Cellular stress response and innate immune signaling: integrating pathways in host defense and inflammation

Toll-like receptors.

Of these PPRs, the TLRs and their downstream signaling pathways have been the most extensively studied [16–21] (Figs. 1 and ​22). The TLR is a membrane-spanning receptor consisting of an extracellular domain containing leucine-rich repeats and a cytoplasmic signaling Toll/IL-1R (TIR) domain. To date, 11 TLRs have been identified, and TLR1 to -10 have been characterized in humans. TLR1, -2, -4, -5, and -6, which recognize bacterial components, such as lipopeptides, LPS, and flagellin, are expressed on the cell surface, and TLR3, -7/8, and -9, which recognize viral double-stranded DNA, single-stranded RNA, and CpG DNA, respectively, are endosomal. In addition to bacterial and viral ligands, TLRs are activated by DAMPs, which are endogenous or exogenous molecules released by cells during stress [22, 23]. For example, Hsps are DAMPs that can bind to TLR2 and -4 to upregulate the expression of costimulatory molecules and induce release of cytokines [24]. TLR engagement by ligands can result in the activation of NADPH oxidase and production of ROS in neutrophils and macrophages [25–27]. ROS, in turn, mediate release of other DAMPs and promote sterile inflammation [28]. For example, ROS-induced oxidation of extracellular matrix proteins also generates DAMPs such as heparan sulfate [29]. TLR4-induced ROS-mediated release of the DAMP HMGB1 protein has been found in liver [30].

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Figure 1.

Interactions of stress proteins and cell surface TLR signaling pathways.

Engagement of cell surface TLR1, -2, -4, -5, or -6 initiates the MyD88-dependent signaling pathway, which results in activation of the downstream IKK and MKK signaling branches. Each of these branches induces expression of proinflammatory cytokines such as TNFα, IL-6, and IL-1β by the transcription factors NF-κB and AP-1. The TRIF-dependent signaling branch downstream of TLR4 results in expression of type I IFNs mediated by IRF3. The regulatory roles of stress proteins important in oxidative stress (yellow), heat shock response (green), UPR (blue), and DDR (pink) in these TLR signaling pathways are illustrated. →, stimulation; ↔, direct interaction; ⫞, inhibition.

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Figure 2.

Interactions of stress proteins and endosomal TLR signaling pathways.

Endosomal TLR7/8 or -9 activation induces the expression of type I IFNs such as IFNα and -β via MyD88-dependent activation of IRF7, in addition to NF-κB- and AP-1-induced proinflammatory cytokines. The TRIF-dependent signaling branch downstream of TLR3 results in expression of type I IFNs mediated by IRF3. The regulatory roles of stress proteins important in oxidative stress (yellow), heat shock response (green), UPR (blue), and DDR (pink) in these TLR signaling pathways are illustrated. →, stimulation; ↔, direct interaction; ⫞, inhibition.

TLRs can be classified based on cellular localization, which correlates with the ligands recognized, as cell surface (membrane-bound) (Fig. 1) and endosomal (Fig. 2). TLR signaling is divided into two distinct intracellular pathways: the MyD88 dependent pathway, which is used by all TLRs except TLR3, and the MyD88-independent pathway, which is downstream of TLR3 and -4 [16, 19].

Membrane-bound TLRs.

