TRIF-Dependent TLR Signaling, Its Functions in Host Defence and Inflammation, and Its Potential As a Therapeutic Target

TRIF-dependent TLR signaling, its functions in host defence and inflammation, and its potential as a therapeutic target

M. Obayed Ullah*†‡1, Matthew J. Sweet†‡§, Ashley Mansell¶, Stuart Kellie*†‡ and Bostjan Kobe*†‡2

*School of Chemistry and Molecular Biosciences, †Institute for Molecular Bioscience, Australian Infectious Diseases Research Centre and ‡Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Queensland 4072, Australia; ¶Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Monash University, Melbourne, Victoria, 3168, Australia.

1Current address: Department of Pharmaceutical Sciences, North South University, Banshundhara, Dhaka, Bangladesh.

2Correspondence: Bostjan Kobe, School of Chemistry and Molecular Biosciences, Cooper Road, University of Queensland, Brisbane, Qld 4072, Australia. E-mail: [email protected].

Summary Sentence: Review of the adaptor TRIF-dependent Toll-like receptor signaling pathways, their positive and negative regulators, and their involvement in disease.

Short running title: TRIF-dependent TLR signaling

KEY WORDS: adaptor protein, inflammation, innate immunity, pattern recognition receptor.

Total character count: 51,027; total number of figures: 4; total number of color figures: 4; total number of references: 220; total number of words in the Abstract: 132; total number of words in the Summary Statement: 19. Abbreviations: ACLF = acute-on-chronic liver failure, AD = autosomal dominant, AP- 1 = activator protein 1, AR = autosomal recessive, ARM motif = armadillo motif, CD16 = cluster of differentiation 16, CHB = chronic hepatitis B, CNS = central nervous system, CSV = coxsackievirus, DUBA = deubiquitinating enzyme A, ELM = endosomal localization motif, EMCV = encephalomyocarditis virus, FADD = Fas- associated death domain, GPI = glycophosphatidylinositol, HBeAg = hepatitis B e antigen, HBV = hepatitis B virus, HCC = hepatocellular carcinoma, HCV = hepatitis C virus, HDL = high-density lipoprotein, HSE = Herpes simplex encephalitis, IAV = influenza A virus, IFN = interferon, IL-1 = interleukin-1, IKKi = inhibitor of nuclear factor κB kinase ε, IRAK = IL-1R-associated kinase, IL-1RA = IL-1R antagonist, IRF = interferon-regulatory factor, ISG = IFN-stimulated gene, ISRE = IFN-stimulated response element, JNK = c-Jun N-terminal kinase, LPS = lipopolysaccharide, LRR = leucine-rich repeat, MAL = MyD88-adaptor like, MAPK = mitogen-activated protein kinase, mDC = myeloid dendritic cell, MKK = MAPK kinase, MyD88 = myeloid differentiation primary response protein 88, MYND domain = myeloid translocation protein 8, Nervy, and DEAF-1 domain, NF-κB = nuclear factor-kappa B, NTD = N- terminal domain, PAMP/DAMP = pathogen/danger-associated molecular pattern, NEMO = NF-κB essential modifier, PBMC = peripheral blood mononuclear cell, PHD = plant homeodomain, PRR = pattern recognition receptor, PIAS = protein inhibitors of activated STAT family, PIP2 = phosphatidylinositol 4,5-biphosphate, RAUL = replication and transcription activator-associated ubiquitin ligase, RIP = receptor- interacting protein, RHIM = RIP homotypic interaction motif, RSV = respiratory syncitial virus, SAM = sterile α-motif, SARM = SAM and ARM motif-containing protein, SH2 = Src homology 2, SHP = SH2-containing tyrosine phosphatase, SOCS3 = suppressor of cytokine signaling-3, STAT = , STING = stimulator of IFN genes, SUMO = small ubiquitin-related modifier, TAG = TRAM adaptor with GOLD domain, TBK1 = TANK-binding kinase 1, TMEV = Theiler's murine encephalomyelitis virus, TICAM-1 = TIR-containing adaptor molecule-1, TIR domain = Toll/interleukin-1 receptor domain, TIRAP = also called TIR domain-containing adaptor, TLR = Toll-like receptor, TMED7 = transmembrane emp24 domain-containing protein 7, TNF = tumor necrosis factor, TRADD = TNF receptor-associated death domain, TRAM = TRIF-related adaptor molecule, TRAF = TNF-associated factor, TRIF = TIR domain-

2 containing adaptor inducing interferon-β, TRIL = TLR4 interactor with LRRs, TRIM = tripartite motif, VACV = vaccinia virus, WNV = West Nile virus

3 ABSTRACT

Toll/interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-β (TRIF)-dependent signaling is required for Toll-like receptor (TLR)-mediated production of type-I-interferon and several other pro-inflammatory mediators. Various pathogens target the signaling molecules and transcriptional regulators acting in the TRIF pathway, thus demonstrating the importance of this pathway in host defence. Indeed, the TRIF pathway contributes to control of both viral and bacterial pathogens through promotion of inflammatory mediators and activation of antimicrobial responses. TRIF signaling also has both protective and pathological roles in different chronic inflammatory disease conditions, as well as an essential function in wound repair processes. Here, we review our current understanding of the regulatory mechanisms that control TRIF-dependent TLR signaling, the role of the TRIF pathway in different infectious and non-infectious pathological states, and the potential for manipulating TRIF-dependent TLR signaling for therapeutic benefit.

4 Introduction The innate immune system acts as a danger-sensing system to maintain homeostasis, and is thus activated by infection, injury and dysregulated cellular processes. It acts as the first line of defence against infection, rapidly initiating inflammatory responses to counter viral, bacterial, fungal and parasitic pathogens. Members of the Toll-like receptor (TLR) family of pattern-recognition receptors (PRRs) recognize pathogen- and danger-associated molecular patterns (PAMPs/DAMPs) [1, 2]. Structurally, TLRs are single-pass transmembrane proteins that are characterized by a PAMP/DAMP-recognizing leucine-rich repeat (LRR) domain, a transmembrane helix and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain [3] involved in TIR:TIR domain interactions [4]. Recognition of specific PAMPs/DAMPs leads to receptor dimerization [5]. The dimerized TLR selectively recruits one or more adaptor proteins and activates a specific downstream signaling cascade, which triggers a broad range of inflammatory and antimicrobial responses, as well as the maturation of the adaptive immune response [5-8]. TLR adaptors are TIR domain-containing proteins that associate with TLRs via TIR:TIR domain interactions, and facilitate downstream signaling [9-11]. Five TIR domain-containing adaptors have been described: myeloid differentiation primary response protein 88 (MyD88); MyD88-adaptor like (MAL), also called TIR domain-containing adaptor (TIRAP); TIR domain-containing adaptor inducing interferon-β (TRIF), also called TIR-containing adaptor molecule-1 (TICAM-1); TRIF- related adaptor molecule (TRAM), also called TICAM-2; and sterile α and armadillo (ARM) motif-containing protein (SARM) (Figure 1). Selective recruitment of these adaptors to specific TLRs activates distinct signaling pathways, orchestrating immune responses for each TLR. TLR signaling pathways are broadly classified into MyD88-dependent and TRIF-dependent pathways, based on specific adaptor recruitment. These two pathways are collectively responsible for the activation of the mitogen-activated protein kinases (MAPKs), as well as a suite of transcription factors such as nuclear factor-kappa B (NF-κB) and members of the interferon-regulatory factor (IRF) family [6, 12-14]. With the exception of TLR3, all TLRs recruit MyD88 as a signaling adaptor. In humans, TLR5, TLR7, TLR8 and TLR9 recruit MyD88 directly, while

