Mol. Cells 2018; 41(8): 705-716
Published online July 30, 2018
https://doi.org/10.14348/molcells.2018.0241
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: sojs@dongguk.ac.kr
The endoplasmic reticulum (ER) is a critical organelle for protein synthesis, folding and modification, and lipid synthesis and calcium storage. Dysregulation of ER functions leads to the accumulation of misfolded- or unfolded-protein in the ER lumen, and this triggers the unfolded protein response (UPR), which restores ER homeostasis. The UPR is characterized by three distinct downstream signaling pathways that promote cell survival or apoptosis depending on the stressor, the intensity and duration of ER stress, and the cell type. Mammalian cells express the UPR transducers IRE1, PERK, and ATF6, which control transcriptional and translational responses to ER stress. Direct links between ER stress and immune responses are also evident, but the mechanisms by which UPR signaling cascades are coordinated with immunity remain unclear. This review discusses recent investigations of the roles of ER stress in immune responses that lead to differentiation, maturation, and cytokine expression in immune cells. Further understanding of how ER stress contributes to the pathogenesis of immune disorders will facilitate the development of novel therapies that target UPR pathways.
Keywords ER stress, immune response, inositol requiring enzyme 1 (IRE1), unfolded protein response (UPR), X-box binding protein 1 (XBP1)
Secreted or transmembrane proteins are synthesized, folded, and modified in the endoplasmic reticulum (ER), which also plays crucial roles in lipid biosynthesis, calcium storage, and detoxification (Braakman and Bulleid, 2011; Chen et al., 2010; Marisa et al., 2018)(Fig. 1). Upon entry into the ER lumen, newly synthesized polypeptides are folded and modified to ensure precise conformations and function. Impairments of this process lead to ER stress due to the accumulation of misfolded- or unfolded-proteins in the ER (Byrd and Brewer, 2012). ER homeostasis is sensitive to multiple physiological and pathological processes, and perturbations have been associated with excessive protein synthesis, abnormal ER calcium content, lipid overload, hypoxia, oxidative stress, iron imbalance, nutrient deprivation, cancer, and infection (Kaufman et al., 2002; Marciniak and Ron, 2006; Martins et al., 2016).
Protein quality control is monitored by a complex system that modulates ER function to resolve ER stress and dysfunction. This protective mechanism is known as the unfolded protein response (UPR) and is an adaptive cellular response that elicits specific intracellular signaling pathways to protect against ER stress (Hotamisligil, 2010)(Fig. 1). The UPR restores ER homeostasis by attenuating protein synthesis or by inducing the expression of various genes encoding molecular chaperones and protein processing enzymes that promote protein folding and post-translational modification. Proteins that remain improperly folded are degraded by the proteasome through a pathway known as ER-associated degradation (ERAD)(Adachi et al., 2008). The UPR also enhances ER capacity by promoting expansions of the ER membrane through increased expression of genes involved in lipid metabolism (Lee et al., 2008; Volmer and Ron, 2015; Walter and Ron, 2011). Finally, when these mechanisms fail to resolve chronic or overwhelming ER stress, the UPR stimulates apoptosis (Hetz, 2012).
Accumulating evidence suggests that ER stress is a major contributor to the pathogenesis of several diseases, including obesity, diabetes, cancer, neurodegenerative disorders, and inflammatory and autoimmune diseases, and can increase the severity of these (Hetz et al., 2013; Hotamisligil, 2010; Marciniak and Ron, 2006; Oyadomari et al., 2008; Özcan et al., 2004; Wang and Kaufman, 2012). Whereas sustained ER stress has been associated with the expression of pro-inflammatory cytokines and potential induction of inflammatory responses, causal relationships between immunity and ER stress remain an incipient area of interest (Smith, 2018). Herein, I discuss the underlying mechanisms by which ER stress affects immune responses, with a focus on differentiation and cytokine expression in immune cells and the etiology of immune disorders.
ER stress is recognized by the ER-resident proteins inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6) (Bettigole and Glimcher, 2015)(Fig. 2). These transmembrane proteins are major transducers of the UPR in mammals and mediate multiple distinct signals from the ER to the cytosol. As a UPR initiating response to perturbations of protein folding, the ER chaperone Grp78 dissociates from the ER luminal domain and binds to the unfolded protein to prevent aggregation (Bertolotti et al., 2000; Ron and Walter, 2007; Shen et al., 2002). Various pharmacological agents have been shown to disturb ER functions. Among these, tunicamycin (Tun) prevents N-linked glycosylation, thapsigargin (Tg) depletes Ca2+ from the ER by inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump, dithiothreitol (DTT) interferes with the formation of protein disulfide bonds, and MG132 inhibits the proteasome (Oslowski and Urano, 2011)(Fig. 1).