Upon recognition of the appropriate PAMP or DAMP, cell surface TLR dimerization occurs, and downstream signaling pathways are activated (Fig. 1). In the MyD88-dependent pathway, TLR activation results in recruitment of MyD88 to the receptor via the TIR domain. MyD88 in turn recruits and activates IRAK-4 and -1 through its death domain. The IRAKs can then phosphorylate and activate TRAF6, which in turn activates a complex containing TAK1 and TAB-1, -2, and -3 [31–33]. This complex triggers the activation of two signaling branches: the NF-κB pathway and the MAPK pathway [34]. The TAK1 complex phosphorylates and activates the IKK complex, which comprises IKKα, IKKβ, and NEMO [31]. The transcription factor NF-κB, sequestered in the cytoplasm by inhibitory IκB, is released and translocated to the nucleus for subsequent activation of gene transcription as a result of phosphorylation of IκB by IKK [35–38]. The MAPK signaling pathway is triggered by the activation of MKK3/6 and -4/7 (by TAK1) and of MEK1/2 (by Tpl2 kinase downstream of IKK), which phosphorylate downstream molecules such as p38, JNK, and ERK—MAPKs that activate the transcription factor AP-1 [39, 40]. The transcription factors NF-κB and AP-1 promote expression of proinflammatory cytokine genes, such as TNFα, IL-6, and IL-1β, and chemokines, such as IL-8, MCP-1, and MIP-1α [41–43]. TLR4 activation can also induce signaling via the MyD88-independent pathway by recruitment of the adaptor molecule TRIF to the TIR domain (Fig. 1) [44]. TLR4 must have the additional adaptor molecule TRAM for the activation of MyD88-independent signaling, which results in induction of the cytokines and chemokines responsible for viral clearance, such as type I IFNs and RANTES [45]. The MyD88-independent signaling pathway, which is also downstream of endosomal TLR3, is more fully described in the following section. Of note, TLR signaling intermediates have been shown to interact with oxidative-stress–mediated pathways. For instance, in phagocytic cells, TLR4 directly interacts with Nox4, an NADPH oxidase family member, and results in induction of ROS production and oxidative stress, culminating in NF-κB activation [46–49]. NADPH oxidase subunit p47^phox can also promote TLR4 expression and trafficking in vascular muscle cells and macrophages [50–52]. ROS also contribute to TLR4-mediated calcium mobilization and calmodulin kinase in monocytic cells [30, 53]. Engagement of TLR2 and -4 activates the ER stress response proteins IRE1α and XBP1 (detailed in the unfolded protein response section), which is necessary for optimal production of inflammatory cytokines by macrophages [54]. Furthermore, MAPKs, including JNK and p38 (also termed SAPKs), are activated downstream of physiological stress including oxidative stress, heat shock, and ER stress in macrophages and fibroblasts [55–58].

Endosomal TLRs.

Activation of viral endosomal TLRs such as TLR7/8 and -9 leads to induction of proinflammatory cytokines via MyD88-dependent NF-κB activation and also type I IFNs via IRF-7 activation, (Fig. 2) [59, 60]. Recruitment of the adaptor MyD88 to the TLR results in activation of IRAK-4 and TRAF6 [45, 60, 61], leading to activation of TRAF3 and subsequent phosphorylation and activation of IKKα and IRAK-1. The transcription factor IRF-7 is phosphorylated by IRAK1 and IKKα, leading to its translocation to the nucleus and induction of type I IFNs, such as IFNα and -β which are necessary for elimination of viral infection [45, 60, 61]. Finally, the MyD88-independent pathway downstream of another endosomal receptor, TLR3, involves the adaptor molecule TRIF, which is recruited to the TIR domain (Fig. 2) [44]. TRIF induces activation of TRAF6 and subsequent downstream NF-κB DNA binding activity [62]. TRIF also mediates activation of TRAF3 and leads to the activation of the noncanonical IKKs TBK1 and IKKε, which phosphorylate and activate IRF3, leading to nuclear translocation and transcription of chemokines such as RANTES and type I IFNs, such as IFNβ [63, 64]. These inflammatory mediators play an important role in the clearance of viral infection. It should be noted that viral infections induce rapid generation of ROS which potentiate activation of downstream TLR signaling in macrophages [65]. ER stress induced by viruses also augments IRF3-mediated production of type I IFN downstream of TLR activation [66, 67]. Cellular oxidative stress can also enhance TLR3 mediated NF-κB in airway epithelial cells, probably by enhancement of TLR3 expression [68]. Modulation of these stress-mediated effects on TLR signaling may be a therapeutic target in viral-induced exacerbations of diseases, including chronic obstructive pulmonary disease.

Hsp90.