5 TLR4, TLR1/TLR2 and TLR2/TLR6 use the bridging adaptor MAL to recruit MyD88. In a similar fashion, TRIF is directly recruited by TLR3, but is indirectly recruited to TLR4 via the bridging adaptor TRAM [4]. TLR4 is the only receptor that uses TRIF, TRAM, MyD88 and MAL, thus it serves as a prototype for both the TRIF- and MyD88- dependent pathways [15-19] (Figure 2). Compared to the other four adaptors, the role of SARM in TLR signaling is less well understood. It was originally shown to play an inhibitory role in TLR signaling by direct interaction with TRIF [9, 20]. However, several groups have reported that mouse SARM, predominantly expressed in neurons in the brain, has a role in neuronal morphology, stress-induced neuronal cell death and axon degeneration [21-25]. More recently, SARM was shown to be required for optimal TLR-inducible production of the chemokine CCL5 in macrophages [26]. Several early studies showed that the adaptor MyD88 is required for NF-κB and p38 MAPK activation downstream of TLR2, TLR5, TLR7 and TLR9 [27-30]. By contrast, TLR3-mediated signaling leads to activation of IRF3 and, to a lesser extent, NF-κB, to enable inducible expression of interferon (IFN)-β [31]. TLR4 signaling is unique in that it robustly activates all of these pathways [32]. The ability of TLR4 to induce IFN-β was initially attributed to MAL as the mediator of the MyD88- independent pathway [32-34]. However, further studies implicated an unknown adaptor molecule, rather than MAL, in the MyD88-independent pathway [15, 16, 32, 35, 36]. Oshiumi and colleagues identified TRIF/TICAM-1 as the adaptor molecule that links TLR3 to IFN-β promoter activation [37, 38]. Using the yeast-two-hybrid system, they showed that the C-terminal region of TLR3 specifically binds to TRIF. Using immunoprecipitation analysis and reporter gene assays in HEK293 cells, TRIF was shown to interact with the TIR domain of TLRs or adaptors, leading to NF-κB, IRF3 and activator protein 1 (AP-1) activation and inducible IFN-β expression [38]. Oshiumi and colleagues also first reported the identification of another adaptor, TRAM/TICAM-2 that physically bridges TLR4 and TRIF to enable lipopolysaccharide (LPS)-mediated activation of TLR4 signaling via TRIF [37].

OVERVIEW OF TRIF-DEPENDENT TLR SIGNALING TRIF is a TIR-containing protein responsible for mediating TLR4-dependent, but

6 MyD88-independent, activation of NF-κB and IFN-β production [18, 39, 40], as well as TLR3-dependent IFN-β activation [37, 38]. While the TIR domain is crucial for TIR:TIR domain interactions, facilitating organization of the TIR signaling complex containing TLR receptors and cytosolic TRAM and TRIF, the N-terminal domain (NTD) orchestrates downstream signaling events via interaction with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and other signaling molecules [41-43]. Human TRIF consists of 712 amino acids, while murine TRIF consists of 732 amino acids. TRIF plays a central role in mediating a sub-set of TLR signaling responses. Interfering with TRIF-dependent signaling may therefore have therapeutic applications, and indeed, a number of pharmacological agents that regulate TRIF-dependent responses have been described (see the final section). It is therefore useful to review the molecular events involved in TRIF signaling. Signaling through TRIF activates several transcription factors including NF- κB, IRF3 and AP-1, which leads to inducible production of cytokines and type-I-IFN, as well as maturation of myeloid dendritic cells (mDCs) [18, 37, 38, 44]. Biological responses emanating from TRIF-dependent signaling depend on both the type of cell responding, as well as the particular TLR that is activated. TRIF is expressed by most cell types, and localizes to the cytoplasm in resting cells [38, 45]. Upon TLR3 activation by dsRNA, TRIF, which initially transiently colocalizes with the receptor, dissociates and redistributes to speckle-like structures that are likely to act as hubs for downstream signaling responses [45, 46]. By contrast, during TLR4 signaling, TRIF associates with internalized TLR4 via endosomal TRAM [47]. At a structural level, TRIF has the NTD involved in autoregulation, a consensus TRAF6-binding motif, a TIR domain and a receptor-interacting protein (RIP) homotypic interaction motif (RHIM) at the C-terminus (Figure 1). The NTD of TRIF interacts with TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor κB kinase ε (IKKi) to enable subsequent phosphorylation and activation of IRF3 [48]. The TIR domain of TRIF interacts with the TRAM TIR domain or the TLR3 TIR domain during its recruitment to endosomal TLR4 and TLR3, respectively. The C- terminal region is involved in NF-κB activation and initiation of apoptosis [49, 50] or, if caspase-8 is concurrently inhibited, necroptosis [51]. It has been reported that the TIR domain and C-terminal region of TRIF, but not the NTD, are responsible for

7 self-association [52], which is a key event in signaling by TIR domain-containing proteins [53]. The NTD has a helical structure and on its own suppresses TRIF- mediated activation of the IFN-β promoter, as well as NF-κB-dependent reporter- gene activity [54]. It appears to do so by interacting directly with the TIR domain to inhibit self-association of TRIF molecules, and it also acts as a negative regulator of downstream signaling molecules such as TRAF6 and TBK1, by blocking their access to binding sites within TRIF [43]. During TLR4 signaling, the TLR4 TIR domain uses TRAM to recruit TRIF to the signaling complex, either operating from the plasma membrane or the endosomes. It has been reported that localization of TRAM to the endosomes is necessary for IRF3 activation in the TRIF-dependent pathway [47]. TRAM has an N- terminal myristoylation site responsible for membrane association [55], and a C- terminal TIR domain involved in TIR:TIR domain interactions (Figure 1). It has a bipartite sorting signal that controls the distribution of TRAM between the plasma membrane and the endosomal system; the minimal localization signal is defined by amino acids 1-20, which also includes a myristoylation site that corresponds to amino acids 1 to 7. Although the myristoylation motif is sufficient for endosomal localization, the full bipartite motif is necessary for plasma membrane targeting [47]. The phosphorylation of serine 16 of TRAM, which is close to the myristoylation site, is also important for TRAM activation [56] (Figure 1). Whether the consequences of TLR signaling are beneficial or detrimental is crucially dependent on different physiological and pathological cues. A plethora of positive and negative regulators control the TRIF pathway, and thus play important roles in TLR3- and TLR4-mediated signaling responses (Figure 3). These regulators can potentially be exploited for selective manipulation of different TLR signaling arms.

POSITIVE REGULATORS OF TRIF-DEPENDENT TLR SIGNALING While most studies have focused on factors promoting signaling downstream of TRIF, there is also evidence of accessory factors that enhance TRIF signaling at the level of the cell surface (Table 1). For example, in human monocytic cells, CD16 (cluster of differentiation 16) positively regulates TRIF-dependent TLR4 responses

8 to LPS, as assessed by both CD16 over-expression and knock-down in monocytic cells. Moreover, it was shown that CD16 down-regulates MyD88-dependent signaling by up-regulating the expression of interleukin-1 receptor (IL-1R)- associated kinase M (IRAK-M) and the IL-1R antagonist (IL-1RA), negative regulators of this signaling arm [57]. Intriguingly, the glycophosphatidylinositol (GPI)-anchored membrane protein CD14, which has a well-characterized function in sensitizing LPS-dependent TLR4 signaling, has also been shown to promote TLR3 signaling [58]. The mechanism appears to involve enhancing ligand uptake and delivery to the TLR3-containing lysosomal compartment. This is consistent with our emerging understanding that CD14 has a rather promiscuous role in TLR signaling, rather than being selective for TLR4 responses [59, 60]. For example, CD14 can interact with TLR1/TLR2 to recognize triacylated lipoproteins [61-63] or with TLR2/TLR6/CD36 to recognize diacylated lipoproteins [64, 65]. CD14 has also been shown to enhance signaling in reponse to poly I/C, imiquimod and CpG DNA, implicating this molecule in TLR3, TLR7 and TLR9 signaling [58, 66]; however, in humans this has only been demonstrated for TLR9 [67]. Several molecules have been identified that enhance TLR responses by regulating endosomal translocation and signalling. For example, TRIL (TLR4 interactor with LRRs) was originally shown to bind to both LPS and TLR4 and enhance TLR4 signaling [68]. More recently, TRIL has also been reported to localize to the endosomal compartment and to enhance TLR3 signaling [69]. Thus, TRIL appears to regulate multiple TLR signalling pathways. Another molecule that regulates the functions of endosomal TLRs is UNC93B, a chaperone molecule that mediates translocation of nucleic acid-sensing TLRs to endolysosomes. Ablation of UNC93B in mice abolishes the signalling function of TLR3, TLR7 and TLR9, and consequently, these animals are highly susceptible to infections [70-73]. Various positive regulators of TRIF signaling act at the level of the adaptor and downstream signaling molecules. Certain members of the tripartite motif (TRIM) family of E3 ubiquitin ligases can positively regulate the TRIF pathway, promoting activation of NF-κB and AP-1. One example is TRIM56, which enhances TRIF-dependent TLR3 signaling via direct interaction with TRIF [74]. Extracellular dsRNA-induced expression of IFN-β and IFN-stimulated genes (ISGs) was