The proteins IRE1 and PERK are central type-I transmembrane proteins of the UPR. IRE1 has Ser/Thr kinase and endoribonuclease (RNase) domains in the cytoplasmic region, and comparisons of yeast IRE1 with the mammalian homologs IRE1α and IRE1β reveal a high level of conservation (Fig. 2A). Whereas IRE1α is expressed in most cells and tissues, IRE1β expression is primarily restricted to epithelial cells of the gastrointestinal tract (Kaufman et al., 2002). The ER-luminal domain of the yeast IRE1 senses misfolded proteins following dissociation of Grp78 and via direct interactions with unfolded proteins, but does not bind to unfolded proteins in mammals (Oikawa et al., 2009). Subsequent IRE1 oligomerization induces autophosphorylation in the kinase domain, and the resulting RNase activity of IRE1 leads to unconventional splicing of yeast HAC1 or mammalian X-box binding protein 1 (XBP1) mRNA (Yoshida et al., 2001). XBP1 mRNA encodes the short unstable protein “unspliced form XBP1” (267-amino acids), and IRE1-mediated excision of 26 intronic nucleotides produces the spliced form that encodes “XBP1s” (371-amino acids). Due to the resulting translational frame-shift, this protein contains a novel carboxyl terminus (Calfon et al., 2002; Lee et al., 2003). Specifically, XBP1s contains a basic leucine zipper (bZIP) domain in the C-terminus and acts as a transcription factor for UPR-related genes, including the ER chaperones ERdj4, HEDJ, Grp58, and p58IPK, the ERAD components EDEM, Herp, and Hrd1, the protein disulfide isomerase PDI-P5, and the ER translocon Sec61α (Byrd and Brewer, 2012; Lee et al., 2003; Yamamoto et al., 2008; Yoshida et al., 2001). XBP1s binds to the UPR element (UPRE) containing the consensus sequence TGACGTGG/A and regulates the transcription of target genes in a cell type- and condition-specific manner (Yamamoto et al., 2004). These genes play distinct roles in various physiological processes, including secretion, lipid metabolism, glucose homeostasis, and inflammation. (Acosta-Alvear et al., 2007; Byrd and Brewer, 2012).
PERK is a protein Ser/Thr kinase with a stress-sensing domain in the ER lumen (Bertolotti et al., 2000) (Fig. 2B). Similar to IRE1, Grp78 dissociates from the luminal domain of PERK under ER stress, which activates oligomerization of PERK and autophosphorylation in the cytosolic kinase domain (Ron and Walter, 2007). PERK then phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α) at Serine 51, leading to general inhibition of protein translation. Although eIF2α phosphorylation decreases global protein synthesis and reduces protein uptake into the ER, it induces the translation of activating transcription factor 4 (ATF4), which controls specific transcriptional programs of the UPR (Walter and Ron, 2011). The 5′-untranslated region (5′-UTR) of ATF4 has two short upstream open reading frames (uORF) that prevent ATF4 translation in unstressed cells. However, eIF2α phosphorylation induces ribosome bypass of inhibitory uORF, resulting in increased translation from the ATF4 ORF (Lu et al., 2004). ATF4 activates the transcription of target genes encoding C/EBP homologous protein (CHOP) and growth arrest and DNA damage-inducible protein 34 (GADD34) (Fawcett et al., 1999; Ma and Hendershot, 2003; Ma et al., 2002). As a negative feedback mechanism, GADD34 then associates with protein phosphatase 1 (PP1) and promotes dephosphorylation of eIF2α to restore protein synthesis upon stress relief (Harding et al., 2009; Novoa et al., 2003). When ER stress remains unmitigated, CHOP induces apoptosis via the genes encoding death receptor 5 (DR5) and Bim (Puthalakath et al., 2007; Raffaella and Cristina, 2015; Yamaguchi and Wang, 2004; Zinszner et al., 1998). DR5 induces extrinsic apoptosis pathways by recruiting the adaptor protein FADD, which activates caspase-8 (Raffaella and Cristina, 2015). Concomitantly, the pro-apoptotic protein Bim activates Bax and Bak and induces permeabilization of the mitochondrial membrane to release cytochrome c (Urra et al., 2013).
ATF6 is a type-II ER transmembrane protein that contains a bZIP domain in the cytosol and a stress-sensing domain in ER lumen (Adachi et al., 2008)(Fig. 2C). Under conditions of protein homeostasis, ATF6 is maintained in the ER through interactions with Grp78, but is translocated from the ER to the Golgi apparatus under conditions of ER stress, where it is sequentially cleaved at two sites by site-1 (S1P) and site-2 (S2P) proteases (Ye et al., 2000). The released aminoterminal of ATF6 (ATF6-N) then migrates to the nucleus and binds to the ER stress response element (ERSE) containing the consensus sequence CCAAT-N9-CCACG to activate genes encoding ER chaperones, ERAD components, and XBP1 (Chen et al., 2010; Yamamoto et al., 2004; Yoshida et al., 2001).
ER stress profoundly affects innate and adaptive immune responses and is associated with autoimmune and inflammatory disorders such as diabetes, atherosclerosis, myositis, and inflammatory bowel disease (Heazlewood et al., 2008; Kaser et al., 2008; Vattemi et al., 2004; Zhang and Kaufman, 2008). Many recent studies show immune roles of the UPR via direct crosstalk between ER stress-induced signaling pathways and immune responses (Bettigole and Glimcher, 2015; Cao et al., 2016; Garg et al., 2012; Janssens et al., 2014; Zhang and Kaufman, 2008).
Increased expression of pro-inflammatory cytokines during ER stress suggests roles of the UPR in inflammatory disorders (Smith, 2018; Wheeler et al., 2008) (Fig. 3A). In agreement, several studies suggest that ER stress induces inflammatory responses by activating UPR transcription factors such as XBP1s, ATF6, and cAMP response element-binding protein H (CREBH) (Kaser et al., 2008; Shkoda et al., 2007; Zhang et al., 2006). A missense mutation in the
Accumulating evidence suggests that ER stress activates inflammatory signaling cascades through the interaction between UPR components and canonical cytokine-regulatory transcription factors (Smith et al., 2008)(Fig. 3B). ER stress reportedly activates NF-κB signaling, and this was considered a mechanism for inducing inflammatory responses (Deng et al., 2004; Hu et al., 2006). ER stress induced the formation of a complex between IRE1α and IκB kinase (IKK) through the adaptor protein TNF-α receptor associated factor 2 (TRAF2), thus promoting TNF-α production by enhancing IκBα degradation and NF-κB activation (Hu et al., 2006). In addition, NF-κB was activated by eIF2α phosphorylation, which induces translational suppression of IκBα (Deng et al., 2004). Following binding of TRAF2 to IRE1 cytoplasmic regions, IRE1α and IRE1β activated c-Jun N-terminal kinase (JNK) (Urano et al., 2000). These data were also confirmed in XBP1-deficient small intestines, with IRE1α hyperactivation and increased phosphorylation of JNK (Kaser et al., 2008). Although these studies collectively indicate immunological roles of the UPR, further studies are required to characterize and identify the related regulatory mechanisms and signaling molecules.