Among the Hsps identified with an immunomodulatory function, Hsp90, -70, and -60 have been extensively studied. The chaperone functions of cell-compartment–specific Hsp90 proteins are predominantly to prevent formation of aggregates of unfolded proteins and to restore stability and function by binding specific client–substrate proteins that are in the late stages of folding. Some client proteins of cytoplasmic Hsp90 that are involved in immune signaling pathways include transcription factors and protein kinases, such as IRF3, IKK, and TBK1 (Figs. 1​1–3) [111]. Hsp90 plays a regulatory role in the activation or repression of these specific substrate client proteins via direct association or indirect regulation. For instance, activation of the IKK complex requires interaction with Hsp90, whereas noncanonical NF-κB is inhibited by Hsp90-mediated stabilization of the protein Monarch-1, which promotes degradation of NF-κB-inducing kinase in macrophages [112–114]. ER-specific Hsp90, also called gp96, is important for the processing and membrane expression of TLRs, including TLR4, -7, and -9 in macrophages [115]. Hsp90 is also essential for optimal function and activation of NK and T cells [116]. The association of extracellular Hsp90β with peptides leads to receptor-mediated endocytosis of the Hsp–peptide complex and enhances presentation of the peptide by MHC I on DCs [117]. These observations point to a complex pro- or anti-inflammatory role of Hsp90 in immune signaling.

Hsp70.

Hsp70 proteins are also implicated in immune responses, and these proteins are also involved in the folding of newly synthesized or stress-denatured proteins, the dissociation of unfolded protein aggregates, and the transport of proteins into different cellular compartments [118, 119]. Of note is the role of extracellular Hsp70 as DAMPs, released after cell death or by active secretion, to activate cell surface receptors on immune cells and induce downstream signaling [120]. Exogenous Hsp70 has been shown to elicit rapid calcium flux, activate NF-κB, and induce the production of inflammatory cytokines and chemokines by monocytes and DCs via the TLR2 and -4 pathways [121]. Extracellular Hsp70 can also associate with peptides and bind to cell surface receptors, such as CD91 and Lox-1, allowing the Hsp–peptide complex to be internalized by endocytosis and thus enhance the cross-presentation of the Hsp-bound peptide by MHC I on DCs [117]. In contrast to these proinflammatory functions, Hsp70 can also dampen the inflammatory response to prevent tissue damage. Heat-shocked macrophages show reduced TNF expression at specific time points that correlate with maximum upregulation of Hsp70 [122]. Overexpression of Hsp70 has been shown to inhibit the LPS-induced production of cytokines such as TNFα and IL-1β by human macrophages [123]. Hsp70 treatment reduces the capacity of DCs to stimulate T cells [124]. The effect of Hsp70 on the NF-κB signaling pathway has been extensively studied over the years: The expression of Hsp70 in macrophages blocks LPS-induced NF-κB activation by binding to TRAF6 and inhibiting its ubiquitination, thus preventing activation of the inflammatory signaling cascade (Figs. 1​1–3) [125]. Binding of Hsp70 to the NEMO protein impedes formation of the active IKK complex, which in turn inhibits NF-κB activation [126, 127]. Hsp70 expression prevents degradation of IkB and inhibits nuclear translocation of the NF-κB complex, suggesting inactivation of the NF-κB pathway as the mechanism of Hsp70-induced LPS tolerance in macrophages [128, 129]. With regard to the MAPK signaling pathways, Hsp70 can bind to and inhibit JNK activation in fibroblasts [130].

Hsp60.

Hsp60, another immunomodulatory stress protein, is expressed in the mitochondria and predominantly plays a role in intracellular protein trafficking, although there is increasing evidence of its expression in the cytoplasm as well. Studies have indicated a possible role for Hsp60 in immune response signaling and have also linked Hsp60 to cancer [131, 132]. On the one hand, Hsp60 can induce a proinflammatory profile by increasing the release of TNFα, IL-12, and other inflammatory cytokines from macrophages mediated by CD14 signaling [133]. On the other hand, treatment of effector T cells with recombinant Hsp60 inhibits activation of NF-κB and production of TNFα in a TLR2-dependent manner, indicating an immunosuppressive role [134]. Stimulation of Tregs with exogenous Hsp60 enhances their immunosuppressive activity on effector T cells in terms of cytokine production and proliferation, and this effect is mediated by TLR2 signaling [135].