9 substantially increased by TRIM56 overexpression in HeLa cells. On the other hand, knock-down of TRIM56 significantly impaired IRF3 activation, as well as induction of IFN-β and ISGs. Moreover, TRIM56 silencing impaired TLR3-mediated antiviral responses and chemokine induction following infection with the hepatitis C virus (HCV). Interestingly, the elevated TLR3 response correlated with a physical interaction between TRIF and the C-terminal region of TRIM56, but was independent of its E3 ubiquitin ligase activity [74]. However, another study reported that TRIM56 facilitates double-stranded DNA-stimulated IFN induction by the ubiquitination of stimulator of IFN genes (STING) [75]. Another TRIM family member, TRIM62, acts as a component of the TRIF-dependent TLR4 signaling pathway to promote inflammatory responses [76]. LPS-stimulated TLR4 signaling in HEK293 cells was substantially reduced in TRIM62-silenced cells, as compared to the wild-type cells. The involvement of TRIM62 in the TRIF branch of TLR4 signaling was confirmed by experiments performed on TRIM62-silenced primary human macrophages, which demonstrated impaired NF-κB and AP-1 activation. Co- expression of Pellino-1 (another E3 ubiquitin ligase) with TRIF potentiated NF-κB responses, whereas ablation of Pellino-1 impaired both MyD88- and TRIF- dependent cytokine gene expression upon TLR activation. Mechanistically, Pellino-1 promotes K63-linked polyubiquitination of IRAK1, TBK1 and TAK1 [77]. Nuclear BS69, a ‘myeloid translocation protein 8, Nervy, and DEAF-1’ (MYND) domain-containing protein, is another regulator that enhances TRIF-dependent NF- κB activation and type-I-IFN production. Originally described as a transcriptional co- repressor, BS69 has a plant homeodomain (PHD), a bromodomain and a PWWP domain, all of which are implicated in recognizing histone modifications [78]. It has now been shown to be a histone 3.3-specific reader, recognizing methylated H3.3k36 [79, 80]. The recruitment of BS69 by oligomerized TRIF was confirmed by yeast- two-hybrid and immunoprecipitation assays. Overexpression of BS69 into HEK293T cells enhanced TRIF-mediated TLR3 signaling (NF-κB activation and IFN-β induction). Furthermore, knockdown of endogenous BS69 in HeLa cells decreased inducible IFN-β expression, thus implicating BS69 in positive regulation of TRIF- mediated TLR3 signaling [81]. BS69 has also been shown to cooperate with TRAF3 in Epstein-Barr virus-mediated NF-κB activation [82].

10 A number of other positive regulators of TRIF signaling have been identified. The low molecular mass polypeptide (LMP) immunoproteasome subunits LMP7, LMP2, and LMP10 (MECL-1) [83, 84] have been shown to regulate TRIF/TRAM- mediated TLR4 signaling. In particular, LMP7 and LMP10 positively regulate LPS- inducible and TRIF-dependent nitric oxide (NO) production [85]. This response was significantly reduced in thioglycollate-elicited macrophages from LMP7, LMP2 and LMP10 knock-out mice, as well as LMP7/LMP10 double knock-out mice, whereas TNFα production remained unaltered. However, treatment with IFN-γ overcame this deficiency, permitting robust LPS-inducible NO production, comparable to wild-type cells. In another report, it was shown that IRF7 directly binds to the LMP2 promoter to induce its expression, thus implicating the immunoproteasome subunits as downstream targets of TRIF-mediated signaling [86]. Finally, the WD repeat and FYVE-domain-containing protein WDFY1 potentiates TLR3 and TLR4-induced activation of NF-κB and IRF3, whereas its depletion reduces these responses. Mechanistically, WDFY1 was reported to recruit TRIF to these receptors, probably by bridging the TLR-TRIF interaction [87]. In summary, TRIF–dependent signaling can be positively regulated by a number of factors acting at various levels including membrane-proximal receptor and co-receptor complexes, endosomal trafficking, and downstream signaling.

NEGATIVE REGULATORS OF TRIF-DEPENDENT TLR SIGNALING As a potent inflammatory signaling module, the TRIF pathway is tightly controlled by numerous host factors. Conversely, several pathogens target TRIF-dependent signaling as a host evasion strategy. Indeed, a strikingly large number of host- and pathogen-derived factors inhibit the TRIF pathway by acting at the receptor and adaptor levels, as well as at the level of downstream signaling (Figure 3, Table 2).

Endogenous negative regulators Numerous host factors can directly antagonize TRIF signaling by preventing it from interacting with proteins required for signal transduction, by targeting it for degradation or by post-translationally modifying TRIF signaling components to inhibit protein function or trigger degradation. A splice variant of TRAM, TRAM

11 adaptor with GOLD domain (TAG), operates via preventing TRIF interactions. TAG is localized to late endosomes and can down-regulate TRIF-dependent signaling by displacing TRIF from TRAM during signaling [88]. Therefore, antagonism of TAG action could potentially selectively boost TRIF-dependent responses. This could promote adjuvanticity without causing excessive inflammation, as the MyD88- independent pathway is required for LPS-triggered adjuvant efects [17]. Another GOLD domain-containing molecule, transmembrane emp24 domain-containing protein 7 (TMED7), can also displace TRIF from TRAM during TLR4 signaling [89]. Similarly, TRAM can be sequestered away from TRIF as a means of preventing engagement of this pathway. Such a mechanism has been described for the high- density lipoprotein (HDL), which suppresses TLR4 signaling by initiating the translocation of TRAM to signaling-incompetent intracellular compartments [90]. A number of regulatory mechanisms that control the TRIF pathway involve post-translational modifications of TRIF and downstream signaling molecules. Several such mechanisms control TRIF protein expression. While some TRIM family members promote TRIF-mediated signaling (see above), others regulate cytosolic TRIF protein levels and localization. TRIM38 negatively regulates TRIF-mediated TLR3 signaling by targeting TRIF for proteasomal degradation. Overexpression of TRIM38 inhibited type-I-IFN induction by decreasing levels of endogenous as well as ectopically expressed TRIF. Conversely, knock-down of TRIM38 had the opposite effect [91]. Coimmunoprecipitation analyses showed that TRIM38 interacts with TRIF, but not with TBK1 or IKKi, thereby suggesting that down-regulation of TRIF- mediated TLR3 signaling occurs by direct targeting of TRIF. Consistent with this, overexpression of TRIM38 induced K48-linked polyubiquitination and proteasomal degradation of TRIF in HEK239T cells [91]. The ubiquitination status of TRIF is also exploited for the regulatory control of TRIF-mediated functions, either through deubiquitination of K63-linked scaffolding complexes to inhibit signaling or through K48-mediated ubiquitination of signaling molecules to target them for degradation. The ovarian tumor domain-containing deubiquitinating enzyme A (DUBA) is a negative regulator of TRIF-dependent type-I-IFN production; it binds and cleaves K63-linked polyubiquitin chains on TRAF3, an E3 ubiquitin ligase. As a result, TRAF3 is dissociated from the downstream signaling complex containing TBK-1, thus

