Toll样受体信号通路中MyD88的研究进展

MyD88是Toll样受体信号通路中的重要转导蛋白,其依赖的信号通路以及调控的基因产物在固有免疫和适应性免疫中均发挥着关键作用。本文对MyD88及其依赖的信号通路做一简要综述,以期为临床预防和治疗疾病提供新的思路和方法。     

MyD88-dependent signaling protects against anthrax lethal toxin-induced impairment of intestinal barrier function

MyD88-deficient mice were previously shown to have increased susceptibility to Bacillus anthracis infection relative to wild-type animals. To determine the mechanism by which MyD88 protects against B. anthracis infection, knockout mice were challenged with nonencapsulated, toxigenic B. anthracis or with anthrax toxins. MyD88-deficient mice had increased susceptibility to B. anthracis and anthrax lethal toxin but not to edema toxin. Lethal toxin alone induced marked multifocal intestinal ulcers in the knockout animals, compromising the intestinal epithelial barrier. The resulting enteric bacterial leakage in the knockout animals led to peritonitis and septicemia. Focal ulcers and erosion were also found in MyD88-heterozygous control mice but with far lower incidence and severity. B. anthracis infection also induced a similar enteric bacterial septicemia in MyD88-deficient mice but not in heterozygous controls. We show that lethal toxin and B. anthracis challenge induce bacteremia as a result of intestinal damage in MyD88-deficient mice. These results suggest that loss of the intestinal epithelial barrier and enteric bacterial septicemia may contribute to sensitizing MyD88-deficient mice to B. anthracis and that MyD88 plays a protective role against lethal toxin-induced impairment of intestinal barrier.     

MyD88-dependent autoimmune disease in Lyn-deficient mice

Recent evidence suggests that systemic autoimmune disease depends on signals from TLR ligands, but little is known about how TLR-dependent pathways lead to the loss of self tolerance in vivo. To address this, we have examined the role of TLR signaling in Lyn-deficient mice, which develop an autoimmune disease similar to SLE. We found that absence of the TLR signaling adaptor molecule MyD88 suppresses plasma cell differentiation of switched and unswitched B cells, and prevents the generation of antinuclear IgG antibodies and glomerulonephritis. In mixed chimeras the increased IgM and IgG antibody secretion in Lyn-deficient mice is at least partially due to B cell-independent effects of Lyn. We now show that MyD88 deficiency blocks the expansion and activation of DC in which Lyn is also normally expressed, and prevents the hypersecretion of proinflammatory cytokines IL-6 and IL-12 by Lyn-deficient DC. These findings further highlight the important role of TLR-dependent signals in both lymphocyte activation and autoimmune pathogenesis.     

Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades

MyD88 is an adaptor molecule essential for signaling via the Toll-like receptor (TLR)/IL-1 receptor family. TLR4 is a member of the TLR family and a point mutation in the Tlr4 gene causes hyporesponsiveness to lipopolysaccharide (LPS) in C3H/HeJ mice. We have previously shown that both TLR4- and MyD88-deficient mice are hyporesponsive to LPS. In this study we examined the responsiveness of these two knockout mice to various bacterial cell wall components. Cells from TLR4-deficient mice responded to several kinds of LPS, peptidoglycan and crude cell wall preparation from Gram-positive bacteria and mycobacterial lysates. In contrast, macrophages and splenocytes from MyD88-deficient mice did not respond to any of the bacterial components we tested. These results show that MyD88 is essential for the cellular response to bacterial cell wall components.     

Toll-like receptor interactions: tolerance of MyD88-dependent cytokines but enhancement of MyD88-independent interferon-beta production

Toll-like receptors (TLRs) signal through two main pathways: a myeloid differentiation factor (MyD)88-dependent pathway that acts via nuclear factor kappaB (NF-kappaB) to induce proinflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and a MyD88-independent pathway that acts via type I interferons to increase the expression of interferon-inducible genes. Repeated signalling through TLR4 and a number of other TLRs has been reported to result in a reduction in the subsequent proinflammatory cytokine response, a phenomenon known as TLR tolerance. In this study we have shown that, whilst NF-kappaB activation and production of TNF-alpha and interleukin-12 by murine RAW264.7 and J774.2 cells in response to stimulation by TLR4, -5, -7 or -9, was reduced by prior stimulation with TLR4, -5, -7 or -9 ligands, the primary stimulation of TLR3, which does not use the MyD88 pathway, did not reduce the TNF-alpha or interleukin-12 responses to subsequent TLR stimulation. The response to TLR3 stimulation was not diminished by prior TLR ligand exposure. Furthermore, the production of interferon-beta (IFN-beta) following stimulation of TLR3 or -4, which is MyD88-independent, was increased by prior activation of TLR4, -5, -7 or -9. In contrast, TLR9 ligand-induced IFN-beta production, which is MyD88-dependent, was tolerized by prior TLR stimulation. These results are consistent with differential regulation of MyD88-dependent and MyD88-independent cytokine production following serial activation of TLRs.     