Hsp110, Hsp40, and sHsps.

Among the other members of the Hsp family, the Hsp110 protein localizes to the nuclear and cytoplasmic compartments in response to hyperthermic exposure [136, 137]. Hsps have adjuvant-like properties, whereby their association with the antigenic peptide elicits a rapid and efficient TLR-mediated immune response to the respective antigen [138]. This phenomenon has been illustrated by the immunization of mice with Hsp110 complexed to melanoma antigen in a model of targeted immunotherapy [139, 140]. Members of the Hsp40 protein family serve as co-chaperones and associate with and activate the ATPase activity of Hsp70 [141, 142]. The DNAJA3 Hsp40 protein family member exerts an anti-inflammatory role via association with IKK and inhibition of its activity, resulting in stabilization of cytoplasmic IκB and inhibition of NF-κB activation, as demonstrated in tumor cells [143]. Among the sHsps, Hsp22 acts as an agonist of TLR4 inducing TNFα and IL-6 by DCs [144]. Hsp27 mediates the interaction between p38 and NF-κB for the induction of proinflammatory mediators during viral infection, and it is also necessary for activation of the TAK1, MKK, and IKK signaling pathways downstream of cytokine stimulation in fibroblasts, illustrating its proinflammatory role [145, 146]. Hsp27 exerts its anti-inflammatory effect by binding to the IKK β subunit and inactivating the IKK complex [147]. Exogenous Hsp27 induces production of the anti-inflammatory cytokine IL-10 from monocytes and promotes differentiation of monocytes into inhibitory DCs [148, 149].

PERK.

Upon ER stress, activated PERK inhibits eIF2α by phosphorylation at serine 51, which blocks the exchange of GDP for GTP that is necessary for eIF2α activation, resulting in overall inhibition of protein translation. Of note, this global inhibition of protein translation initiated by the PERK-eIF2α pathway can result in activation of NF-κB, thus promoting a proinflammatory immune profile, as is seen in fibroblasts [163]. Phosphorylation of eIF2α leads to increased translation of ATF4, which in turn induces expression of genes such as CHOP and Gadd34 [164]. It has been shown that stimulation of TLR2 or -4 inhibits ER stress-mediated activation of PERK and expression of CHOP in macrophages [54]. ER-stress–induced expression of CHOP in peritoneal macrophages is also inhibited by TLR3 or -4 activation in a TRIF-dependent manner [165]. In contrast, a recent study showed that stimulation of human monocytes by TLR4 directly upregulates expression and nuclear translocation of ATF4, which is dependent on MyD88 and JNK, resulting in production of IL-1β, -6 and -8 and RANTES [166]. However, ATF4 inhibits TBK1/IKKε-mediated phosphorylation and activation of IRF7, thereby suppressing expression of type I IFNs (Figs. 1 and ​and2)2) [167]. These observations indicate complex crosstalk in the PERK–eIF2α–ATF4 pathway and innate immune signaling, which merits further study.

ATF6.

ATF6, another ER stress sensor, activates transcription of target genes including XBP1; chaperone proteins, such as Grp78; and components of the ERAD. The ERAD targets misfolded proteins from the ER into the cytosol for proteasomal degradation. The induction of acute-phase response proteins by UPR is mediated by interaction of ATF6 with CREBH, to induce transcription of the target genes [162]. On the one hand, ER-stress–induced ATF6 mediates transient Akt phosphorylation and NF-κB activation in a short timeframe but subsequently blunts NF-κB activation in the later phase in an Akt-mTOR- and C/EBPβ-dependent manner [168, 169]. On the other hand, TLR2 or -4 inhibits ER-stress–induced ATF6 processing in macrophages (Fig. 1) [54]. Viruses such as human cytomegalovirus and hepatitis C induce and regulate the 3 branches of UPR to potentiate viral replication and viral protein folding and prevent early cellular apoptosis [170–172]. Virus-induced ER stress allows the pathogen to evade antiviral type I IFN responses by ATF6/IRE1-mediated inhibition of signaling downstream of interferons in fibroblasts [173].