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