Cutting edge: TLR9 and TLR2 signaling together account for MyD88-dependent control of parasitemia in Trypanosoma cruzi infection

Activation of innate immune cells by Trypanosoma cruzi-derived molecules such as GPI anchors and DNA induces proinflammatory cytokine production and host defense mechanisms. In this study, we demonstrate that DNA from T. cruzi stimulates cytokine production by APCs in a TLR9-dependent manner and synergizes with parasite-derived GPI anchor, a TLR2 agonist, in the induction of cytokines by macrophages. Compared with wild-type animals, T. cruzi-infected Tlr9(-/-) mice displayed elevated parasitemia and decreased survival. Strikingly, infected Tlr2(-/-)Tlr9(-/-) mice developed a parasitemia equivalent to animals lacking MyD88, an essential signaling molecule for most TLR, but did not show the acute mortality displayed by MyD88(-/-) animals. The enhanced susceptibility of Tlr9(-/-) and Tlr2(-/-)Tlr9(-/-) mice was associated with decreased in vivo IL-12/IFN-gamma responses. Our results reveal that TLR2 and TLR9 cooperate in the control of parasite replication and that TLR9 has a primary role in the MyD88-dependent induction of IL-12/IFN-gamma synthesis during infection with T. cruzi.     

Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus

We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c(+) dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-alpha) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-alpha by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.     

TLR9 and MyD88 are crucial for the development of protective immunity to malaria

Effective resolution of malaria infection by avoiding pathogenesis requires regulated pro- to anti-inflammatory responses and the development of protective immunity. TLRs are known to be critical for initiating innate immune responses, but their roles in the regulation of immune responses and development of protective immunity to malaria remain poorly understood. In this study, using wild-type, TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) mice infected with Plasmodium yoelii, we show that TLR9 and MyD88 regulate pro/anti-inflammatory cytokines, Th1/Th2 development, and cellular and humoral responses. Dendritic cells from TLR9(-/-) and MyD88(-/-) mice produced significantly lower levels of proinflammatory cytokines and higher levels of anti-inflammatory cytokines than dendritic cells from wild-type mice. NK and CD8(+) T cells from TLR9(-/-) and MyD88(-/-) mice showed markedly impaired cytotoxic activity. Furthermore, mice deficient in TLR9 and MyD88 showed higher Th2-type and lower Th1-type IgGs. Consequently, TLR9(-/-) and MyD88(-/-) mice exhibited compromised ability to control parasitemia and were susceptible to death. Our data also show that TLR9 and MyD88 distinctively regulate immune responses to malaria infection. TLR9(-/-) but not MyD88(-/-) mice produced significant levels of both pro- and anti-inflammatory cytokines, including IL-1β and IL-18, by other TLRs/inflammasome- and/or IL-1R/IL-18R-mediated signaling. Thus, whereas MyD88(-/-) mice completely lacked cell-mediated immunity, TLR9(-/-) mice showed low levels of cell-mediated immunity and were slightly more resistant to malaria infection than MyD88(-/-) mice. Overall, our findings demonstrate that TLR9 and MyD88 play central roles in the immune regulation and development of protective immunity to malaria, and have implications in understanding immune responses to other pathogens.     

CpG oligonucleotides induce an immune response of odontoblasts through the TLR9, MyD88 and NF-κB pathways

Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-α expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-κB or IκBα markedly inhibited CpG ODN-induced IL-6 and TNF-α expression. Moreover, the CpG ODN-mediated increase of κB-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9, MyD88 and IκBα, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-κB are involved in this process.     

