Protective T cell response against intracellular pathogens in the absence of Toll-like receptor signaling via myeloid differentiation factor 88

Toll-like receptors (TLR) have been indicated as germline-encoded receptors for sensing a variety of pathogens. Although the role of TLR in innate immunity is beyond question, their function in acquired immunity, in particular in T cell immunity, is less clear. Here, we used experimental Listeria monocytogenes infection of mice to analyze requirements for TLR2, TLR4 and the central TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the generation of specific T cell responses. We demonstrate that following L. monocytogenes infection, mice deficient in TLR2, TLR4 and MyD88 can generate Listeria-specific CD8+ and CD4+ Th1 responses. These T cell responses are sufficient to control secondary infection with a high dose of L. monocytogenes even in the absence of TLR signaling via MyD88. Thus, TLR2-, TLR4- and MyD88-dependent signals are not essential for the generation of CD4+ Th1 and CD8+ T cells, and T cells can protect mice against infection in the absence of these signals.     

TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses through signaling pathways mediated by Toll-interleukin 1 receptor (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. Here we have identified a fourth TIR domain-containing adaptor, TRIF-related adaptor molecule (TRAM), and analyzed its physiological function by gene targeting. TRAM-deficient mice showed defects in cytokine production in response to the TLR4 ligand, but not to other TLR ligands. TLR4- but not TLR3-mediated MyD88-independent interferon-beta production and activation of signaling cascades were abolished in TRAM-deficient cells. Thus, TRAM provides specificity for the MyD88-independent component of TLR4 signaling.     

Suppression of allergic reaction by λ-carrageenan: Toll-like receptor 4/MyD88-dependent and -independent modulation of immunity

BACKGROUND: Recognition of foreign substances by innate immunity through pattern recognition receptors (PRRs) regulates acquired immunity such as allergic reaction. Because PRRs recognize heterogeneous ligands, daily food intake can potentially regulate immune allergic reaction. OBJECTIVE: Elucidation of the effect of lambda-carrageenan on allergic reactions was aimed. METHOD: IFN-gamma and IL-4 was measured in in vitro T cell-stimulated culture. Cytokine production from macrophages in response to lambda-carrageenan was measured as indicator for innate immunity activation. Mice were immunized with OVA in alum to induce specific IgE, and then histamine release was induced by systemic injection of OVA. RESULTS: Activation of innate immunity by lambda-carrageenan is dependent on Toll-like receptor-4 (TLR4) and MyD88, in which induction of pro-inflammatory cytokines such as TNF-alpha and IL-6 was largely impaired in macrophages from TLR4- and MyD88-deficient mice. Footpad oedema, a model for in vivo inflammatory reactions, was significantly reduced in these mice. Similar to recent evidence showing a preference for the stimulation of Th1 via TLR/MyD88 signalling, lambda-carrageenan showed enhanced IFN-gamma and decreased IL-4 in stimulated T cell cultures. Interestingly, increased IFN-gamma production was still seen in TLR4- and MyD88-deficient splenocytes. Oral administration of lambda-carrageenan to immunized mice successfully decreased OVA-specific IgE, and lambda-carrageenan was also effective in previously immunized mice. Further, serum histamine release upon systemic challenge of OVA was significantly inhibited. Neither OVA-specific IgG1/IgG2a nor cytokine secretion from in vitro cultures were altered, suggesting the involvement of multiple PRRs as demonstrated by TLR4/MyD88-independent IFN-gamma up-regulation. The simultaneous feeding of OVA with lipopolysaccharide abrogated oral tolerance, but lambda-carrageenan was not only devoid of such an effect but was also found to promote oral tolerance in the absence of TLR4. CONCLUSION: lambda-Carrageenan was suggested to be a useful dietary supplement to ameliorate allergic reactions while maintaining oral tolerance-dependent intestinal homeostasis.     

TLR signaling pathways

Toll-like receptors (TLRs) have been established to play an essential role in the activation of innate immunity by recognizing specific patterns of microbial components. TLR signaling pathways arise from intracytoplasmic TIR domains, which are conserved among all TLRs. Recent accumulating evidence has demonstrated that TIR domain-containing adaptors, such as MyD88, TIRAP, and TRIF, modulate TLR signaling pathways. MyD88 is essential for the induction of inflammatory cytokines triggered by all TLRs. TIRAP is specifically involved in the MyD88-dependent pathway via TLR2 and TLR4, whereas TRIF is implicated in the TLR3- and TLR4-mediated MyD88-independent pathway. Thus, TIR domain-containing adaptors provide specificity of TLR signaling.     