12 suppressing type-I-IFN production [92]. Conversely, the E3 ubiquitin ligase family member suppressor of cytokine signaling-3 (SOCS3) negatively regulates TRAF- dependent inflammatory responses by mediating the degradation of TRAF proteins [93]. Similarly, the replication and transcription activator-associated ubiquitin ligase (RAUL) [94], as well as the peptidyl-propyl isomerase Pin1 [95], both negatively regulate type-I-IFN production by promoting ubiquitination of IRF family members and consequently their degradation. The protein inhibitors of activated STAT family proteins with E3 small ubiquitin-related modifier (SUMO) ligase activity (PIAS) family are key regulators of signal transduction [96]. They primarily act through negative regulation of transcription [97]. In mammals, five PIAS family members have been identified, PIAS1, PIASxα, PIASxβ, PIAS3 and PIAS4 [96-99]. Three of these (PIAS1, PIAS3 and PIAS4) negatively regulate NF-κB-dependent responses, and one of these has also been shown to control the TRIF pathway. PIAS4 was reported to interact with TRIF, IRF3 and IRF7, and to inhibit TRIF-induced NF-κB- and IFN-stimulated response element (ISRE)-dependent transcriptional responses in HEK293 cells [100]. Its regulation of TRIF responses is selective, as PIAS4 has no effect on TRIF-induced apoptosis. SUMOylation is likely involved in PIAS4-mediated inhibition of TRIF signaling, but this has yet to be formerly demonstrated. In support of this original study, a PIAS4 homologue from zebrafish was shown to negatively regulate TRIF- mediated NF-κB activation, as well as INF-φ expression [101]. Other studies have also demonstrated that other PIAS family members target NF-κB proteins. PIAS3 was reported to suppress NF-κB activation, and to interfere with Rel homology domain of the p65 subunit (RelA) binding to transcriptional co-activators [102]. Consistent with this study, experiments performed in murine 3T3, HEK293 and H1299 cells showed that PIAS3 negatively regulates NF-κB activity by E3 SUMO ligase-dependent SUMOylation of the RelA subunit [103]. PIAS1 has also been reported to negatively regulate NF-κB transcriptional activity, again by targeting the RelA subunit [104]. While the above studies on PIAS1 and PIAS3 did not specifically focus on TRIF signaling, the latter study did show that LPS-inducible expression of a sub-set of TLR4 target genes was enhanced in PIAS1-deficient macrophages. Thus, it

13 is quite possible that other PIAS proteins, in addition to PIAS4, regulate TRIF- dependent inflammatory responses. Caspase-1-mediated cleavage of TRIF is also crucial in controlling type-I-IFN production, autophagy and inflammasome activation. Direct cleavage of TRIF by caspase-1 diminished TRIF-dependent signaling, reducing type-I-IFN production and inhibited autophagy [105]. Conversely, preventing TRIF cleavage by caspase-1 in an in vivo model of Pseudomonas aeruginosa infection resulted in enhanced autophagy, attenuated IL-1β production, and increased bacterial clearance [105]. While these data implicate the TRIF pathway in promoting autophagy, autophagy has also been reported to negatively regulate TRIF responses. Specifically, the autophagy receptor NDP52 was reported to interact with TRIF, as assessed by coimmunoprecipitation experiments, and to negatively regulate TRIF-dependent, LPS-induced IRF3 and NF- κB activation via autophagic degradation of TRIF and TRAF6 [106]. The ubiquitin- like protein ubiquilin-1 was further shown to negatively regulate TRIF-mediated TLR3 signaling. Specifically, ubiquilin-1 directly interacts with TRIF, and co-localizes, along with TRIF, with the autophagosome marker LC3 in punctate subcellular compartments [107]. Thus, ubiquilin-1 may provide a molecular mechanism by which TRIF can be targeted for autophagic degradation, at least in the context of TLR3 signaling. Collectively, the above studies demonstrate that there is an array of mechanisms targeting TRIF protein expression and cellular localization as a means of regulating TRIF-dependent signaling pathways. Consistent with many other signaling pathways, TRIF-dependent signaling is regulated by protein phosphorylation. One example of such a mechanism involves the CD300 family of cell-surface proteins that limit TRIF signaling via the actions of Src homology 2 (SH2)-containing tyrosine phosphatase (SHP) family members. Members of the CD300 family, as well as synthetic peptides based on their sequences, can suppress both TRIF- and MyD88-dependent signaling [108-110]. For example, CD300f blocked MyD88- and TRIF-mediated activation of NF-κB, with TRIF-dependent signaling being targeted by the combined activation of SHP-1 and SHP-2 [109]. Lee and colleagues reported similar findings [108] and demonstrated that synthetic peptides containing immunoreceptor tyrosine-based inhibitory motif- like sequences derived from CD300f had a similar effect [110]. As SHP-2 negatively

14 regulates TLR signaling by interacting with the kinase domain of TBK1 through its C- terminal domain [111], this likely explains the mechanism by which CD300f targets TRIF signaling. Thus, the host employs a plethora of distinct mechanisms to tightly regulate this potent pro-inflammatory signaling pathway, thus highlighting the potential danger that dysregulated TRIF signaling and subsequent inflammatory responses is likely to have on the host.

Pathogen-derived negative regulators Given the importance of the TRIF pathway in type-I-IFN production and anti-viral defence (see below), it is not surprising that TRIF is the target of viral immunomodulatory strategies to suppress this signaling pathway. For example, viral proteases have been shown to inhibit TRIF-dependent signaling. Many do so by directly cleaving the TRIF protein. This mechanism of action applies to the hepatitis A virus protease polymerase-processing intermediate called 3CD [112], the enterovirus 71 3C protease [113], the 3C protease from coxsackievirus (CSV) B3 [114] and the HCV protease NS3/4A [115], which all can cleave TRIF.

A number of viral proteins negatively regulate TRIF-dependent signaling by interacting with adaptors to interfere with their function, or by targeting downstream signaling. For example, A46 from vaccinia virus (VACV) interacts with TRIF, TRAM, MyD88 and MAL, thereby preventing NF-κB, MAPK and IRF3 activation [116, 117]. A52R, another VACV protein, negatively regulates TLR signaling by interacting with TRAF6 and IRAK2 [118]. The hepatitis B e antigen (HBeAg) can suppress TLR signaling by interacting with TRAM and MAL, leading to down- regulation of TLR signaling [119]. Finally, the HIV-encoded proteins VPR and Vif negatively regulate TLR signaling by targeting IRF3 for degradation [120]. In addition to viruses, bacterial pathogens have also evolved mechanisms to target TLR signaling. One such mechanism involves subversion of TLR responses by bacterial TIR-containing proteins, as exemplified by TlpA from Salmonella enterica serovar Enteritidis [121]. Similarly, TcpC, a TIR domain-containing protein from the uropathogenic Escherichia coli strain CFT073, can inhibit TLR4 signaling by interacting directly with both TRIF and MyD88 [122, 123]. A related protein, TcpB

15 from Brucella melitensis, antagonizes TLR4 signaling by interfering with the TLR- MAL interaction [124]; as yet however, there is no direct evidence to suggest that TcpB also targets the TRIF pathway. Given the essential role of the TRIF pathway in host defence against many bacterial pathogens (see below), as well as the observation that TIR-domain-containing proteins are widespread among both pathogenic and non-pathogenic bacteria [125], it is quite likely that TIR-domain- containing proteins from other pathogenic bacteria may be identified that target the TRIF signaling nexus through competing with interactions required for signalling. This remains to be determined. Nonetheless, there is abundant evidence that many pathogens have utilized the diverse mechanisms of regulatory control employed by the host to evolve similar strategies to target TRIF-mediated inflammatory responses.

THE ROLE OF TRIF-DEPENDENT TLR SIGNALING IN HOST DEFENCE TRIF-dependent TLR signaling contributes to host defence responses by driving type-I-IFN production, and regulating caspase activation, apoptosis, necroptosis and homeostasis, thus enabling effective bacterial clearance and inhibition of viral replication during infections. In these contexts, TRIF also plays a role in neuro- virulence, neuro-protection and cardio-protection (Figure 4). Below, we discuss the role of TRIF-dependent pathways in the host response to pathogen challenge.

