In this study we demonstrate a novel link between ER stress and the innate immune response to microbial products that induce type I IFN. Specifically, UPR activation synergistically augments IFN-β induction by TLR3 and TLR4 agonists as well as cytoplasmic dsRNA. This effect is robust in that it occurs in multiple cell types, including primary macrophages, macrophage-like cell lines (RAW264.7), fibroblasts, and HEK293 cells. Synergy does not require autocrine IFN-α/β, but is dependent on XBP-1, implying a previously unrecognized role for this transcription factor. Moreover, we show that synergistic IFN-β induction occurs in rat macrophages expressing HLA-B27, an MHC class I allele that has been shown to misfold, and is strongly linked to spondyloarthritis. These results have implications for the pathogenesis of protein misfolding diseases.
Several lines of evidence support a critical role for XBP-1 in synergistic IFN-β induction. Genetic ablation or RNAi-mediated knockdown of XBP-1 mRNA prevents synergy, and XBP-1 splicing is dramatically increased during LPS stimulation of stressed B27-Tg macrophages. Furthermore, restoring expression of XBP-1s in XBP-1−/− cells permits synergy, and overexpression in RAW264.7 macrophages enhances IFN-β induction in response to LPS. While these data clearly implicate XBP-1s in the UPR component of synergistic IFN-β induction, it should be noted that overexpression of XBP-1s does not substitute entirely for ER stress, indicating that it is necessary but not sufficient.
XBP-1u encodes a truncated protein lacking the transactivation domain found in XBP-1s, and turns over rapidly during non-stress situations [20
]. However, there is evidence that XBP-1u protein can act as a dominant negative when forced to accumulate by inhibiting proteasomal degradation [37
], and as a negative feedback regulator of XBP-1s protein during the recovery phase of ER stress when it accumulates naturally [38
]. While our data imply that XBP-1s protein mediates synergistic upregulation of IFN-β, whether or not XBP-1u plays any role is not clear. In preliminary experiments we do not observe any inhibition of IFN-β expression or upregulation when XBP-1u is transiently overexpressed (unpublished observations). However, it is not possible to draw conclusions about effects of XBP-1u overexpression on synergistic upregulation since these experiments require UPR activation, and IRE1-mediated XBP-1u mRNA splicing is strongly activated under these conditions.
In macrophages undergoing a UPR, synergistic IFN-β induction was observed with activators of TLR3, TLR4, and MDA5, but not TLR2 or TLR9. TLR3/4 use the TRIF-dependent pathway to activate IRF3 via TBK-1 (Tank binding kinase 1) and induce IFN-β, whereas TLR2 signals solely through MyD88, and does not cause significant upregulation of type I IFN [39
]. MDA5 recognizes cytoplasmic dsRNA (poly(I:C)) and activates IRF3 via IPS-1 and TBK-1, suggesting that ER stress exerts its effect downstream of TRIF [10
]. TLR9 agonists (e.g. Type B CpG DNA) stimulate robust IFN-β induction in myeloid DCs and macrophages through MyD88 and IRF-1 independently of IRF3 or IRF7 [12
]. Lack of synergy with the TLR9 agonist is consistent with specificity of IFN-β synergy for the IRF3 activating pathways.
IFN-β expression is regulated at transcriptional and post-transcriptional levels. Transcriptional regulation is complex, requiring promoter access via deacetylation of histones and coordinated assembly of several transcription factors including IRF3, IRF7, CEBP/p300, AP-1 (ATF-2/c-Jun), and NF-κB [42
]. We have not found any XBP-1 binding sites in the IFN-β gene promoter region using TFSEARCH and the TRANSFAC database, suggesting that XBP-1 is unlikely to directly activate IFN-β gene transcription. Nevertheless, in preliminary experiments using HEK293 cells expressing CD14 and TLR4, we find some increased activation of an IFN-β promoter reporter construct with the combination of ER stress and LPS stimulation (unpublished observations). Another possibility is that UPR activation prolongs IFN-β mRNA stability, which is regulated by an AU-rich element in the 3′ untranslated region and sequences in the coding region [46
]. Further studies will be necessary to define the molecular mechanism of synergistic induction of IFN-β.
The sensitization of cells by ER stress to PRR agonists has implications for diseases where the UPR plays a role in pathogenesis. For example, ischemia-reperfusion, which results in inflammatory injury, is associated with induction of UPR target genes and XBP-1 splicing [48
]. In the liver, damage has been shown to occur through a TLR4-mediated IRF3-dependent mechanism implicating endogenous TLR4 ligands, and raising the possibility that synergistic IFN-β induction might contribute to ischemia reperfusion injury [50
]. The UPR is also activated in inflammatory myopathies, where there is evidence for an IFN-induced gene expression signature that may contribute to disease chronicity [51
]. In addition, infection with many viruses activates the UPR, which could enhance cytoplasmic dsRNA-induced type I IFN production through MDA5/RIG-I. It is noteworthy that Hepatitis C and human CMV have evolved mechanisms to inhibit XBP-1 activity [53
]. This has been hypothesized to inhibit viral protein degradation by ERAD, but it might also limit type I IFN production and thus be an important viral strategy for subverting the innate immune response.
Our demonstration that HLA-B27-induced UPR activation can enhance TLR-induced IFN-β has implications for the pathogenesis of SpA. In the rat model, inflammation begins in the gastrointestinal tract shortly after weaning and requires normal flora [55
], which may provide a rich source of TLR ligands. IFN-β has recently been shown to play a crucial role in promoting macrophage survival after TLR stimulation [56
]. Thus, we suggest that enhanced type I IFN production by HLA-B27-expressing macrophages could promote survival of activated macrophages via autocrine and paracrine effects, thus sustaining production of cytokines that promote T cell activation and IFN-γ synthesis [57
]. This in turn could contribute to a positive feedback loop where IFN-mediated upregulation of HLA-B27 further enhances misfolding, UPR activation, and type I IFN production.
It should be emphasized that the expression of HLA-B27 in transgenic rats surpasses normal levels found in humans carrying this allele. However, rat SpA is not a consequence of class I overexpression per se
, since HLA-B7 transgenic rats expressing comparable amounts of heavy chain remain healthy [24
]. There is evidence that the UPR is activated in cells from the synovial fluid of patients with spondyloarthritis [58
]. However, more studies will need to be done to determine the conditions leading to this response in cells from humans, and the role of HLA-B27 misfolding in disease pathogenesis.
In summary, we demonstrate that intracellular stress can magnify IFN-β induction in response to exogenous and endogenous danger signals recognized by Toll-like and viral dsRNA receptors. These results suggest an additional novel function for XBP-1 coupling UPR signaling to IFN-β induction, and support the idea that ER homeostasis may play an important role in influencing the production of inflammatory cytokines. A better understanding of how the various processes encompassed by the integrated stress response impact the immune system may aid in elucidating the pathogenesis of a variety of disease states.