Deficiencies of myeloid differentiation factor 88, Toll-like receptor 2 (TLR2), or TLR4 produce specific defects in macrophage cytokine secretion induced by Helicobacter pylori

Helicobacter pylori is a gram-negative microaerophilic bacterium that colonizes the gastric mucosa, leading to disease conditions ranging from gastritis to cancer. Toll-like receptors (TLRs) play a central role in innate immunity by their recognition of conserved molecular patterns on bacteria, fungi, and viruses. Upon recognition of microbial components, these TLRs associate with several adaptor molecules, including myeloid differentiation factor 88 (MyD88). To investigate the contribution of the innate immune system to H. pylori infection, bone marrow-derived macrophages from mice deficient in TLR2, TLR4, TLR9, and MyD88 were infected with H. pylori SS1 and SD4 for 24 or 48 h. We demonstrate that MyD88 was essential for H. pylori induction of all cytokines investigated except alpha interferon (IFN-alpha). The secretion of IFN-alpha was substantially increased from cells deficient in MyD88. H. pylori induced interleukin-12 (IL-12) and IL-10 through TLR4/MyD88 signaling. In addition, H. pylori induced less IL-6 and IL-1beta in TLR2-deleted macrophages, suggesting that the MyD88 pathway activated by TLR2 stimulation is responsible for H. pylori induction of the host proinflammatory response (IL-6 and IL-1beta). These observations are important in light of a recent report on IL-6 and IL-1beta playing a role in the development of H. pylori-related gastric cancer. In conclusion, our study demonstrates that H. pylori activates TLR2 and TLR4, leading to the secretion of distinct cytokines by macrophages.     

Toll-like receptor]

Toll-like receptors (TLRs) have been revealed to recognize specific patterns of microbial components. Recognition of microbial components by TLRs initiates signal transduction pathways, triggering expression of genes, which products control innate immune responses and further instruct development of antigen-specific acquired immunity. TIR domain-containing adaptors, such as MyD88, TIRAP, TRIF, and TRAM, play pivotal roles in TLR signaling pathways. Differential utilization of these TIR domain-containing adaptors provides specificity of individual TLR-mediated signaling pathways. TLR-mediated activation of innate immunity, when in excess, leads to immune disorders such as inflammatory bowel diseases. Therefore, several mechanisms that negatively control TLR signaling pathways and thereby prevent overactivation of innate immunity have been elucidated. Nuclear IkappaB proteins, such as Bcl-3 and IkappaBNS, have been revealed to be responsible for this process, by differentially inhibiting TLR-dependent cytokine production.     

TIR domain-containing adaptors define the specificity of TLR signaling

The concept that Toll-like receptors (TLRs) recognize specific molecular patterns in various pathogens has been established. In signal transduction via TLRs, MyD88, which harbors a Toll/IL-1 receptor (TIR)-domain and a death domain, has been shown to link between TLRs and MyD88-dependent downstream events leading to proinflammatory cytokine production and splenocyte proliferation. However, recent studies using MyD88-deficient mice have revealed that some TLRs possess a MyD88-independent pathway, which is represented by interferon (IFN)-beta production induced by LPS stimulation. This indicates that additional signaling molecules other than MyD88 exist in the TLR signaling pathway. Indeed, two additional TIR domain-containing adaptors, TIRAP/Mal and TRIF, have recently been identified. Both define the specific biological responses of each TLR.     

Binding specificity of Toll-like receptor cytoplasmic domains

MyD88 participates in signal transduction by binding to the cytoplasmic Toll/IL-1 receptor (TIR) domains of activated Toll-like receptors (TLR). Yeast two-hybrid experiments reveal that the TIR domains of human TLR differ in their ability to associate with MyD88: The TIR of TLR2 binds to MyD88 but the TIR of the closely related TLR1, 6, or 10 do not. Using chimeric TIR domains, we define the critical region responsible for differential MyD88 binding, and use a computational analysis of the critical region to reveal the amino acids that differ between MyD88 binders and non-binders. Remarkably, a single missense mutation created in TLR1 (N672D) confers on it the ability to bind MyD88, without affecting its association with other proteins. Mutations identified as critical for MyD88 binding also affect signaling of TLR pairs in mammalian cells. To investigate the difference between MyD88 binders and non-binders, we identify novel interacting proteins for each cytoplasmic domain of TLR1, 2, 6, and 10. For example, heat shock protein (HSP)60 binds to TLR1 but not to TLR2, and HSP60 and MyD88 appear to bind the same region of the TIR domain. In summary, interactions between the TLR, MyD88, and novel associated proteins have been characterized.     