Several studies show that inflammation augments ER stress responses (Fig. 3C). In particular, injections of pro-inflammatory cytokines such as IL-1β and IL-6, induced ER stress and activated the UPR in the livers of mice (Zhang et al., 2006). The inflammatory cytokine TNF-α also increased eIF2α phosphorylation and XBP1s expression by elevating reactive oxygen species (ROS) levels in L929 murine fibrosarcoma cells (Xue et al., 2005). The immunostimulant lipopolysaccharide (LPS) increases the production of inflammatory cytokines, and intraperitoneal injections of LPS into mice activated the transcription of ER stress-related genes, including
Because the UPR is required for ER expansion and protein secretion, it is essential for development, differentiation, and the precise function of highly secretory cells, including pancreatic β cells, exocrine acinar cells, hepatocytes, and salivary gland epithelial cells (Bettigole and Glimcher, 2015; Harding et al., 2001; Lee et al., 2005; Lipson et al., 2006; Marisa et al., 2018; Reimold et al., 2000; Rutkowski and Hegde, 2010)(Fig. 4A). The UPR also has crucial roles in the development of immune cells, such as plasma cells, dendritic cells (DCs), and eosinophils (Bettigole et al., 2015; Iwakoshi et al., 2007; Lee et al., 2003; Reimold et al., 2001)(Fig. 4B). Many recent studies of various immune cell types show roles of ER stress in immune processes, including differentiation, immune activation, and cytokine expression.
Depending on microenvironments, naive CD4+ T cells differentiate into one of several lineages of helper T (Th) cells, including T helper type 1 (Th1), Th2, Th9, Th17, and regulatory T cells (Tregs) (Stadhouders et al., 2018). Effector T cell subsets secrete distinguishing cytokines and coordinately regulate different aspects of immune responses. Potentially, the UPR plays fundamental roles in T cell differentiation upon antigen recognition. This hypothesis is supported by observations of activated ER stress responses during differentiation of Th2 cells, and the PERK-eIF2α axis has been implicated in this process (Scheu et al., 2006). Specifically, primed Th2 cells showed increased phosphorylation of eIF2α and upregulation of ATF4 and its target genes
A recent study showed that ER stress responses are important for CD4+ T cell activation (Thaxton et al., 2017). In their study, T cell receptor (TCR) ligation-mediated ER stress increased Grp94 expression in CD4+ T cells, whereas deletion of Grp94 resulted in a critical activation defect. In addition, Grp94-deficient T cells showed impaired Ca2+ mobilization upon TCR engagement and failed to accomplish activation-induced glycolysis. ER molecular chaperone Grp94, upregulated by ER stress, has diverse immunological activities (Ansa-Addo et al., 2016).
The effects of ER stress on differentiation have been investigated in Tregs that maintain tolerance to self-antigens and prevent autoimmune disease (Franco et al., 2010). Specifically, T cells from patients with Kawasaki disease produced high levels of the anti-inflammatory cytokines IL-10 and TGF-β, and Foxp3, which is a critical transcription factor for Tregs (Franco et al., 2010). Treatments of these cells with thapsigargin induced the expression of the ER stress-response genes
In the presence of pathogens, CD8+ cytotoxic T cells secrete various cytokines and cytotoxic molecules to eliminate infected cells. In a study of CD8+ T cells by Kamimura and Bevan, the IRE1–XBP1 pathway was activated by acute infection and was required for differentiation into effector T cells (Kamimura and Bevan, 2008). These investigators also showed that viral or bacterial infection induces IRE1-mediated splicing and increases XBP1s mRNA expression in Ag-specific CD8+ T cells (Kamimura and Bevan, 2008). Finally, splicing of XBP1 mRNA was enriched in terminally differentiated effector cells expressing killer cell lectin-like receptor G1 (KLRG1), whereas XBP1-deficiencies in CD8+ T cells decreased the proportion of KLRG1high effector cells.