Cell death as a host defence mechanism While TRIF is best characterized for its role in mediating TLR-induced inflammation via NF-κB and IRF3 activation, it is also apparent that TRIF plays a role in inducing cell death via either apoptosis or necroptosis, independent of its role in activating pro-inflammatory transcription factors. Shu and colleagues demonstrated that TRIF induced apoptosis through a RIP/FADD (Fas-associated death domain)/caspase-8- dependent (mitochondrion-independent) pathway, which could be inhibited by co- expression with a mutated FADD dominant-negative construct [126]. TRIF directly interacts with RIP1 via homotypic RHIM domains, following ligand binding to TLR3 and TLR4. RIP1 in turn interacts with RIP3 via death-domain interactions, triggering

16 cell death [51]. When caspase-8 function is inhibited, which could potentially occur as a pathogen subversion strategy, TLR-mediated TRIF signaling drives necroptosis [51, 127]. Therefore, it would appear that TRIF may also play a crucial role in removing infected cells as a means of potentially inhibiting pathogen dissemination, presumably in a cell-specific manner.

TRIF-dependent anti-viral responses In contrast to our rather limited understanding of the role of the TRIF pathway in host protection against bacterial pathogens in humans, the role of this pathway in viral infections has been much more extensively studied through genetic approaches. Analysis of autosomal dominant negative mutations and SNPs has identified TRIF-dependent signaling as a key component of anti-viral defence in humans. Although TRIF-mediated TLR3 signaling is generally associated with host defence against double-stranded RNA viruses, it is also important for resistance to infection with DNA viruses and single-stranded RNA viruses [128]. Our understanding of the role of TLR3 signaling in response to infection has been further informed by the use of mice deficient in TLR3 and related signaling pathway components. Such mice represent important models to investigate the complexity of TRIF-dependent signaling in different viral infections, and have defined roles in host defence during viral infections of the central nervous system (CNS), respiratory tract, liver, heart and pancreas [129]. As discussed below, the susceptibility of TLR3-deficient mice has been reported to a number of viruses including the West Nile virus (WNV), poliovirus, Theiler's murine encephalomyelitis virus (TMEV), HSV-1 and HSV-2 and Sindbis virus.

Neurological infections. The role of TLR3 in viral infections that target the CNS and cause neuropathology has been studied widely in mice. Initial studies reported that TRIF-mediated signaling actually promoted the development of encephalitis, by facilitating the entry of WNV into the CNS. TRIF-dependent inflammatory responses resulted in a breakdown of the blood-brain barrier, thus facilitating the invasion of WNV into the CNS [130]. However, later studies reported that TRIF-dependent TLR3 signaling was essential for the reduction of viral burden in the CNS during WNV

17 infection [131, 132]. These differences highlight the complexity in studying virus infection, where the balance between chronology of infection, viral load, innate response and cytokine production can all affect the pathological outcomes. Herpes simplex encephalitis (HSE) is a rare form of CNS encephalitis caused by the herpes simplex virus (HSV-1) in children. Autosomal deficiencies in TLR3 and in UNC93B, which is required for TLR3 signaling, have both been linked to this condition. Using induced stem cells from normal, TLR3- or UNC93B-deficient patients, it was shown that impaired TLR3- and UNC93B-dependent IFN-α/β responses to HSV-1 in neurons and oligodendrocytes in the CNS are linked to HSE in TLR3-deficient children [133, 134]. Moreover, TRIF polymorphisms were associated with pediatric HSV-induced encephalitis in a study of HSE in children with autosomal recessive (AR) or autosomal dominant (AD) TRIF deficiency (Sancho- Shimizu et al., 2011). The AR form of the disease was found to be due to a homozygous nonsense mutation that results in a complete absence of the TRIF protein, leading to the elimination of both TLR3- and TLR4-mediated TRIF signaling. The AD form of disease is a result of heterozygous missense mutations, resulting in a dysfunctional TRIF protein. In this form of the disease, the TLR3 signaling pathway is impaired [135]. TLR3 deficiency was also linked to susceptibility to influenza A virus (IAV)-induced encephalopathy. A missense mutation (F303S) in the TLR3 gene was found in a patient with this condition [136]. Given the clinical associations between deficiency in the TLR3/TRIF pathway and HSE, HSV infections have been studied in TLR3-deficient mice. These studies have focused on HSV-2, which can cause meningitis, sacral radiculitis and myelitis in humans [137]. TLR3-deficient mice were hyper-susceptible to HSV-2 infection in the CNS, after vaginal inoculation [138]. TLR3-deficient astrocytes were found to be defective in HSV-induced type-I- IFN production, without any global defect in the immune response to HSV. Thus, immediate sensing of HSV-2 by astrocyte TLR3 after entry into the CNS may help to prevent HSV from spreading beyond the neurons mediating entry into the CNS [138]. TRIF-dependent signaling also contributes to defence against the Sindbis virus and poliovirus infections. The TRIF-dependent type-I-IFN pathway plays a host protective role in preventing Sindbis virus-induced neurological disease [139].

18 Similarly, TRIF-deficient mice displayed an increased mortality rate upon infection with poliovirus, due to impaired type-I-IFN production [134, 140]. Thus, the TLR3/TRIF pathway appears to have a particularly important role in host protection against viral pathogens that target the CNS.

Respiratory infections. In addition to the role in IAV-induced encephalopathy, children with the TLR3 rs5743313/CT polymorphism showed an increased risk of pneumonia caused by the pandemic A/H1N1/2009 influenza virus [141]. Mechanistically, the host protective function of TLR3/TRIF signaling during respiratory infections likely relates to its roles in the production of inflammatory mediators leading to damaged airway mucosa [142], as well as the production of host factors that blocks viral replication, such as Mx-GTPase [143]. In mouse models, TRIF-dependent TLR3 signaling appears to have detrimental effects for infections caused by many viruses targeting the respiratory system, for example, rhinovirus, IAV, VACV and respiratory syncitial virus (RSV) infections [129]. In many cases, this likely relates to a hyper-inflammatory response and immunopathology driven by the TRIF pathway. For example, IAV-infected TLR3- deficient mice display increased survival rates as compared to wild-type mice, even though the viral burden in lungs is elevated [144]. A similar study examining VACV infection reported that TLR3-deficient mice not only displayed reduced lung inflammation and neutrophil recruitment, but remarkably, also showed reduced viral replication in the respiratory tract, as well as dissemination [145]. Another study suggested that TLR3-mediated signaling contributed to pulmonary edema in mice [146], which is characteristic of several respiratory viruses including IAV and RSV. Thus, by triggering excessive inflammation, the TLR3/TRIF pathway appears to be detrimental to the host in many respiratory virus infections, counter to what may be expected of an innate immune sensor. The precise molecular mechanisms that account for these effects are unclear and warrant further investigation.

Hepatic disease. TLR3/TRIF-mediated signaling can have both beneficial and detrimental effects in hepatic viral infections. As expected, TLR3- and TRIF-deficient mice challenged with poliovirus, and TLR3-/- mice challenged with