MyD88-dependent signaling contributes to protection following Bacillus anthracis spore challenge of mice: implications for Toll-like receptor signaling

Bacillus anthracis is a spore-forming, gram-positive organism that is the causative agent of the disease anthrax. Recognition of Bacillus anthracis by the host innate immune system likely plays a key protective role following infection. In the present study, we examined the role of TLR2, TLR4, and MyD88 in the response to B. anthracis. Heat-killed Bacillus anthracis stimulated TLR2, but not TLR4, signaling in HEK293 cells and stimulated tumor necrosis factor alpha (TNF-alpha) production in C3H/HeN, C3H/HeJ, and C57BL/6J bone marrow-derived macrophages. The ability of heat-killed B. anthracis to induce a TNF-alpha response was preserved in TLR2-/- but not in MyD88-/- macrophages. In vivo studies revealed that TLR2-/- mice and TLR4-deficient mice were resistant to challenge with aerosolized Sterne strain spores but MyD88-/- mice were as susceptible as A/J mice. We conclude that, although recognition of B. anthracis occurs via TLR2, additional MyD88-dependent pathways contribute to the host innate immune response to anthrax infection.     

黄芩苷对结核分枝杆菌作用下TLR2-MyD88信号通路的影响

目的:观察黄芩苷是否存在对TLR2和MyD88的调节,以探寻其抗结核的可能作用机制。方法:取黄芩苷单体作用于感染结核杆菌的U937单核巨噬细胞,分别采用RT-QPCR、Western blot和FCM方法检测用药前后TLR2和MyD88的表达。结果:与对照组相比,模型组TLR2及MyD88表达均显著降低(P<0.05);而与模型组相比,黄芩苷组TLR2及MyD88 mRNA表达则显著增高(P<0.05),TLR2蛋白表达亦显著增高(P<0.05)。结论:黄芩苷具有上调TLR2和MyD88的作用,从而为黄芩苷抗结核机制的揭示提供了依据。     

Mucus clearance, MyD88-dependent and MyD88-independent immunity modulate lung susceptibility to spontaneous bacterial infection and inflammation

It has been postulated that mucus stasis is central to the pathogenesis of obstructive lung diseases. In Scnn1b-transgenic (Scnn1b-Tg⁺) mice, airway-targeted overexpression of the epithelial Na⁺ channel β subunit causes airway surface dehydration, which results in mucus stasis and inflammation. Bronchoalveolar lavage from neonatal Scnn1b-Tg⁺ mice, but not wild-type littermates, contained increased mucus, bacteria, and neutrophils, which declined with age. Scnn1b-Tg⁺ mice lung bacterial flora included environmental and oropharyngeal species, suggesting inhalation and/or aspiration as routes of entry. Genetic deletion of the Toll–interleukin-1 receptor adapter molecule MyD88 in Scnn1b-Tg⁺ mice did not modify airway mucus obstruction, but caused defective neutrophil recruitment and increased bacterial infection, which persisted into adulthood. Scnn1b-Tg⁺ mice derived into germ-free conditions exhibited mucus obstruction similar to conventional Scnn1b-Tg⁺ mice and sterile inflammation. Collectively, these data suggest that dehydration-induced mucus stasis promotes infection, compounds defects in other immune mechanisms, and alone is sufficient to trigger airway inflammation.     

Necrotic neurons enhance microglial neurotoxicity through induction of glutaminase by a MyD88-dependent pathway

Background  

Microglia are macrophage-like cells that constantly sense the microenvironment within the central nervous system (CNS). In the event of neuronal stress or injury, microglial cells rapidly react and change their phenotype. This response may lead to a deleterious type of microglial activation, which is often associated with neuroinflammation and neurotoxicity in several neuropathological conditions. We investigated the molecular mechanisms underlying triggering of microglial activation by necrotic neuronal damage.     