The physiological importance of the UPR was first described in B cells that were differentiating into plasma cells. In these studies, the UPR was activated during differentiation of B cells, and XBP1s was deemed essential for antibody production from plasma cells (Gass et al., 2002; Reimold et al., 2001; Shaffer et al., 2004; Todd et al., 2009). Accordingly, XBP1s induced ER expansion in plasma cells, therefore accommodating the synthesis of large amounts of immunoglobulin (Shaffer et al., 2004). Whereas XBP1-deficient B cells developed normally and formed germinal centers upon activation, they failed to secrete immunoglobulins (Reimold et al., 2001; Taubenheim et al., 2012). As a transcription factor, Blimp-1 necessary for plasma cell differentiation, and Blimp-1-deficient B cells failed to activate transcription of most plasma cell-related genes, including
ER stress also upregulated the production of pro-inflammatory cytokines in B cells, and expression of Ag with the ER-retention motif KDEL in B cells induced the ER stress-responsive genes
Macrophages are crucial effector cells of the innate immune system that phagocytose pathogens and produce various inflammatory mediators (Hirayama et al., 2018). XBP1s is a positive regulator of toll-like receptor (TLR) responses in macrophages, and TLR2 and TLR4 activated the IRE1α–XBP1s axis, but not the PERK or ATF6α pathways (Martinon et al., 2010). Moreover, XBP1s was required to produce pro-inflammatory cytokines, such as IL-6, TNF, and INF-β. TLR-mediated splicing of XBP1 mRNA was dependent on signaling via TIRAP, MyD88, TRIF, TRAF6, and NADPH oxidase 2 (NOX2) as a TRAF-bound enzyme complex. Under condition of XBP1-deficiency, IL-6 production was reduced in macrophages, and the bacterial burden was increased in mice infected with an intracellular pathogen. Conversely, overexpression of XPB1s or thapsigargin treatments increased LPS-stimulated IFN-β production in RAW267.4 cells (Smith et al., 2008).
Prolonged ischemia induces the expression of pro-inflammatory cytokines in liver Kupffer cells, primarily through ER stress-activated ATF6 (Rao et al., 2014). Accordingly, knockdown of ATF6 in macrophages suppressed the expression of TNF-α and IL-6 by limiting NF-κB activity. In agreement, alleviation of ER stress using the chemical chaperone 4-phenylbutyrate (4-PBA) or ATF6 siRNA led to decreased inflammatory responses in Kupffer cells against ischemia-reperfusion.
DCs are professional antigen-presenting cells, and developmental and survival roles of XBP1s have been demonstrated in these cells (Iwakoshi et al., 2007). In agreement, XBP1s expression was greater in plasmacytoid DCs than in immature conventional DCs, and XBP1-deficient chimeric mice had decreased numbers of conventional and plasmacytoid DCs. Under conditions of ER stress, treatment with TLR agonists significantly enhanced IL-23p19 expression in DCs (Goodall et al., 2010). The gene
A recent study associated tumor microenvironments with ER stress in DCs, and showed that XBP1s is important for the suppression of anti-tumor immunity (Cubillos-Ruiz et al., 2015). Specifically, XBP1s promoted the triglyceride biosynthetic program in DCs, which led to abnormal lipid accumulation and impaired antigen presentation. Conversely, DC-specific deletion of XBP1 restored immunostimulatory activities and increased anti-tumor responses.
Several recent studies show that IRE1 recognizes unspliced XBP1 mRNA and various other mRNAs as substrates to be degraded, and the associated process is called regulated IRE1-dependent decay (RIDD) (Coelho and Domingos, 2014; Hollien and Weissman, 2006; Hollien et al., 2009)(Fig. 5). Because IRE1 mediates the degradation of mRNAs encoding ER-targeted proteins, RIDD was originally thought to reduce ER protein loads during ER stress (Hollien and Weissman, 2006). In mammalian cells, RIDD targets have specific motifs with the consensus sequence CUGCAG and secondary structures that are similar to the stem-loop of XBP1 mRNA (Moore and Hollien, 2015; Oikawa et al., 2010; So et al., 2015)(Fig. 5A). Emerging evidence indicates that RIDD targets various mRNAs and therefore has diverse physiological functions, particularly in drug and lipid metabolism, and ER homeostasis in goblet cells and pancreatic β-cells (Han et al., 2009; Hur et al., 2012; Iqbal et al., 2008; Lipson et al., 2008; So et al., 2012; Tsuru et al., 2013)(Fig. 5B).
RIDD has also been implicated in the regulation of immune responses, with critical functions in the cross-presentation of cell-derived antigens via MHC class I between CD8+ T cells and CD8α+ conventional DCs (cDCs) (Osorio et al., 2014). Deletion of XBP1 in CD8α+ cDCs resulted in hyperactivation of IRE1α, and the RNase activity of IRE1α limited the expression of CD18 integrins and components of the MHC class I machinery by degrading
Several miRNAs have been identified as RIDD targets, and these reportedly increase the expression of other mRNAs during ER stress (Upton et al., 2012). IRE1α improved the stability of thioredoxin-interacting protein (TXNIP) mRNA by inducing cleavage of miR17, which destabilizes TXNIP (Lerner et al., 2012). Consequent augmentation of TXNIP likely activates the NLRP3 inflammasome, thus inducing caspase-1 cleavage and IL-1β secretion, and promoting inflammation and programmed cell death.
The ER is an essential organelle for protein homeostasis, and perturbations of ER homeostasis by physiological or pathological stressors cause accumulations of misfolded proteins in the ER. ER stress-induced UPR restores ER homeostasis through dynamic intracellular signaling pathways but gains its relationship with various diseases by regulating cell-specific functions. Much remains unknown regarding the impacts of ER stress on immune responses, although recent studies suggest that ER stress has specific immune regulatory activities in specialized cells and tissues. These studies collectively suggest that the UPR is a fundamental mediator of the pathogenesis of inflammatory disorders. In line with this suggestion, ER stress is known to induce inflammatory responses through UPR signaling, and immune cells are extensively dependent on the signaling branches of the UPR to perform their specific functions. Improved understanding of the molecular mechanisms underlying ER stress-regulated immune responses may lead to the discovery of new therapeutic targets for immune disorders. However, investigations of the UPR branches and their associated molecules in specific immune cells and under specific immune disorders remain challenging.
Mol. Cells 2018; 41(8): 705-716
Published online August 31, 2018 https://doi.org/10.14348/molcells.2018.0241
Copyright © The Korean Society for Molecular and Cellular Biology.