19 encephalomyocarditis virus (EMCV) and CSV B4, display both increased viral loads in the liver and increased mortality [134, 147, 148]. Interestingly, EMCV-infected mice displayed reduced cytokine and chemokine production, yet IFN-β levels were unaffected. It is important to remember however that the RIG-like receptor (RLR) pathway is also a major sensor of EMCV, poliovirus and CSV, and this may account for the lack of effect on IFN-β production. Contrary to these findings however, infection of TLR3-deficient mice with the Punta Toro virus (a Phlebovirus) resulted in resistance to lethal infection and reduced liver disease [149]. Indeed, in TLR3- deficient mice, Punta Toro virus was cleared faster, whereas cytokine and chemokine production was decreased, suggesting that TLR3-mediated inflammatory responses are detrimental to disease outcome for this particular virus. An important caveat in these studies, however, is the nature of the actual viruses used; they are not liver-specific viruses, infecting multiple organs and have different pathologies and specificity depending on serotype and infective units. Indeed, a clearer role for TLR/TRIF-dependent signaling is apparent in EMCV and CSV-induced disease, as described below. Furthermore, given the broad range of organs these viruses may infect, redundancy and specificity of TLR versus RLR sensors and their respective responses must also be considered and may contribute to the variability and conflicting aspects of these studies. While multiple PRRs have been implicated in detection and innate immune responses to hepatitis B virus (HBV) and HCV, TLR3 and TRIF-dependent signalling has been identified as key drivers of the host response. TLR3 plays a key role in recognizing viral dsRNA. Point mutations in the dsRNA-binding region of TLR3 ablated cytokine and chemokine responses in reconstituted hepatoma cells, demonstrating that TLR3 was the PRR responsible for responding to HCV dsRNA [150, 151]. TLR3-mediated signaling induces NF-κB activation and pro- inflammatory cytokine and chemokine secretion in infected cultured hepatoma cells, as well as in primary human hepatocytes [150]. TLR3 polymorphisms have been associated with chronic hepatitis B and hepatitis B-related hepatocellular carcinoma [152, 153], while TLR3 recognition of HCV dsRNA has been linked to the induction of chronic hepatitis and hepatocellular carcinoma [154]. The lack of a mouse model of HBV infection has limited our understanding of the role of TLR3/TRIF signaling;

20 however, genetic evidence has provided critical insights. Analysis of Saudi Arabian patients with HBV found an association between a particular SNP in TLR3 and the outcome of HBV infection [155]. Further studies revealed that TLR3 polymorphisms could be a risk factor for chronic hepatitis B (CHB)-related acute-on-chronic liver failure (ACLF) [152] and hepatocellular carcinoma (HCC) [153]. Analysis of TLR expression in peripheral blood mononuclear cells (PBMCs) from patients with varying phases of clinical chronic HBV infection found that TLR3 (in addition to other TLRs) was enhanced in active stage CHB and CHB-related liver failure. Furthermore, reduced levels of TRIF expression were noted in HBV-infected patients compared to healthy controls. Together, these findings suggest TLR3 and TRIF- mediated signaling and inflammation may play a clear role in HBV infection and that dysregulated or inadequate TLR3/TRIF immune responses may predispose patients to detrimental outcomes.

Other viral infections. TRIF-mediated TLR3 signaling also has a role in regulating virus-induced cardiac and pancreatic pathology. TLR3-deficient mice were found to be highly susceptible to EMCV [147, 156] and CSV B3 and B4 [148, 157, 158] infections, resulting in significant mortality. TLR3-deficient mice infected with EMCV showed an impaired pro-inflammatory response in the heart and the liver, along with enhanced cardiac damage. A similar impaired pro-inflammatory response was observed in the pancreas of TLR3-deficient mice infected with the EMCV variant, facilitating increased viral burdens, as well as pancreatic β-cell damage leading to diabetes [147, 156]. Similarly, TLR3-deficient mice displayed enhanced cardiac damage and increased viral loads in the heart, liver and circulation, as compared to wild-type mice infected with CSV B3 and B4 [148, 157, 158]. In another report, TRIF- dependent signaling was also linked with protection against CSV group B-induced myocarditis, as TRIF-deficient mice have a higher mortality rate [159]. Taken together, these studies suggest that the TLR3/TRIF pathway very often has a cardioprotective function during many viral infections.

TRIF-dependent anti-bacterial responses

21 Although genetic studies primarily link the TRIF pathway to anti-viral defence in humans, emerging studies also support a role in responses to bacterial pathogens. Indeed it must be remembered that TRIF was discovered for mediating MyD88- independent activation of NF-κB and inducible IFN-β expression following TLR4 recognition of Gram-negative bacterial LPS. Cuenca and colleagues demonstrated that TRIF-deficient neonates are highly susceptible to Escherichia coli-mediated peritonitis and bacteremia, and that TRIF signaling is important for TLR4-protective adjuvanticity during Gram-negative infections of neonates [160]. Genetic association studies also link susceptibility to tuberculosis with TRIF-dependent signaling. SNP analysis of individuals from Uganda linked polymorphisms in TRAM to susceptibility to tuberculosis [161]. Further to this, Mycobacterium tuberculosis Hsp70-mediated induction of inflammatory responses in dendritic cells, as well as differentiation of T cells, were found to be dependent upon TRIF-dependent signaling [162], supporting a potential molecular mechanism for TRIF function in mycobacterial infection.

Lung infections. Contrary to the contrasting roles in different viral infections of the lung, the role of TRIF in bacterial lung pathogenesis appears more clear-cut. TRIF- deficient mice display reduced survival and higher bacterial burdens in the lung, compared to wild-type mice when infected with Klebsiella pneumoniae, probably due to impaired local cytokine responses and neutrophil influx [163]. Similarly, TRIF- dependent signaling pathways are also important for efficient clearance of other Gram-negative bacteria such as Escherichia coli, Campylobacter jejuni and Pseudomonas aeruginosa during lung infections [164-166]. Therefore, it would appear that contrary to viral lung infections, TRIF-dependent signaling initiates an effective innate immune inflammatory response to clear bacterial infections in the lung.

Intestinal infections. TRIF signaling was recently shown to be essential for homeostatic epithelial cell function in the intestine [167]. In the absence of TRIF, Paneth cell numbers were reduced, as was the production of key antimicrobial peptides. Indeed, in response to intestinal infection with Yersinia enterocolitica, TRIF-deficient mice displayed increased mortality and bacterial dissemination,

22 correlating with defective macrophage functions of phagocytosis and IFN expression [8]. TRIF-dependent signaling is essential for the expression and or activation of the non-canonical caspase-11 inflammasome and the maturation of pro-IL-1β into its pyrogenic mature form, thus enabling effective host defence against Salmonella enterica serovar Typhimurium (S. Typhimurium) and other enteric bacterial pathogens [168-170].

Systemic infections. Comparisons of host responses to a pathogen during localized and systemic infections shed some light on specific roles for TRIF during systemic responses. TRIF-deficient mice responded differently during intestinal versus systemic infection with Y. enterocolitica. Remarkably, TRIF appeared to play no major role in protection against systemic Y. enterocolitica infection, despite its essential role in responses to this pathogen after intestinal challenge [8, 171]. By contrast, as described previously, TRIF was required for the control of S. Typhimurium during the early phase of systemic infection, and increased bacterial loads in TRIF-deficient mice were attributed to deficient neutrophil function [172]. As mentioned above, the TRIF pathway ‘licenses’ expression and activation of caspase-11 [170] in response to Gram-negative bacteria, itself a critical component of LPS-mediated systemic inflammatory responses [173]. Given the role of the inflammasome in maturing adaptive immunity, this therefore places TRIF- dependent signaling at the intersection of innate anti-bacterial inflammatory responses and adaptive immunity.

THE ROLE OF TRIF-DEPENDENT TLR SIGNALING IN INFLAMMATION-LINKED DISEASE Intestinal inflammation PRR signaling pathways, including the TRIF pathway, play important roles in homeostatic control. For example, TLR4-mediated TRIF signaling is required for the maintenance of steady-state neutrophil homeostasis [174]. As described above, TRIF signaling is also required for intestinal homeostasis. In particular, it is required for the production of antimicrobial factors such as Reg3γ and antimicrobial peptides from Paneth cells, and the generation and differentiation of Paneth cells [167]. In a

23 mouse model of colitis, TRIF deficiency led to increased intestinal inflammation, which correlated with abnormal regulation of IFN-γ-expressing Th17 cells in the lamina propria, as a result of defective expression of the regulatory cytokine IL- 27p28 by macrophages [175]. However, in the absence of the autophagy pathway, colitis was exacerbated in a mouse model, with TRIF-dependent signaling driving enhanced IL-1β production [176]. One study has also provided evidence for a role for TRIF-mediated TLR4 responses in driving excessive inflammatory responses in ulcerative colitis [177]. Thus, TRIF may have either protective or pathological roles in intestinal inflammation, depending on the specific environment and inflammatory mechanisms involved.

