TIR domain--containing adaptors regulate TLR-mediated signaling pathways

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses via signaling pathways mediated by several Toll/IL-1R (TIR) domain-containing adaptors such as MyD88, TIRAP, and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4 that is responsible for type I interferon production in response to double-stranded RNA and LPS, respectively. TIRAP specifically participates in the MyD88-dependent pathways shared by TLR2 and TLR4, and TRAM is essential for the TLR4-mediated MyD88-independent pathway. Thus, TIR domain-containing adaptors play an important role in the TLR mediated signaling pathways.     

Gout: new insights into an old disease

Gout is an autoinflammatory disorder associated with deposition of monosodium urate (MSU) crystals in joints and periarticular tissues. Recent advances suggest that the innate immune system may drive the gouty inflammatory response to MSU. These findings prompt questions concerning how the innate immune system recognizes MSU and the identities of the receptors involved. In this issue of the JCI, Chen et al. show that the IL-1 receptor and its signaling protein myeloid differentiation primary response protein 88 (MyD88) but not the "classical" innate immune receptors, TLRs, are central for MSU-induced inflammation (see the related article beginning on page 2262).     

MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide, and IL-1alpha

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll-IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88-/-) mice and TRIF-deficient (TRIF-/-) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1alpha stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF-/- mice, but not MyD88-/- mice. These factors stimulated receptor activator of nuclear factor-kappaB ligand mRNA expression in TRIF-/- osteoblasts, but not MyD88-/- osteoblasts. LPS stimulated IL-6 production in TRIF-/- osteoblasts, but not TRIF-/- macrophages. LPS and IL-1alpha enhanced the survival of TRIF-/- osteoclasts, but not MyD88-/- osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88-/- mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.     

Pathological mechanisms of gouty arthritis

Gout is a disease caused by the deposition of monosodium urate monohydrate (MSU) crystals. Precise mechanisms underlying the initiation of acute gout, however, are not known. Recent investigations provided novel evidence in the pathology of acute gout. A number of studies indicated that MSU crystals can act as a "danger signal" which resembles exogenous adjuvants, and toll-like receptor(TLR)-mediated pathways and/or MyD88-dependent IL-1 receptor pathways are involved in acute gout. Up-regulation of the triggering receptor expressed on myeloid cells 1(TREM-1) in phagocytes by the stimulation with MSU crystals has been demonstrated. Furthermore, pathological significance of NALP 3 inflammasome in gout has been also demonstrated. These findings provide a new insight into the mechanisms underlying the initiation of MSU crystal-induced acute inflammation.     

IL-1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice

The molecular mechanisms of acute lung injury resulting in inflammation and fibrosis are not well established. Here we investigate the roles of the IL-1 receptor 1 (IL-1R1) and the common adaptor for Toll/IL-1R signal transduction, MyD88, in this process using a murine model of acute pulmonary injury. Bleomycin insult results in expression of neutrophil and lymphocyte chemotactic factors, chronic inflammation, remodeling, and fibrosis. We demonstrate that these end points were attenuated in the lungs of IL-1R1– and MyD88-deficient mice. Further, in bone marrow chimera experiments, bleomycin-induced inflammation required primarily MyD88 signaling from radioresistant resident cells. Exogenous rIL-1β recapitulated a high degree of bleomycin-induced lung pathology, and specific blockade of IL-1R1 by IL-1 receptor antagonist dramatically reduced bleomycin-induced inflammation. Finally, we found that lung IL-1β production and inflammation in response to bleomycin required ASC, an inflammasome adaptor molecule. In conclusion, bleomycin-induced lung pathology required the inflammasome and IL-1R1/MyD88 signaling, and IL-1 represented a critical effector of pathology and therapeutic target of chronic lung inflammation and fibrosis.     