Jae-Seon So
Department of Medical Biotechnology, Dongguk University Gyeongju, Gyeongju 38066, Korea
Correspondence to:*Correspondence: sojs@dongguk.ac.kr
The endoplasmic reticulum (ER) is a critical organelle for protein synthesis, folding and modification, and lipid synthesis and calcium storage. Dysregulation of ER functions leads to the accumulation of misfolded- or unfolded-protein in the ER lumen, and this triggers the unfolded protein response (UPR), which restores ER homeostasis. The UPR is characterized by three distinct downstream signaling pathways that promote cell survival or apoptosis depending on the stressor, the intensity and duration of ER stress, and the cell type. Mammalian cells express the UPR transducers IRE1, PERK, and ATF6, which control transcriptional and translational responses to ER stress. Direct links between ER stress and immune responses are also evident, but the mechanisms by which UPR signaling cascades are coordinated with immunity remain unclear. This review discusses recent investigations of the roles of ER stress in immune responses that lead to differentiation, maturation, and cytokine expression in immune cells. Further understanding of how ER stress contributes to the pathogenesis of immune disorders will facilitate the development of novel therapies that target UPR pathways.
Keywords: ER stress, immune response, inositol requiring enzyme 1 (IRE1), unfolded protein response (UPR), X-box binding protein 1 (XBP1)
Secreted or transmembrane proteins are synthesized, folded, and modified in the endoplasmic reticulum (ER), which also plays crucial roles in lipid biosynthesis, calcium storage, and detoxification (Braakman and Bulleid, 2011; Chen et al., 2010; Marisa et al., 2018)(Fig. 1). Upon entry into the ER lumen, newly synthesized polypeptides are folded and modified to ensure precise conformations and function. Impairments of this process lead to ER stress due to the accumulation of misfolded- or unfolded-proteins in the ER (Byrd and Brewer, 2012). ER homeostasis is sensitive to multiple physiological and pathological processes, and perturbations have been associated with excessive protein synthesis, abnormal ER calcium content, lipid overload, hypoxia, oxidative stress, iron imbalance, nutrient deprivation, cancer, and infection (Kaufman et al., 2002; Marciniak and Ron, 2006; Martins et al., 2016).
Protein quality control is monitored by a complex system that modulates ER function to resolve ER stress and dysfunction. This protective mechanism is known as the unfolded protein response (UPR) and is an adaptive cellular response that elicits specific intracellular signaling pathways to protect against ER stress (Hotamisligil, 2010)(Fig. 1). The UPR restores ER homeostasis by attenuating protein synthesis or by inducing the expression of various genes encoding molecular chaperones and protein processing enzymes that promote protein folding and post-translational modification. Proteins that remain improperly folded are degraded by the proteasome through a pathway known as ER-associated degradation (ERAD)(Adachi et al., 2008). The UPR also enhances ER capacity by promoting expansions of the ER membrane through increased expression of genes involved in lipid metabolism (Lee et al., 2008; Volmer and Ron, 2015; Walter and Ron, 2011). Finally, when these mechanisms fail to resolve chronic or overwhelming ER stress, the UPR stimulates apoptosis (Hetz, 2012).
Accumulating evidence suggests that ER stress is a major contributor to the pathogenesis of several diseases, including obesity, diabetes, cancer, neurodegenerative disorders, and inflammatory and autoimmune diseases, and can increase the severity of these (Hetz et al., 2013; Hotamisligil, 2010; Marciniak and Ron, 2006; Oyadomari et al., 2008; Özcan et al., 2004; Wang and Kaufman, 2012). Whereas sustained ER stress has been associated with the expression of pro-inflammatory cytokines and potential induction of inflammatory responses, causal relationships between immunity and ER stress remain an incipient area of interest (Smith, 2018). Herein, I discuss the underlying mechanisms by which ER stress affects immune responses, with a focus on differentiation and cytokine expression in immune cells and the etiology of immune disorders.
ER stress is recognized by the ER-resident proteins inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6) (Bettigole and Glimcher, 2015)(Fig. 2). These transmembrane proteins are major transducers of the UPR in mammals and mediate multiple distinct signals from the ER to the cytosol. As a UPR initiating response to perturbations of protein folding, the ER chaperone Grp78 dissociates from the ER luminal domain and binds to the unfolded protein to prevent aggregation (Bertolotti et al., 2000; Ron and Walter, 2007; Shen et al., 2002). Various pharmacological agents have been shown to disturb ER functions. Among these, tunicamycin (Tun) prevents N-linked glycosylation, thapsigargin (Tg) depletes Ca2+ from the ER by inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump, dithiothreitol (DTT) interferes with the formation of protein disulfide bonds, and MG132 inhibits the proteasome (Oslowski and Urano, 2011)(Fig. 1).