Molecular analysis of the binding mode of Toll/interleukin-1 receptor (TIR) domain proteins during TLR2 signaling

Toll-like receptor (TLR) signaling is initiated by the binding of various adaptor proteins through ligand-induced oligomerization of the Toll/interleukin-1 receptor (TIR) domains of the TLRs. TLR2, which recognizes peptidoglycans, lipoproteins or lipopeptides derived from Gram-positive bacteria, is known to use the TIR domain-containing adaptor proteins myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal). Molecular analyses of the binding specificity of MyD88, Mal, and TLR2 are important for understanding the initial defenses mounted against Gram-positive bacterial infections such as Streptococcus pneumoniae. However, the detailed molecular mechanisms involved in the multiple interactions of these TIR domains remain unclear. Our study demonstrates that the TIR domain proteins MyD88, Mal, TLR1, and TLR2 directly bind to each other in vitro. We have also identified two binding interfaces of the MyD88 TIR domain for the TLR2 TIR domain. A residue at these interfaces has recently been found to be mutated in innate immune deficiency patients. These novel insights into the binding mode of TIR proteins will contribute to elucidation of the mechanisms underlying innate immune deficiency diseases, and to future structural studies of hetero-oligomeric TIR-TIR complexes.     

Endotoxin can induce MyD88-deficient dendritic cells to support T(h)2 cell differentiation

Toll-like receptor (TLR) signaling activates dendritic cells (DC) to secrete proinflammatory cytokines and up-regulate co-stimulatory molecule expression, thereby linking innate and adaptive immunity. A TLR-associated adapter protein, MyD88, is essential for cytokine production induced by TLR. However, in response to a TLR4 ligand, lipopolysaccharide (LPS), MyD88-deficient (MyD88(-/-)) DC can up-regulate co-stimulatory molecule expression and enhance their T cell stimulatory activity, indicating that the MyD88-independent pathway through TLR4 can induce some features of DC maturation. In this study, we have further characterized function of LPS-stimulated, MyD88(-/-) DC. In response to LPS, wild-type DC could enhance their ability to induce IFN-gamma production in allogeneic mixed lymphocyte reaction (alloMLR). In contrast, in response to LPS, MyD88(-/-) DC augmented their ability to induce IL-4 instead of IFN-gamma in alloMLR. Impaired production of T(h)1-inducing cytokines in MyD88(-/-) DC cannot fully account for their increased T(h)2 cell-supporting ability, because absence of T(h)1-inducing cytokines in DC caused impairment of IFN-gamma, but did not lead to augmentation of IL-4 production in alloMLR. In vivo experiments with adjuvants also revealed T(h)2-skewed immune responses in MyD88(-/-) mice. These results demonstrate that the MyD88-independent pathway through TLR4 can confer on DC the ability to support T(h)2 immune responses.     

Molecular analysis of the binding mode of Toll/interleukin-1 receptor (TIR) domain proteins during TLR2 signaling

Toll-like receptor (TLR) signaling is initiated by the binding of various adaptor proteins through ligand-induced oligomerization of the Toll/interleukin-1 receptor (TIR) domains of the TLRs. TLR2, which recognizes peptidoglycans, lipoproteins or lipopeptides derived from Gram-positive bacteria, is known to use the TIR domain-containing adaptor proteins myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal). Molecular analyses of the binding specificity of MyD88, Mal, and TLR2 are important for understanding the initial defenses mounted against Gram-positive bacterial infections such as Streptococcus pneumoniae. However, the detailed molecular mechanisms involved in the multiple interactions of these TIR domains remain unclear. Our study demonstrates that the TIR domain proteins MyD88, Mal, TLR1, and TLR2 directly bind to each other in vitro. We have also identified two binding interfaces of the MyD88 TIR domain for the TLR2 TIR domain. A residue at these interfaces has recently been found to be mutated in innate immune deficiency patients. These novel insights into the binding mode of TIR proteins will contribute to elucidation of the mechanisms underlying innate immune deficiency diseases, and to future structural studies of hetero-oligomeric TIR–TIR complexes.     