Toll-like receptor 9 mediates CpG oligonucleotide-induced cellular invasion

Toll-like receptor 9 (TLR9) belongs to the innate immune system and recognizes microbial and vertebrate DNA. We showed previously that treatment with the TLR9-agonistic ODN M362 (a CpG sequence containing oligonucleotide) induces matrix metalloproteinase-13-mediated invasion in TLR9-expressing human cancer cell lines. Here, we further characterized the role of the TLR9 pathway in this process. We show that CpG oligonucleotides induce invasion in macrophages from wild-type C57/B6 and MyD88 knockout mice and in human MDA-MB-231 breast cancer cells lacking MyD88 expression. This effect was significantly inhibited in macrophages from TLR9 knockout mice and in human MDA-MB-231 breast cancer cells stably expressing TLR9 small interfering RNA or dominant-negative tumor necrosis factor receptor-associated factor 6 (TRAF6). Sequence modifications to the CpG oligonucleotides that targeted the stem loop and other secondary structures were shown to influence the invasion-inducing effect in MDA-MB-231 cells. In contrast, methylation of the cytosine residues of the parent CpG oligonucleotide did not affect the TLR9-mediated invasion compared with the unmethylated parent CpG oligonucleotide. Finally, expression of TLR9 was studied in clinical breast cancer samples and normal breast epithelium with immunohistochemistry. TLR9 staining localized in epithelial cells in both cancer and normal samples. The mean TLR9 staining intensity was significantly increased in the breast cancer cells compared with normal breast epithelial cells. In conclusion, our results suggest that TLR9 expression is increased in breast cancer and CpG oligonucleotide-induced cellular invasion is mediated via TLR9 and TRAF6, independent of MyD88. Further, our findings suggest that the structure and/or stability of DNA may influence the induction of TLR9-mediated invasion in breast cancer.     

Proinflammatory cytokine production in liver regeneration is Myd88-dependent, but independent of Cd14, Tlr2, and Tlr4

TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.     

Toll-like receptor 9 acts at an early stage in host defence against pneumococcal infection

Toll-like receptor 9 (TLR9) induces an inflammatory response by recognition of unmethylated CpG dinucleotides, mainly present in prokaryotic DNA. So far, TLR9-deficient mice have been shown to be more sensitive than wild-type mice to viral, but not to bacterial infections. Here, we show that mice deficient in TLR9 but not in TLR1, TLR2, TLR4 and TLR6 or IL-1R/IL-18R are more susceptible to a respiratory tract bacterial infection caused by Streptococcus pneumoniae. Intranasal challenge studies revealed that TLR9 plays a protective role in the lungs at an early stage of infection prior to the entry of circulating inflammatory cells. Alveolar as well as bone marrow-derived macrophages deficient in either TLR9 or the myeloid adaptor differentiation protein MyD88 were impaired in pneumococcal uptake and in pneumococcal killing. Our data suggest that in the airways, pneumococcal infection triggers a TLR9 and MyD88-dependent activation of phagocytic activity from resident macrophages leading to an early clearance of bacteria from the lower respiratory tract.     

Th1-like cytokine induction by heat-killed Brucella abortus is dependent on triggering of TLR9

In this report we provide evidence, for the first time, that bacterial DNA in the context of heat-killed Brucella abortus (HKBA) engages TLR9 in dendritic cells (DC), resulting in a Th1-like cytokine response. This is based on the findings that HKBA induction of IL-12p40 is: 1) abolished in DC from TLR9(-/-) mice; 2) blocked by suppressive oligodeoxynucleotides; 3) simulated by bacterial DNA derived from HKBA; and 4) abrogated by DNase or methylation of the DNA from HKBA. Furthermore, the effect of HKBA can be inhibited by chloroquine, indicating that endosomal acidification is required and supporting the notion that DNA from HKBA is interacting with TLR9 at the level of the endosome, as is the case with CpG oligodeoxynucleotides. In addition to DC, HKBA can elicit IL-12p40 secretion from macrophages, in which case the effect is wholly MyD88 dependent but only partially TLR9 dependent. This probably explains why HKBA effects in vivo are only partially reduced in TLR9(-/-), but absent in MyD88(-/-) mice. Because of their intimate interactions with T cells, the DC response is most likely to be critical for linking innate and adaptive immune responses, whereas the macrophage reaction may play a role in enhancing NK cell and bystander immune responses. In addition to IL-12p40, HKBA induces other Th1-like cytokines, namely, IFN-alpha and IFN-gamma, in a TLR9-dependent manner. These cytokines are important in protection against viruses and bacteria, and their induction enhances HKBA as a potential carrier for vaccines.     