The proteins IRE1 and PERK are central type-I transmembrane proteins of the UPR. IRE1 has Ser/Thr kinase and endoribonuclease (RNase) domains in the cytoplasmic region, and comparisons of yeast IRE1 with the mammalian homologs IRE1α and IRE1β reveal a high level of conservation (Fig. 2A). Whereas IRE1α is expressed in most cells and tissues, IRE1β expression is primarily restricted to epithelial cells of the gastrointestinal tract (Kaufman et al., 2002). The ER-luminal domain of the yeast IRE1 senses misfolded proteins following dissociation of Grp78 and via direct interactions with unfolded proteins, but does not bind to unfolded proteins in mammals (Oikawa et al., 2009). Subsequent IRE1 oligomerization induces autophosphorylation in the kinase domain, and the resulting RNase activity of IRE1 leads to unconventional splicing of yeast HAC1 or mammalian X-box binding protein 1 (XBP1) mRNA (Yoshida et al., 2001). XBP1 mRNA encodes the short unstable protein “unspliced form XBP1” (267-amino acids), and IRE1-mediated excision of 26 intronic nucleotides produces the spliced form that encodes “XBP1s” (371-amino acids). Due to the resulting translational frame-shift, this protein contains a novel carboxyl terminus (Calfon et al., 2002; Lee et al., 2003). Specifically, XBP1s contains a basic leucine zipper (bZIP) domain in the C-terminus and acts as a transcription factor for UPR-related genes, including the ER chaperones ERdj4, HEDJ, Grp58, and p58IPK, the ERAD components EDEM, Herp, and Hrd1, the protein disulfide isomerase PDI-P5, and the ER translocon Sec61α (Byrd and Brewer, 2012; Lee et al., 2003; Yamamoto et al., 2008; Yoshida et al., 2001). XBP1s binds to the UPR element (UPRE) containing the consensus sequence TGACGTGG/A and regulates the transcription of target genes in a cell type- and condition-specific manner (Yamamoto et al., 2004). These genes play distinct roles in various physiological processes, including secretion, lipid metabolism, glucose homeostasis, and inflammation. (Acosta-Alvear et al., 2007; Byrd and Brewer, 2012).
PERK is a protein Ser/Thr kinase with a stress-sensing domain in the ER lumen (Bertolotti et al., 2000) (Fig. 2B). Similar to IRE1, Grp78 dissociates from the luminal domain of PERK under ER stress, which activates oligomerization of PERK and autophosphorylation in the cytosolic kinase domain (Ron and Walter, 2007). PERK then phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α) at Serine 51, leading to general inhibition of protein translation. Although eIF2α phosphorylation decreases global protein synthesis and reduces protein uptake into the ER, it induces the translation of activating transcription factor 4 (ATF4), which controls specific transcriptional programs of the UPR (Walter and Ron, 2011). The 5′-untranslated region (5′-UTR) of ATF4 has two short upstream open reading frames (uORF) that prevent ATF4 translation in unstressed cells. However, eIF2α phosphorylation induces ribosome bypass of inhibitory uORF, resulting in increased translation from the ATF4 ORF (Lu et al., 2004). ATF4 activates the transcription of target genes encoding C/EBP homologous protein (CHOP) and growth arrest and DNA damage-inducible protein 34 (GADD34) (Fawcett et al., 1999; Ma and Hendershot, 2003; Ma et al., 2002). As a negative feedback mechanism, GADD34 then associates with protein phosphatase 1 (PP1) and promotes dephosphorylation of eIF2α to restore protein synthesis upon stress relief (Harding et al., 2009; Novoa et al., 2003). When ER stress remains unmitigated, CHOP induces apoptosis via the genes encoding death receptor 5 (DR5) and Bim (Puthalakath et al., 2007; Raffaella and Cristina, 2015; Yamaguchi and Wang, 2004; Zinszner et al., 1998). DR5 induces extrinsic apoptosis pathways by recruiting the adaptor protein FADD, which activates caspase-8 (Raffaella and Cristina, 2015). Concomitantly, the pro-apoptotic protein Bim activates Bax and Bak and induces permeabilization of the mitochondrial membrane to release cytochrome c (Urra et al., 2013).
ATF6 is a type-II ER transmembrane protein that contains a bZIP domain in the cytosol and a stress-sensing domain in ER lumen (Adachi et al., 2008)(Fig. 2C). Under conditions of protein homeostasis, ATF6 is maintained in the ER through interactions with Grp78, but is translocated from the ER to the Golgi apparatus under conditions of ER stress, where it is sequentially cleaved at two sites by site-1 (S1P) and site-2 (S2P) proteases (Ye et al., 2000). The released aminoterminal of ATF6 (ATF6-N) then migrates to the nucleus and binds to the ER stress response element (ERSE) containing the consensus sequence CCAAT-N9-CCACG to activate genes encoding ER chaperones, ERAD components, and XBP1 (Chen et al., 2010; Yamamoto et al., 2004; Yoshida et al., 2001).
ER stress profoundly affects innate and adaptive immune responses and is associated with autoimmune and inflammatory disorders such as diabetes, atherosclerosis, myositis, and inflammatory bowel disease (Heazlewood et al., 2008; Kaser et al., 2008; Vattemi et al., 2004; Zhang and Kaufman, 2008). Many recent studies show immune roles of the UPR via direct crosstalk between ER stress-induced signaling pathways and immune responses (Bettigole and Glimcher, 2015; Cao et al., 2016; Garg et al., 2012; Janssens et al., 2014; Zhang and Kaufman, 2008).
