatory responses to ER stress in FLS have been scarcely studied so far. Contrary to freshly isolated synovial fluid macrophages, cultured RA FLS do not show signs of increased ER stress [20], although they upregulate UPR-related genes more readily than OA FLS in response to a variety of stimuli [14]. Analogously to similar observations in macrophages [9], a possible effect of TLR-dependent XBP1 activation on gene expression has been proposed in RA FLS [20]. However, following stimulation with LPS alone, we have observed only minor differences in the amount and fraction of XBP1 existing in the spliced form, indicating no significant changes in UPR signaling (data not shown).

Our data suggest that enhanced mRNA stability is a major contributor to the increased level of gene expression during ER stress. A growing number of reports underscore the importance of mRNA stability regulation during chronic synovitis in RA. In particular, Loupasakis et al. [27] recently demonstrated mRNA stabilization as a crucial factor shaping the FLS transcriptome during long-term exposure to TNF, with a strong influence on IL6, IL8, CCL2, PTGS2, and other genes with pathogenic potential.

Intriguingly, ER stress has long been known to impact the mRNA stability of certain genes via regulated IRE1α-dependent degradation [28]. In such cases, activated IRE1α was shown to splice not only XBP1 but also several other mRNAs, resulting in their accelerated decay. However, the idea that ER stress might conversely contribute to inflammation by stabilizing cytokine mRNA has not previously been explored. Regulation of cytokine expression through changes in mRNA stability depends primarily on the presence of adenylate- and uridylate-rich elements in their sequences. These are recognized by adenylate- and uridylate-rich element-binding proteins (ABPs) whose expression and activity are tightly regulated and can lead to both positive and negative regulation of mRNA half-life [29]. We have screened possible candidate ABPs, including BRF1 (ZFP36L1), BRF2 (ZFP36L2), AUF1 (HNRNPD), TTP (ZFP36), HuR (ELAVL1) and KHSRP, using small interfering RNA-mediated knockdown (data not shown), but we were unsuccessful in mimicking or significantly modulating the effects of combined LPS and thapsigargin stimulation by their independent targeting. Additionally, inhibition of conventional pathways involved in ABP-mediated decay [26], such as p38, ERK, and JNK, did not block a positive effect of ER stress on mRNA stability. These observations indicate that additional regulatory layers, such as microRNAs or components of nonsense-mediated decay, may be implicated.

The observation that ER stress regulates mRNA stability in DF is similarly novel, suggesting a shared mode of stromal cell response to suspected injury by preparing to mount a rapid inflammatory response if further danger signals appear in the environment. This may be relevant to rheumatic diseases other than RA characterized by skin involvement, such as psoriatic arthritis and systemic sclerosis. In this regard, the role of TLR ligands and ER stress in systemic sclerosis has been described extensively (reviewed in [30, 31]), and it will be of interest to determine whether ER stress-dependent regulation of gene expression contributes to the acquisition of the profibrotic phenotype in these patients.

Our inability to observe a similar effect of ER stress on mRNA stability in macrophages was surprising, given the available literature. For example, the IRE1α-XBP1 signaling pathway was shown to be a critical element of macrophage responses to TLR ligation [9], and myeloid-specific knockout of IRE1α ameliorated disease severity in the K/BxN serum-induced arthritis model [16]. Although the primary focus of these previous studies was the role of IRE1α during TLR stimulation alone, an enhancement of LPS-induced cytokine expression during ER stress in murine bone marrow-derived macrophages was noted, and a similar finding was observed in human macrophages [9]. The discrepancy in macrophage responses to ER stress between these studies and ours, where we also noted a sensitivity of macrophages to LPS and ER stress-induced apoptosis, may be a result of differences in tissue- and polarization-specific macrophage responses. In line with this, it was previously observed that resident and thioglycolate-elicited peritoneal macrophages show opposite patterns of regulation of CXCL1 during stimulation with LPS and thapsigargin [32]. Our results suggest that in RA synovial tissue, the IRE1α-XBP1 axis might contribute to macrophage responses to TLR signaling in the absence of induction of ER stress, whereas in stromal cells, TLR stimulation in the presence of ER stress amplifies cytokine and chemokine production.

Whereas a strong ER stress signature is a distinguishing feature of RA synovium, the understanding of its capacity to influence pathological processes was incomplete. Specifically in the case of stromal cells, the effects of ER stress reported in the literature that could be relevant in a disease setting are linked to the RA FLS intrinsic resistance to apoptosis and were not known to contain an inflammatory component. In our present study, however, we identify a novel regulatory mechanism relying on interactions between stromal cells, ER stress, and molecular danger signals with potential to profoundly affect the course of the locally developed inflammation. We propose a model in which cytokine transcription is initiated by an external trigger rather than by ER stress itself, with the latter being instead responsible for promoting mRNA stability. Combination of both inputs leads to significant augmentation of the overall response in stressed cells. Further characterization of this mechanism may lead to identification of molecular targets relevant for a range of immune-mediated inflammatory diseases characterized by synovial and connective tissue involvement.