Toll-like receptors in innate immunity

Functional characterization of Toll-like receptors (TLRs) has established that innate immunity is a skillful system that detects invasion of microbial pathogens. Recognition of microbial components by TLRs initiates signal transduction pathways, which triggers expression of genes. These gene products control innate immune responses and further instruct development of antigen-specific acquired immunity. TLR signaling pathways are finely regulated by TIR domain-containing adaptors, such as MyD88, TIRAP/Mal, TRIF and TRAM. Differential utilization of these TIR domain-containing adaptors provides specificity of individual TLR-mediated signaling pathways. Several mechanisms have been elucidated that negatively control TLR signaling pathways, and thereby prevent overactivation of innate immunity leading to fatal immune disorders. The involvement of TLR-mediated pathways in autoimmune and inflammatory diseases has been proposed. Thus, TLR-mediated activation of innate immunity controls not only host defense against pathogens but also immune disorders.     

Nonredundant roles of TIRAP and MyD88 in airway response to endotoxin, independent of TRIF, IL-1 and IL-18 pathways

Inhaled endotoxins induce an acute inflammatory response in the airways mediated through Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). However, the relative roles of the TLR4 adaptor proteins TIRAP and TRIF and of the MyD88-dependent IL-1 and IL-18 receptor pathways in this response are unclear. Here, we demonstrate that endotoxin-induced acute bronchoconstriction, vascular damage resulting in protein leak, Th1 cytokine and chemokine secretion and neutrophil recruitment in the airways are abrogated in mice deficient for either TIRAP or MyD88, but not in TRIF deficient mice. The contribution of other TLR-independent, MyD88-dependent signaling pathways was investigated in IL-1R1, IL-18R and caspase-1 (ICE)-deficient mice, which displayed normal airway responses to endotoxin. In conclusion, the TLR4-mediated, bronchoconstriction and acute inflammatory lung pathology to inhaled endotoxin critically depend on the expression of both adaptor proteins, TIRAP and MyD88, suggesting cooperative roles, while TRIF, IL-1R1, IL-18R signaling pathways are dispensable.     

Toll-like receptors control activation of adaptive immune responses

Mechanisms that control the activation of antigen-specific immune responses in vivo are poorly understood. It has been suggested that the initiation of adaptive immune responses is controlled by innate immune recognition. Mammalian Toll-like receptors play an essential role in innate immunity by recognizing conserved pathogen-associated molecular patterns and initiating the activation of NF-kappaB and other signaling pathways through the adapter protein, MyD88. Here we show that MyD88-deficient mice have a profound defect in the activation of antigen-specific T helper type 1 (TH1) but not TH2 immune responses. These results suggest that distinct pathways of the innate immune system control activation of the two effector arms of adaptive immunity.     

Toll-like receptor 9 is required for full host resistance to Mycobacterium avium infection but plays no role in induction of Th1 responses

To investigate the role of Toll-like receptor 9 (TLR9) in innate immunity to Mycobacterium avium, TLR9, TLR2, and MyD88 knockout (KO) mice were infected with this bacterium. Bacterial burdens were higher in the spleens, livers, and lungs of infected TLR9 KO mice than in those of C57BL/6 mice, indicating that TLR9 is required for efficient control of M. avium infection. However, TLR9 KO or TLR2 KO spleen cells displayed normal M. avium-induced tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) responses. This finding was confirmed by determining the number of splenic CD4(+) T cells producing IFN-γ by flow cytometry. Furthermore, TLR2 and MyD88, but not TLR9, played a major role in interleukin-12 and TNF-α production by M. avium-infected macrophages and dendritic cells (DCs). We also found that major histocompatibility complex class II molecule expression on DCs is regulated by TLR2 and MyD88 signaling but not by TLR9. Finally, lack of TLR9, TLR2, or MyD88 reduced the numbers of macrophages, epithelioid cells, and lymphocytes in M. avium-induced granulomas but only MyD88 deficiency affected the number of liver granulomas. In summary, our data demonstrated that the involvement of TLR9 in the control of M. avium infection is not related to the induction of Th1 responses.