Increased expression of pro-inflammatory cytokines during ER stress suggests roles of the UPR in inflammatory disorders (Smith, 2018; Wheeler et al., 2008) (Fig. 3A). In agreement, several studies suggest that ER stress induces inflammatory responses by activating UPR transcription factors such as XBP1s, ATF6, and cAMP response element-binding protein H (CREBH) (Kaser et al., 2008; Shkoda et al., 2007; Zhang et al., 2006). A missense mutation in the
Accumulating evidence suggests that ER stress activates inflammatory signaling cascades through the interaction between UPR components and canonical cytokine-regulatory transcription factors (Smith et al., 2008)(Fig. 3B). ER stress reportedly activates NF-κB signaling, and this was considered a mechanism for inducing inflammatory responses (Deng et al., 2004; Hu et al., 2006). ER stress induced the formation of a complex between IRE1α and IκB kinase (IKK) through the adaptor protein TNF-α receptor associated factor 2 (TRAF2), thus promoting TNF-α production by enhancing IκBα degradation and NF-κB activation (Hu et al., 2006). In addition, NF-κB was activated by eIF2α phosphorylation, which induces translational suppression of IκBα (Deng et al., 2004). Following binding of TRAF2 to IRE1 cytoplasmic regions, IRE1α and IRE1β activated c-Jun N-terminal kinase (JNK) (Urano et al., 2000). These data were also confirmed in XBP1-deficient small intestines, with IRE1α hyperactivation and increased phosphorylation of JNK (Kaser et al., 2008). Although these studies collectively indicate immunological roles of the UPR, further studies are required to characterize and identify the related regulatory mechanisms and signaling molecules.
Several studies show that inflammation augments ER stress responses (Fig. 3C). In particular, injections of pro-inflammatory cytokines such as IL-1β and IL-6, induced ER stress and activated the UPR in the livers of mice (Zhang et al., 2006). The inflammatory cytokine TNF-α also increased eIF2α phosphorylation and XBP1s expression by elevating reactive oxygen species (ROS) levels in L929 murine fibrosarcoma cells (Xue et al., 2005). The immunostimulant lipopolysaccharide (LPS) increases the production of inflammatory cytokines, and intraperitoneal injections of LPS into mice activated the transcription of ER stress-related genes, including
Because the UPR is required for ER expansion and protein secretion, it is essential for development, differentiation, and the precise function of highly secretory cells, including pancreatic β cells, exocrine acinar cells, hepatocytes, and salivary gland epithelial cells (Bettigole and Glimcher, 2015; Harding et al., 2001; Lee et al., 2005; Lipson et al., 2006; Marisa et al., 2018; Reimold et al., 2000; Rutkowski and Hegde, 2010)(Fig. 4A). The UPR also has crucial roles in the development of immune cells, such as plasma cells, dendritic cells (DCs), and eosinophils (Bettigole et al., 2015; Iwakoshi et al., 2007; Lee et al., 2003; Reimold et al., 2001)(Fig. 4B). Many recent studies of various immune cell types show roles of ER stress in immune processes, including differentiation, immune activation, and cytokine expression.
Depending on microenvironments, naive CD4+ T cells differentiate into one of several lineages of helper T (Th) cells, including T helper type 1 (Th1), Th2, Th9, Th17, and regulatory T cells (Tregs) (Stadhouders et al., 2018). Effector T cell subsets secrete distinguishing cytokines and coordinately regulate different aspects of immune responses. Potentially, the UPR plays fundamental roles in T cell differentiation upon antigen recognition. This hypothesis is supported by observations of activated ER stress responses during differentiation of Th2 cells, and the PERK-eIF2α axis has been implicated in this process (Scheu et al., 2006). Specifically, primed Th2 cells showed increased phosphorylation of eIF2α and upregulation of ATF4 and its target genes
A recent study showed that ER stress responses are important for CD4+ T cell activation (Thaxton et al., 2017). In their study, T cell receptor (TCR) ligation-mediated ER stress increased Grp94 expression in CD4+ T cells, whereas deletion of Grp94 resulted in a critical activation defect. In addition, Grp94-deficient T cells showed impaired Ca2+ mobilization upon TCR engagement and failed to accomplish activation-induced glycolysis. ER molecular chaperone Grp94, upregulated by ER stress, has diverse immunological activities (Ansa-Addo et al., 2016).
The effects of ER stress on differentiation have been investigated in Tregs that maintain tolerance to self-antigens and prevent autoimmune disease (Franco et al., 2010). Specifically, T cells from patients with Kawasaki disease produced high levels of the anti-inflammatory cytokines IL-10 and TGF-β, and Foxp3, which is a critical transcription factor for Tregs (Franco et al., 2010). Treatments of these cells with thapsigargin induced the expression of the ER stress-response genes
In the presence of pathogens, CD8+ cytotoxic T cells secrete various cytokines and cytotoxic molecules to eliminate infected cells. In a study of CD8+ T cells by Kamimura and Bevan, the IRE1–XBP1 pathway was activated by acute infection and was required for differentiation into effector T cells (Kamimura and Bevan, 2008). These investigators also showed that viral or bacterial infection induces IRE1-mediated splicing and increases XBP1s mRNA expression in Ag-specific CD8+ T cells (Kamimura and Bevan, 2008). Finally, splicing of XBP1 mRNA was enriched in terminally differentiated effector cells expressing killer cell lectin-like receptor G1 (KLRG1), whereas XBP1-deficiencies in CD8+ T cells decreased the proportion of KLRG1high effector cells.