Cardiovascular disease The role of TRIF signaling in cardiovascular disease models is also complex. One study showed that deletion of TLR3 accelerated atherosclerosis in hyperlipidemic ApoE-deficient mice [178], indicating an athero-protective function. Another study also reported that TLR3 had a small, but significant, effect in protecting hyperlipidemic LDL-receptor knockout mice from atherosclerosis development [179]. However, in the same mouse model, impairing TRIF function had an athero- protective effect [179]. This led the authors to conclude that TLR4-mediated TRIF signaling is pro-atherogenic, whereas the reverse is true for TLR3-dependent TRIF responses. Nonetheless, TLR3 and TLR4 signaling in bone-marrow cells had pro- atherogenic effects in LDL receptor-deficient mice [180]. Thus, it seems likely again that TRIF signaling can be either pro-atherogenic or athero-protective, depending on the specific cellular context or upstream receptor (TLR3 or TLR4) that is activated.

Other inflammation-linked conditions There are surprisingly few reports examining the role of the TRIF pathway in autoimmune disease models. One study, investigating an IL-17-dependent arthritis model, showed that the TRIF pathway had a protective role in preventing bone erosion and IL-17 production [181]. This finding is consistent with the known role of type-I-IFNs in suppressing inflammatory arthritis [182]. Interestingly, in a limb ischemia model, TRIF signaling was reported to be required for muscle regeneration

24 [183]. TRIF signaling was also reported to be required for the clearance of axonal debris microglia, thus presumably contributing to repair after neural damage [184]. Finally, TLR3 signaling has been implicated in wound repair. TLR3-deficient mice or silencing of TRIF both showed delayed wound healing [185]. Similarly, TLR3- deficient mice showed a delayed repair response after UV damage to the skin [186]. Collectively, these studies provide examples of the TRIF pathway contributing to tissue repair processes in different settings.

PHARMACOLOGICAL MODULATION OF TRIF-DEPENDENT SIGNALING Approaches to manipulating TRIF signaling Pharmacological modulation of the TRIF pathway is possible through a variety of approaches. For example, poly (I:C) is a well-characterized TLR3 agonist that activates the TRIF pathway. However, it needs to be pointed out that poly(I:C) can also activate RLRs if it gains access to the cytosol [187]. Certain TLR4 agonists are also relatively selective in activating the TRIF pathway over the MAL/MyD88 signaling arm; monophosphoryl lipid A is an example [188]. Finally, type-I-IFNs are a major product of TRIF-mediated signaling, so they can partially mimic the biological effects of TRIF agonism. Conversely, a number of strategies can be employed to antagonize TRIF responses. At the receptor level, lipid A analogues such as Eritoran inhibit TLR4- dependent TRIF signaling [189]. Similarly, a small molecule TLR3 antagonist that competes with dsRNA binding to the receptor has been reported [190]. The crystal structure of the NTD of TRIF, which inhibits TRIF-mediated signaling via autoinhibition, has recently been determined [54]. The knowledge of the structure could potentially be exploited in the future for the development of peptide and small molecule inhibitors of the TRIF pathway. In a similar fashion, the vast array of host- and pathogen-derived negative regulators of TRIF signaling that have been identified (Figure 3, Table 2) may ultimately lead to the development of new approaches for selective targeting of this pathway. For example, a TLR4-inhibitory peptide derived from the VACV protein A46 has been shown to bind directly to MAL and TRAM [191], providing proof-of-concept in specific targeting of TIR-mediated signaling to decrease inflammation.

Therapeutic potential for TRIF modulation in infectious and inflammatory diseases TRIF-mediated signaling contributes to the clearance of a number of viral and bacterial pathogens, and thus TRIF agonists could potentially be exploited in a number of infectious diseases. Certainly, such an approach has been considered for acquired immune deficiency syndrome (AIDS), because HIV suppresses IRF3 to evade host innate defence, and TRIF-mediated signaling directly activates IRF3. Consequently, the TLR3 agonist Ampligen has been trialed as a monotherapy for AIDS patients [192, 193]. Poly(I:C) protected mice against the Gram-negative bacterial pathogens Y. enterocolitica and S. Typhimurium [8]. TRIF agonism is a particularly attractive approach for vaccine adjuvants, and indeed, several TRIF- targeted adjuvants have already been tested in vaccines against a number of pathogens, including tuberculosis, influenza viruses and HIV [193-195]. The TRIF- biased TLR4 agonist monophosphoryl lipid A is also used as a vaccine adjuvant against hepatitis B and cervical cancer [196, 197]. Thus, there is certainly precedent for manipulating the TRIF pathway to protect against, or treat, at least some infectious diseases. The protective functions of the TRIF signaling arm appear to extend well beyond infectious diseases, with beneficial roles reported in animal models of cardiovascular disease, arthritis, colitis and tissue repair (see above). Hence, trials of TRIF agonists in animal models of such diseases are clearly warranted. Indeed, beneficial effects of the TLR3 agonist poly(I:C) have already been reported in mouse models of hyperlipidemic arterial injury [178], arthritis [182], colitis [198] and wound repair [199, 200]. However, it should also be noted that the literature is sometimes conflicting as to the role of the TRIF pathway in individual inflammation- related disease settings, and in many cases, further studies are required to determine whether TRIF agonism is likely to have beneficial effects. On the other hand, TRIF antagonists probably have the most potential in the context of viral infections of the respiratory system, where antagonism of TLR3 signaling could reduce immunopathology. As an example, two peptides derived from the TIR domain of TRIF have been found to block TRIF signaling [201, 202]. One peptide was shown

26 to bind most strongly to TLR4, whereas the other binds TRAM. One of the peptides protected mice from a lethal LPS challenge in a mouse model of TLR4-driven inflammation. However, the beneficial effects of TRIF signaling in controlling bacterial infections targeting the respiratory tract somewhat tempers the enthusiasm for this potential application. Selective antagonism of TLR4-dependent TRIF signaling may also hold some promise for cardiovascular disease [179, 180], although further studies are required to clarify the contribution of this specific signaling arm to atherogenesis.

CONCLUSIONS TRIF-dependent signaling clearly has a central role in host defence, as evidenced by the numerous pathogen subversion strategies that have evolved to target this pathway (Figure 3). In the future, this knowledge, along with an increased understanding of host regulatory mechanisms that control TRIF signaling, is likely to provide exciting new opportunities for modulating TRIF-dependent responses. TRIF antagonists may have potential for reducing immunopathology in some infectious diseases, particularly viral infections of the respiratory tract. Conversely, the host protective functions of TRIF in numerous infectious and non-infectious settings suggest that agonism of the TRIF pathway may have beneficial effects in a number of disease settings. Such beneficial effects have been observed in pre-clinical trials involving animal models of pathologies, including wound and tissue repair. A case- in-point for successful targeting of the TRIF pathways has been the clinical development of TRIF-activating vaccine adjuvants. However, given the contrasting roles of TRIF and TLR3 signaling in tissue-specific (e.g. lung, liver) versus systemic functions in host defence and inflammation, future strategies aimed at modulating the TRIF pathway for therapeutic applications will need to be carefully considered and delivery strategies tailored to prevent any detrimental or unintended side- effects.

27 Acknowledgements The research in the authors’ laboratories was supported by the National Health and Medical Research Council (NHMRC grants 1003326 and 1107804 to BK and AM). BK is an NHMRC Research Fellow (1003325 and 1110971). MJS is the recipient of an Australian Research Council Future Fellowship (FT100100657), and an Honorary NHMRC Senior Research Fellowship (1003470).