Epithelial-specific blockade of MyD88-dependent pathway causes spontaneous small intestinal inflammation

Accumulating evidence suggests a role for Toll-like receptor (TLR) signaling at the intestinal epithelial cells (IECs) level for intestinal protection against exogenous injury or pathogenic infection. We hypothesized that MyD88 dependent TLR signaling at intestinal epithelium is critical for mucosal immune homeostasis. In the current study, a transgenic mouse model was generated in which a dominant-negative mutant of MyD88 (dnMyD88) was driven by an intestinal epithelial-specific murine villin promoter. Aged transgenic mice spontaneously developed chronic small intestinal inflammation, as revealed by increased CD4+ and CD8+ lymphocytes, neutrophil and macrophage infiltration, increased production of cytokines as TNF-α, IFN-γ, IL-1β, and IL-17, crypt abscesses, lymphedema, and Goblet cell depletion. The chronic inflammation was not due to increased epithelial apoptosis or permeability, but to a decreased Paneth cell-derived α-defensins (cryptdins) and RegIII-γ and increased commensal bacteria translocation. Thus, epithelial MyD88-dependent pathway plays an essential role in limiting mucosal microflora penetration and preventing mucosal immunoregulation disturbance in vivo.     

CD14 is required for MyD88-independent LPS signaling

The recessive mutation 'Heedless' (hdl) was detected in third-generation N-ethyl-N-nitrosourea-mutated mice that showed defective responses to microbial inducers. Macrophages from Heedless homozygotes signaled by the MyD88-dependent pathway in response to rough lipopolysaccharide (LPS) and lipid A, but not in response to smooth LPS. In addition, the Heedless mutation prevented TRAM-TRIF-dependent signaling in response to all LPS chemotypes. Heedless also abolished macrophage responses to vesicular stomatitis virus and substantially inhibited responses to specific ligands for the Toll-like receptor 2 (TLR2)-TLR6 heterodimer. The Heedless phenotype was positionally ascribed to a premature stop codon in Cd14. Our data suggest that the TLR4-MD-2 complex distinguishes LPS chemotypes, but CD14 nullifies this distinction. Thus, the TLR4-MD-2 complex receptor can function in two separate modes: one in which full signaling occurs and one limited to MyD88-dependent signaling.     

TLR9 triggers MyD88-independent anti-inflammatory signaling in lupus

Activation of Toll-like receptor 7 (TLR7) can induce lupus in mice, whereas activation of TLR9 can prevent it, even though both receptors interact with myeloid differentiation primary response gene 88 (MyD88) for downstream signaling. How TLR9 triggers anti-inflammatory responses in autoimmunity is unclear. Leibler et al. recently reported that TLR9 initiates anti-inflammatory signaling and inhibits lupus pathogenesis in a MyD88-independent but ligand-dependent manner.     

MyD88-dependent pathways mediate resistance to Cryptosporidium parvum infection in mice

Cryptosporidium spp. cause diarrheal disease worldwide. Innate immune responses mediating resistance to this parasite are not completely understood. To determine whether MyD88-dependent pathways play a role in resistance to Cryptosporidium parvum, we compared the course of infection in MyD88(-/-) mice to that in their wild-type (WT) littermate controls. Three- to 4-week-old mice were infected with C. parvum, and infection was monitored by quantifying fecal oocyst shedding. Twelve days postinfection, the histology of the intestines was examined to quantify intestinal parasite burden and to determine if there were any pathological changes. Fecal oocyst shedding and intestinal parasite burden were significantly greater in MyD88(-/-) mice than in littermate controls. Nonetheless, both WT and MyD88(-/-) mice cleared the infection within 3 weeks. These results indicate that MyD88-dependent pathways are involved in mediating initial resistance to C. parvum. Since gamma interferon (IFN-gamma) is known to mediate resistance to C. parvum, we also studied infection in MyD88(-/-) mice and WT controls in which this cytokine was temporarily neutralized. Fecal oocyst shedding, as well as intestinal parasite burden, intestinal inflammation, and mortality, was significantly greater in MyD88(-/-) mice in which IFN-gamma was neutralized than in IFN-gamma-neutralized WT mice or in MyD88(-/-) mice in which this cytokine was active. These results suggest that MyD88 and IFN-gamma had an additive effect in conferring protection from C. parvum infection. While this study confirms the importance of IFN-gamma in conferring resistance to infection with C. parvum, it suggests that MyD88-mediated pathways also play a role in innate immunity to this parasite.     