Leukotriene B4 amplifies NF-κB activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

Activation of NF-κB and 5-lipoxygenase-mediated (5-LO-mediated) biosynthesis of the lipid mediator leukotriene B4 (LTB4) are pivotal components of host defense and inflammatory responses. However, the role of LTB4 in mediating innate immune responses elicited by specific TLR ligands and cytokines is unknown. Here we have shown that responses dependent on MyD88 (an adaptor protein that mediates signaling through all of the known TLRs, except TLR3, as well as IL-1β and IL-18) are reduced in mice lacking either 5-LO or the LTB4 receptor BTL1, and that macrophages from these mice are impaired in MyD88-dependent activation of NF-κB. This macrophage defect was associated with lower basal and inducible expression of MyD88 and reflected impaired activation of STAT1 and overexpression of the STAT1 inhibitor SOCS1. Expression of MyD88 and responsiveness to the TLR4 ligand LPS were decreased by Stat1 siRNA silencing in WT macrophages and restored by Socs1 siRNA in 5-LO-deficient macrophages. These results uncover a pivotal role in macrophages for the GPCR BLT1 in regulating activation of NF-κB through Stat1-dependent expression of MyD88.     

Dual function of MyD88 in RAS signaling and inflammation, leading to mouse and human cell transformation

Accumulating evidence points to inflammation as a promoter of carcinogenesis. MyD88 is an adaptor molecule in TLR and IL-1R signaling that was recently implicated in tumorigenesis through proinflammatory mechanisms. Here we have shown that MyD88 is also required in a cell-autonomous fashion for RAS-mediated carcinogenesis in mice in vivo and for MAPK activation and transformation in vitro. Mechanistically, MyD88 bound to the key MAPK, Erk, and prevented its inactivation by its phosphatase, MKP3, thereby amplifying the activation of the canonical RAS pathway. The relevance of this mechanism to human neoplasia was suggested by the finding that MyD88 was overexpressed and interacted with activated Erk in primary human cancer tissues. Collectively, these results show that in addition to its role in inflammation, MyD88 plays what we believe to be a crucial direct role in RAS signaling, cell-cycle control, and cell transformation.     

Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like-interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain-containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain-containing adaptor inducing IFN-beta (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor kappaB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-beta by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence.     

Role of MyD88 and TLR9 in the innate immune response elicited by serotype 5 adenoviral vectors

A replication-incompetent adenoviral (Ad) vector is generating interest for both gene therapy and immunotherapy. A major limitation of the use of Ad vectors is the innate immune response, which causes inflammatory cytokine production and tissue damage; however, the precise mechanism of the innate immune response remains to be clarified. Here, we show that serotype 5 human Ad vectors elicit innate immune responses through a myeloid differentiating factor 88 (MyD88)/Toll-like receptor (TLR)-9-dependent and/or -independent manner according to cell type. After stimulation with Ad vectors, the production of interleukin (IL)-6 and IL-12 was significantly decreased in MyD88- or TLR9-deficient dendritic cells (DCs), compared with wild-type DCs. In addition, the surface expression of maturation marker proteins, such as CD40, CD80, CD86, and MHC class II, in MyD88- or TLR9-deficient granulocyte-macrophage colony-stimulating factor (GM-CSF)-DCs was similar to that in wild-type DCs. On the other hand, MyD88- or TLR9-deficient peritoneal macrophages produced the same level of IL-6 as wild-type macrophages after infection with Ad vectors. We did not find any differences in the mRNA expression levels of the molecules involved in innate immunity, such as MyD88, TLR3, TLR7, and TLR9, between DCs and macrophages. The intravenous injection of luciferase-expressing Ad vectors into MyD88- or TLR9-deficient mice resulted in almost comparable levels of IL-6 and IL-12 production and luciferase expression with wild-type mice. These results suggest that Ad vectors can activate innate immunity via MyD88/TLR9-dependent and -independent mechanisms.     

LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF

Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM) is the fourth TIR domain-containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-kappaB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-alpha/beta, regulated on activation, normal T cell expressed and secreted (RANTES), and gamma interferon-inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.     