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TABLE 1. Positive regulators of TRIF-dependent signaling pathways

Positive Description Origin Target Mechanism References Regulators SARM TLR adaptor Endogenous TRIF Interaction [9, 20] with TRIF CD16 Antigen in Endogenous Down-stream Activation of [57] human of TRIF Syk, IRF3, and monocytes STAT1 pathways CD14 Co-receptor in Endogenous Up-stream of Ligand delivery [58] TLR4 signaling TRIF to the lysosome, interaction with TLR3 TRIL TLR4 interactor Endogenous Up-stream of Interaction [69] with LRRs TRIF with TLR3 UNC93B Transmembrane Endogenous Up-stream of Chaperone [70] protein from the TRIF involved in TLR endoplasmic translocation to reticulum endosomes TRIM56, Members of Endogenous TRIF Acceleration of [74-76] TRIM62 tripartite motif the activation (TRIM) family of of IRF3, E3 ubiquitin induction of ligases IFN-β and ISGs Pellino E3 ubiquitin Endogenous Down-stream K63-linked [77] ligase of TRIF polyubiquitination of IRAK1, TBK1, and TAK1 BS69 A MYND Endogenous TRIF Induction of [81] (myeloid IFN-β promoter translocation and NF-κB protein 8, Nervy, activation

51 and DEAF-1) domain- containing protein LMP7, LMP2, Immuno- Endogenous IRF7, down- Interference [83-86] LMP10 proteasome stream of with the (MECL-1) subunits TRIF production of NO and expression of IRF7 WDFY1 WD repeat and Endogenous TRIF Recruitment of [87] FYVE-domain- TRIF to TLRs containing protein

52 TABLE 2. Negative regulators of TRIF-dependent signaling pathways

Negative Description Origin Target Mechanism Reference Regulators TAG TRAM adaptor Endogenous TRIF Displacement [88] with GOLD of TRIF from domain (TAG), a TRAM during splice variant of signaling TRAM adaptor TMED7 A GOLD domain- Endogenous TRIF Displacement [89] containing of TRIF from molecule TRAM during signaling HDL High density Endogenous TRAM Translocation [90] lipoprotein of TRAM into intracellular compartments TRIM38 Member of Endogenous TRIF Interaction [91] tripartite motif with TRIF and (TRIM) family of induces the E3 ubiquitin K48-linked ligases polyubiquitination and proteasomal degradation of TRIF DUBA Ovarian tumor Endogenous TRAF3 Binding and [92] domain- cleavage of containing K63-linked deubiquitinating poly-ubiquitin enzyme A chains on TRAF3 SOCS3 Suppressor of Endogenous TRAF Degradation of [93] cytokine proteins TRAF proteins signaling-3

53 (SOCS3), E3 ubiquitin lyase RAUL replication and Endogenous IRF3/7 Ubiquitination [94] transcription of IRF3/7 activator- associated ubiquitin ligase Pin1 Peptidyl-propyl Endogenous IRF3 Promotion of [95] isomerase polyubiquitination and proteasomal degradation of activated IRF3 PIAS1, Protein Endogenous TRIF Inhibition of [100-103] PIAS3, inhibitors of NF-κB and IFN- PIAS4 activated STAT β promoter (PIAS) family activity by with E3 SUMO interacting (small ubiquitin- with TRIF related modifier) ligase activity NDP52 Autophagy Endogenous TRIF Inhibition of [106] receptor IRF3 and NF- κB activation by interacting with TRIF Ubiquilin-1 Ubiquitin-like Endogenous TRIF Inhibition of [107] protein IFN-β promoter activation by interacting with TRIF CD300f Member of Endogenous Down-stream Activation of [108-110] CD300 family of TRIF SHP and PI3K and synthetic peptides based

54 on the sequence 3CD Hepatitis A virus Pathogen- TRIF Proteasomal [112] protease derived cleavage of polymerase- TRIF processing intermediate 3C protease Protease from Pathogen- TRIF Proteasomal [113] enterovirus 71 derived cleavage of TRIF 3C protease Protease from Pathogen- TRIF Proteasomal [114] CSV B3 derived cleavage of TRIF NS3/4A Protease from Pathogen- TRIF Proteasomal [115] HCV derived cleavage of TRIF A46 Viral protein Pathogen- TRIF, TRAM, Interaction [116, 117] from VACV derived MyD88, MAL with adaptors to prevent the activation of IRF3, NF-κB and MAPK A52R Viral protein Pathogen- TRAF6, Interaction (Harte et al., from VACV derived IRAK2 with TRAF6 2003) and IRAK2 HBeAg Antigen from Pathogen- TRAM, MAL Interaction [119] hepatitis B e derived with adaptors (HBeAg) VPR, Vif HIV proteins Pathogen- IRF3 Degradation of [120] derived IRF3 TcpC TIR domain- Pathogen- TRIF, MyD88 Interaction [122, 123] containing derived with TRIF and protein from MyD88 Escherichia coli CF073

55 Figure 1. Schematic representation of the primary structure of TIR domain- containing adaptor molecules, highlighting structural and functional motifs. MyD88 contains an N-terminal death domain, an IRAK-binding domain, an intermediate domain and a C-terminal TIR domain [203, 204]. MAL contains a PIP2 (phosphatidylinositol 4,5-biphosphate)-binding domain and PEST domain in the N- terminal region, and a TIR domain in the C-terminal region, which contains a TRAF6- binding motif [33, 34, 205]. TRIF has a globular helical NTD [54], a TBK1-binding motif [43], a TRAF6-binding motif and a TRAF2-binding motif [42], a TIR domain and a C-terminal RHIM (receptor-interacting protein homotypic interaction motif) [49]. TRAM contains an N-terminal endosomal localization (ELM) signal including a myristoylation motif and a C-terminal TIR domain [55, 206]. SARM has an N- terminal ARM-repeat region, two central sterile α-motifs (SAM) and a C-terminal TIR domain [207].

Figure 2. TRIF-dependent and MyD88-dependent TLR signaling pathways. TRIF-dependent signaling is responsible for the production of type-I-IFNs. TRIF interacts with different molecules and recruits TRAF3 (TNF receptor associated factor 3), TANK (TRAF family member-associated NF-κB), RIP1 and RIP3. TRAF3 undergoes self-ubiquitination via non-canonical K63-linked polyubiquitination, leading to complex formation with TBK1 and IKK-i/IKK-ε [41, 208-211]. This complex then phosphorylates TBK1 or IKK-I, which activates IRF3 by phosphorylation [212, 213]. Phosphorylated IRF3 forms homo-dimers and heterodimers with IRF7 and translocates to the nucleus. The dimer complex of IRFs binds to DNA target sequences to transcribe IFN and IFN-inducible genes [212, 213]. TRAF6 is involved in TRIF-dependent NF-κB activation. It can activate NF-κB by forming a complex with RIP1, FADD and TRADD (TNF receptor-associated death domain) [41, 208, 211]. The MyD88-dependent signaling pathways are responsible for the production of inflammatory cytokines. The activated TLR4 recruits MyD88 through MAL, which subsequently recruits IRAK family members [214, 215]. As a result, IRAK4 and IRAK1/2 are sequentially phosphorylated. IRAK1 or IRAK2 then associate with TRAF6 and form a complex, which activates TAK1 and IKK subunit NEMO (NF-κB essential modifier) by polyubiquitination. TAK1 then activates the IKK

56 and MAPK pathways [216-218]. In one pathway, TAK1 activates the IKK complex, which subsequently phosphorylates the inhibitory IκB protein. The phosphorylated IκB undergoes proteosomal degradation to activate and translocate NF-κB into the nucleus. NF-κB then induces the transcription of broad range of pro-inflammatory genes [219]. In the other pathway, TAK1 phosphorylates the MKK (MAPK kinase) family members [220]. The phosphorylated MKK3/6 and MKK4/7 subsequently activate p38 and JNK (c-Jun N-terminal kinase), respectively. Finally, through these two signaling pathways, AP1 is activated [220].

Figure 3. Positive and negative regulators of TRIF-dependent TLR signaling. Various host factors that promote TRIF-dependent signaling are displayed in blue, while host factors that negatively regulate this pathway are shown in dark yellow. Pathogen-derived factors that interfere with TRIF signaling are displayed in red.

Figure 4. The role of TRIF-dependent TLR signaling pathways in different pathological settings. TRIF-dependent TLR signaling pathways maintain host defence against bacterial and viral infections, particularly in mucosal organs such as the lung and the intestine. These signaling pathways are also important for host defence during systemic infections, myocarditis, neurological infections and neuro- protection.