The physiological importance of the UPR was first described in B cells that were differentiating into plasma cells. In these studies, the UPR was activated during differentiation of B cells, and XBP1s was deemed essential for antibody production from plasma cells (Gass et al., 2002; Reimold et al., 2001; Shaffer et al., 2004; Todd et al., 2009). Accordingly, XBP1s induced ER expansion in plasma cells, therefore accommodating the synthesis of large amounts of immunoglobulin (Shaffer et al., 2004). Whereas XBP1-deficient B cells developed normally and formed germinal centers upon activation, they failed to secrete immunoglobulins (Reimold et al., 2001; Taubenheim et al., 2012). As a transcription factor, Blimp-1 necessary for plasma cell differentiation, and Blimp-1-deficient B cells failed to activate transcription of most plasma cell-related genes, including
ER stress also upregulated the production of pro-inflammatory cytokines in B cells, and expression of Ag with the ER-retention motif KDEL in B cells induced the ER stress-responsive genes
Macrophages are crucial effector cells of the innate immune system that phagocytose pathogens and produce various inflammatory mediators (Hirayama et al., 2018). XBP1s is a positive regulator of toll-like receptor (TLR) responses in macrophages, and TLR2 and TLR4 activated the IRE1α–XBP1s axis, but not the PERK or ATF6α pathways (Martinon et al., 2010). Moreover, XBP1s was required to produce pro-inflammatory cytokines, such as IL-6, TNF, and INF-β. TLR-mediated splicing of XBP1 mRNA was dependent on signaling via TIRAP, MyD88, TRIF, TRAF6, and NADPH oxidase 2 (NOX2) as a TRAF-bound enzyme complex. Under condition of XBP1-deficiency, IL-6 production was reduced in macrophages, and the bacterial burden was increased in mice infected with an intracellular pathogen. Conversely, overexpression of XPB1s or thapsigargin treatments increased LPS-stimulated IFN-β production in RAW267.4 cells (Smith et al., 2008).
Prolonged ischemia induces the expression of pro-inflammatory cytokines in liver Kupffer cells, primarily through ER stress-activated ATF6 (Rao et al., 2014). Accordingly, knockdown of ATF6 in macrophages suppressed the expression of TNF-α and IL-6 by limiting NF-κB activity. In agreement, alleviation of ER stress using the chemical chaperone 4-phenylbutyrate (4-PBA) or ATF6 siRNA led to decreased inflammatory responses in Kupffer cells against ischemia-reperfusion.
DCs are professional antigen-presenting cells, and developmental and survival roles of XBP1s have been demonstrated in these cells (Iwakoshi et al., 2007). In agreement, XBP1s expression was greater in plasmacytoid DCs than in immature conventional DCs, and XBP1-deficient chimeric mice had decreased numbers of conventional and plasmacytoid DCs. Under conditions of ER stress, treatment with TLR agonists significantly enhanced IL-23p19 expression in DCs (Goodall et al., 2010). The gene
A recent study associated tumor microenvironments with ER stress in DCs, and showed that XBP1s is important for the suppression of anti-tumor immunity (Cubillos-Ruiz et al., 2015). Specifically, XBP1s promoted the triglyceride biosynthetic program in DCs, which led to abnormal lipid accumulation and impaired antigen presentation. Conversely, DC-specific deletion of XBP1 restored immunostimulatory activities and increased anti-tumor responses.
Several recent studies show that IRE1 recognizes unspliced XBP1 mRNA and various other mRNAs as substrates to be degraded, and the associated process is called regulated IRE1-dependent decay (RIDD) (Coelho and Domingos, 2014; Hollien and Weissman, 2006; Hollien et al., 2009)(Fig. 5). Because IRE1 mediates the degradation of mRNAs encoding ER-targeted proteins, RIDD was originally thought to reduce ER protein loads during ER stress (Hollien and Weissman, 2006). In mammalian cells, RIDD targets have specific motifs with the consensus sequence CUGCAG and secondary structures that are similar to the stem-loop of XBP1 mRNA (Moore and Hollien, 2015; Oikawa et al., 2010; So et al., 2015)(Fig. 5A). Emerging evidence indicates that RIDD targets various mRNAs and therefore has diverse physiological functions, particularly in drug and lipid metabolism, and ER homeostasis in goblet cells and pancreatic β-cells (Han et al., 2009; Hur et al., 2012; Iqbal et al., 2008; Lipson et al., 2008; So et al., 2012; Tsuru et al., 2013)(Fig. 5B).
RIDD has also been implicated in the regulation of immune responses, with critical functions in the cross-presentation of cell-derived antigens via MHC class I between CD8+ T cells and CD8α+ conventional DCs (cDCs) (Osorio et al., 2014). Deletion of XBP1 in CD8α+ cDCs resulted in hyperactivation of IRE1α, and the RNase activity of IRE1α limited the expression of CD18 integrins and components of the MHC class I machinery by degrading
Several miRNAs have been identified as RIDD targets, and these reportedly increase the expression of other mRNAs during ER stress (Upton et al., 2012). IRE1α improved the stability of thioredoxin-interacting protein (TXNIP) mRNA by inducing cleavage of miR17, which destabilizes TXNIP (Lerner et al., 2012). Consequent augmentation of TXNIP likely activates the NLRP3 inflammasome, thus inducing caspase-1 cleavage and IL-1β secretion, and promoting inflammation and programmed cell death.
The ER is an essential organelle for protein homeostasis, and perturbations of ER homeostasis by physiological or pathological stressors cause accumulations of misfolded proteins in the ER. ER stress-induced UPR restores ER homeostasis through dynamic intracellular signaling pathways but gains its relationship with various diseases by regulating cell-specific functions. Much remains unknown regarding the impacts of ER stress on immune responses, although recent studies suggest that ER stress has specific immune regulatory activities in specialized cells and tissues. These studies collectively suggest that the UPR is a fundamental mediator of the pathogenesis of inflammatory disorders. In line with this suggestion, ER stress is known to induce inflammatory responses through UPR signaling, and immune cells are extensively dependent on the signaling branches of the UPR to perform their specific functions. Improved understanding of the molecular mechanisms underlying ER stress-regulated immune responses may lead to the discovery of new therapeutic targets for immune disorders. However, investigations of the UPR branches and their associated molecules in specific immune cells and under specific immune disorders remain challenging.
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