Toll-like receptor-mediated tyrosine phosphorylation of paxillin via MyD88-dependent and -independent pathways

Toll-like receptor (TLR)-mediated recognition of pathogens represents one of the most important mechanisms of innate immunity. A proximal signaling event of TLR is the direct binding of an adaptor protein MyD88 to TLR and recruitment of the IL-1R-associated kinase (IRAK). In the present study, we examined the effect of several TLR ligands on protein tyrosine phosphorylation in rat macrophages. Macrophage-activating lipopeptide-2 kDa (MALP2) and lipoarabinomannan were used as activators of TLR2, while lipopolysaccharides (LPS) and lipoteichoic acid were used as TLR4 ligands. All these ligands induced tyrosine phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) and its substrate paxillin, an integrin-associated focal adhesion adaptor protein, in the macrophages. PP2, an inhibitor of Src family tyrosine kinases, prevented the TLR-induced phosphorylation of paxillin and Pyk2 without affecting TLR-induced IRAK activation. MALP2 failed to induce paxillin phosphorylation in the macrophages from MyD88-knockout mice. In contrast, the effect of LPS weakened, but was still observed even in the MyD88-deficient cells. Thus, TLR regulate the function of paxillin in an Src family-dependent mechanism through both MyD88-dependent and MyD88-independent pathways.     

Early MyD88-dependent induction of interleukin-17A expression during Salmonella colitis

The development of T helper 17 (T(H)17) cells is a well-established adaptive mechanism for the production of interleukin-17A (IL-17A), a cytokine involved in neutrophil recruitment. However, pathways contributing to mucosal expression of IL-17A during the initial phase of a bacterial infection have received less attention. Here we used the mouse colitis model of Salmonella enterica serotype Typhimurium infection to investigate the contribution of myeloid differentiation primary response protein 88 (MyD88) to inflammation and mucosal IL-17A expression. Expression of IL-23 in the cecal mucosa during S. Typhimurium colitis was dependent on the presence of MyD88. Furthermore, initial expression of IL-17A at 24 h after S. Typhimurium infection was dependent on MyD88 and the receptor for IL-1β. IL-23 and IL-1β synergized in inducing expression of IL-17A in splenic T cells in vitro. In the intestinal mucosa, IL-17A was produced by three distinct T cell populations, including δγ T cells, T(H)17 cells, and CD4(-)CD8(-) T cells. The absence of IL-1β signaling or IL-17 signaling reduced CXC chemokine expression but did not alter the overall severity of pathological lesions in the cecal mucosa. In contrast, cecal pathology and neutrophil recruitment were markedly reduced in Myd88-deficient mice during the initial phases of S. Typhimurium infection. Collectively, these data demonstrate that MyD88-dependent mechanisms, including an initial expression of IL-17A, are important for orchestrating early inflammatory responses during S. Typhimurium colitis.     

MyD88-dependent dendritic and epithelial cell crosstalk orchestrates immune responses to allergens

Sensitization to inhaled allergens is dependent on activation of conventional dendritic cells (cDCs) and on the adaptor molecule, MyD88. However, many cell types in the lung express Myd88, and it is unclear how signaling in these different cell types reprograms cDCs and leads to allergic inflammation of the airway. By combining ATAC-seq with RNA profiling, we found that MyD88 signaling in cDCs maintained open chromatin at select loci even at steady state, allowing genes to be rapidly induced during allergic sensitization. A distinct set of genes related to metabolism was indirectly controlled in cDCs through MyD88 signaling in airway epithelial cells (ECs). In mouse models of asthma, Myd88 expression in ECs was critical for eosinophilic inflammation, whereas Myd88 expression in cDCs was required for Th17 cell differentiation and consequent airway neutrophilia. Thus, both cell-intrinsic and cell-extrinsic MyD88 signaling controls gene expression in cDCs and orchestrates immune responses to inhaled allergens.     

Influenza H2 haemagglutinin activates B cells via a MyD88-dependent pathway

Influenza viruses are serious respiratory pathogens, responsible for half a million deaths each year. The viral surface haemagglutinin (HA) protein has been shown to be an important determinant of viral pathogenicity. HA is the virion attachment and fusion protein, and the major target for neutralizing antibodies; however, it is also involved in triggering innate responses that may have an important impact on the disease course. We have examined the role of the toll-like receptor (TLR) family in innate responses to influenza virus and influenza HA. TLR7 has recently been found to mediate recognition of influenza RNA. Here, we show for the first time that influenza HA of the H2 subtype induces innate responses in murine B lymphocytes via a MyD88-dependent pathway distinct from that involved in sensing viral RNA. We also show that inactivated influenza virus induces activation of human B cells. Our findings suggest that the molecule mediating these responses may be a novel member of the TLR family.