MyD88-dependent induction of allergic Th2 responses to intranasal antigen

MyD88 is a common Toll-like receptor (TLR) adaptor molecule found to be essential for induction of adaptive Th1 immunity. Conversely, innate control of adaptive Th2 immunity has been shown to occur in a MyD88-independent manner. In this study, we show that MyD88 is an essential innate component in the induction of TLR4-dependent Th2 responses to intranasal antigen; thus we demonstrate what we believe to be a novel role for MyD88 in pulmonary Th2 immunity. Induction of the MyD88-independent type I IFN response to LPS is defective in the pulmonary environment. Moreover, in the absence of MyD88, LPS-induced upregulation of costimulatory molecule expression on pulmonary DCs is defective, in contrast to what has been observed with bone marrow-derived DCs (BMDCs). Reconstitution of Th2 responses occurs upon adoptive pulmonary transfer of activated BMDCs to MyD88-deficient recipients. Furthermore, the dependence of Th2 responses on MyD88 is governed by the initial route of antigen exposure; this demonstrates what we believe are novel site-specific innate mechanisms for control of adaptive Th2 immunity.     

Interleukin 1 receptor dependence of serum transferred arthritis can be circumvented by toll-like receptor 4 signaling

Inflammatory arthritis is associated with the release of a network of key cytokines. In T cell receptor transgenic K/BxN mice interleukin (IL)-1 plays a key role in joint swelling and destruction, as suggested by the ability of anti-IL-1receptor (IL-1R) antibody treatment to delay the onset and slow the progression of this disease. This mechanism is dependent on the signaling pathway intermediary myeloid differentiation factor 88 (MyD88), such that neither IL-1R nor MyD88-deficient mice developed visually detectable synovitis after transfer of arthritogenic sera. The Toll-like receptors (TLRs) share the same signaling pathway through MyD88 as the IL-1R. The administration of a TLR-4 ligand, lipopolysaccharide, concomitant with arthritogenic serum in IL-1 receptor-deficient mice resulted in acute paw swelling, but not in MyD88-deficient mice. Also, serum transferred arthritis was not sustained in TLR-4 mutant mice compared with controls. These results suggest that innate immune functions via TLR-4 might perpetuate inflammatory mechanisms and bypass the need for IL-1 in chronic joint inflammation.     

髓样分化因子88多态性的研究进展

髓样分化因子88(MYD88)是细胞内传递信号的关键衔接蛋白,可介导多种Toll样受体(TLR)、白细胞介素1受体(IL-1R)及白细胞介素-18受体(IL-18R)的信号传递,在固有免疫中发挥显著作用。MYD88依赖通路在多种病原体致病过程中发挥作用,与肿瘤、感染性疾病、自身免疫性疾病等密切相关。MYD88依赖通路被认为是这些疾病治疗的关键靶点。MYD88基因L265P突变导致MYD88蛋白Toll-白细胞介素1受体域(TIR)的265位氨基酸发生改变。该突变可通过加强核因子-κB(NF-κB)等转录因子促进酪氨酸激酶-信号转导子和转录激活子3(JAK-STAT3)信号通路传导,介导白细胞介素(IL)-6、IL-10及β-干扰素(IFN-β)等炎症因子的产生。文章从MYD88的结构、基本功能、在信号传导通路中的作用以及MYD88基因L265P突变与疾病的关联等方面进行综述。     

Fatal Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88

Toll-like receptors (TLRs) such as TLR2 and TLR4 have been implicated in host response to mycobacterial infection. Here, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with Mycobacterium tuberculosis (MTB). While primary MyD88(-/-) macrophages and DCs are defective in TNF, IL-12, and NO production in response to mycobacterial stimulation, the upregulation of costimulatory molecules CD40 and CD86 is unaffected. Aerogenic infection of MyD88(-/-) mice with MTB is lethal within 4 weeks with 2 log(10) higher CFU in the lung; high pulmonary levels of cytokines and chemokines; and acute, necrotic pneumonia, despite a normal T cell response with IFN-gamma production to mycobacterial antigens upon ex vivo restimulation. Vaccination with Mycobacterium bovis bacillus Calmette-Guerin conferred a substantial protection in MyD88(-/-) mice from acute MTB infection. These data demonstrate that MyD88 signaling is dispensable to raise an acquired immune response to MTB. Nonetheless, this acquired immune response is not sufficient to compensate for the profound innate immune defect and the inability of MyD88(-/-) mice to control MTB infection.     

Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6

Transition of immature antigen presenting cells (APCs) to the state of professional APCs is essential for initiation of cell-mediated immune responses to pathogens. Signal transduction via molecules of the Toll-like receptor (TLR)/interleukin 1 receptor (IL-1R) pathway is critical for activation of APCs either by pathogen-derived pattern ligands like lipopolysaccharides (LPS) or by CD40 ligation through T helper cells. The capacity of bacterial DNA (CpG-DNA) to induce APCs to differentiate into professional APCs represents an interesting discovery. However, the signaling pathways involved are poorly understood. Here we show that CpG-DNA activates the TLR/IL-1R signaling pathway via the molecules myeloid differentiation marker 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6), leading to activation of kinases of the IkappaB kinase complex and the c-jun NH(2)-terminal kinases. Moreover, cells of TLR2- and TLR4-deficient mice are activated by CpG-DNA, whereas cells of MyD88-deficient mice do not respond. The data suggest that CpG-DNA initiates signaling via the TLR/IL-1R pathway in APCs in a manner similar to LPS and to T helper cell-mediated CD40 ligation. Activation of the TLR/IL-1R signaling pathway by foreign bacterial DNA may be one way to initiate innate defense mechanisms against infectious pathogens in vivo.     

Haptoglobin activates innate immunity to enhance acute transplant rejection in mice

Immune tolerance to transplanted organs is impaired when the innate immune system is activated in response to the tissue necrosis that occurs during harvesting and implantation procedures. A key molecule in this immune pathway is the intracellular TLR signal adaptor known as myeloid differentiation primary response gene 88 (MyD88). After transplantation, MyD88 induces DC maturation as well as the production of inflammatory mediators, such as IL-6 and TNF-α. However, upstream activators of MyD88 function in response to transplantation have not been identified. Here, we show that haptoglobin, an acute phase protein, is an initiator of this MyD88-dependent inflammatory process in a mouse model of skin transplantation. Necrotic lysates from transplanted skin elicited higher inflammatory responses in DCs than did nontransplanted lysates, suggesting DC-mediated responses are triggered by factors released during transplantation. Analysis of transplanted lysates identified haptoglobin as one of the proteins upregulated during transplantation. Expression of donor haptoglobin enhanced the onset of acute skin transplant rejection, whereas haptoglobin-deficient skin grafts showed delayed acute rejection and antidonor T cell priming in a MyD88-dependent graft rejection model. Thus, our results show that haptoglobin release following skin necrosis contributes to accelerated transplant rejection, with potential implications for the development of localized immunosuppressive therapies.     

Homeostatic MyD88-dependent signals cause lethal inflamMation in the absence of A20

Toll-like receptors (TLRs) on host cells are chronically engaged by microbial ligands during homeostatic conditions. These signals do not cause inflammatory immune responses in unperturbed mice, even though they drive innate and adaptive immune responses when combating microbial infections. A20 is a ubiquitin-modifying enzyme that restricts exogenous TLR-induced signals. We show that MyD88-dependent TLR signals drive the spontaneous T cell and myeloid cell activation, cachexia, and premature lethality seen in A20-deficient mice. We have used broad spectrum antibiotics to demonstrate that these constitutive TLR signals are driven by commensal intestinal flora. A20 restricts TLR signals by restricting ubiquitylation of the E3 ligase tumor necrosis factor receptor-associated factor 6. These results reveal both the severe proinflammatory pathophysiology that can arise from homeostatic TLR signals as well as the critical role of A20 in restricting these signals in vivo. In addition, A20 restricts MyD88-independent TLR signals by inhibiting Toll/interleukin 1 receptor domain-containing adaptor inducing interferon (IFN) beta-dependent nuclear factor kappaB signals but not IFN response factor 3 signaling. These findings provide novel insights into how physiological TLR signals are regulated.