CpG-induced tyrosine phosphorylation occurs via a TLR9-independent mechanism and is required for cytokine secretion

Toll-like receptors (TLRs) recognize molecular patterns preferentially expressed by pathogens. In endosomes, TLR9 is activated by unmethylated bacterial DNA, resulting in proinflammatory cytokine secretion via the adaptor protein MyD88. We demonstrate that CpG oligonucleotides activate a TLR9-independent pathway initiated by two Src family kinases, Hck and Lyn, which trigger a tyrosine phosphorylation–mediated signaling cascade. This cascade induces actin cytoskeleton reorganization, resulting in cell spreading, adhesion, and motility. CpG-induced actin polymerization originates at the plasma membrane, rather than in endosomes. Chloroquine, an inhibitor of CpG-triggered cytokine secretion, blocked TLR9/MyD88-dependent cytokine secretion as expected but failed to inhibit CpG-induced Src family kinase activation and its dependent cellular responses. Knock down of Src family kinase expression or the use of specific kinase inhibitors blocked MyD88-dependent signaling and cytokine secretion, providing evidence that tyrosine phosphorylation is both CpG induced and an upstream requirement for the engagement of TLR9. The Src family pathway intersects the TLR9–MyD88 pathway by promoting the tyrosine phosphorylation of TLR9 and the recruitment of Syk to this receptor.     

FADD negatively regulates lipopolysaccharide signaling by impairing interleukin-1 receptor-associated kinase 1-MyD88 interaction

Lipopolysaccharide (LPS) engages Toll-like receptor 4 (TLR4) on various cells to initiate inflammatory and angiogenic pathways. FADD is an adaptor protein involved in death receptor-mediated apoptosis. Here we report a role for FADD in regulation of TLR4 signals in endothelial cells. FADD specifically attenuates LPS-induced activation of c-Jun NH(2)-terminal kinase and phosphatidylinositol 3'-kinase in a death domain-dependent manner. In contrast, FADD-null cells show hyperactivation of these kinases. Examining physical associations of endogenous proteins, we show that FADD interacts with interleukin-1 receptor-associated kinase 1 (IRAK1) and MyD88. LPS stimulation increases IRAK1-FADD interaction and recruitment of the IRAK1-FADD complex to activated MyD88. IRAK1 is required for FADD-MyD88 interaction, as FADD does not associate with MyD88 in IRAK1-null cells. By shuttling FADD to MyD88, IRAK1 provides a mechanism for controlled and limited activation of the TLR4 signaling pathway. Functionally, enforced FADD expression inhibited LPS- but not vascular endothelial growth factor-induced endothelial cell sprouting, while FADD deficiency led to enhanced production of proinflammatory cytokines induced by stimulation of TLR4 and TLR2, but not TLR3. Reconstitution of FADD reversed the enhanced production of proinflammatory cytokines. Thus, FADD is a physiological negative regulator of IRAK1/MyD88-dependent responses in innate immune signaling.     

Microbial DNA induces a host defense reaction of human respiratory epithelial cells

Epithelial cells represent the initial site of bacterial colonization in the respiratory tract. TLR9 has been identified in B cells and CD 123(+) dendritic cells and found to be involved in the recognition of microbial DNA. It was the aim of the study to investigate the role of TLR9 in the host defense reactions of the respiratory epithelium. Respiratory epithelial cell lines (IHAEo(-), Calu-3) or fully differentiated primary human cells as air-liquid interface cultures were stimulated with bacterial DNA or synthetic oligonucleotides containing CpG motifs (CpG oligodeoxynucleotides). Expression of TLR9, cytokines, and human beta-defensin 2 was determined by quantitative RT-PCR or by ELISA. We found that TLR9 is expressed by respiratory epithelial cell lines and fully differentiated primary epithelial cells at low levels. Stimulation of the above-mentioned cells with bacterial DNA or CpG oligodeoxynucleotide resulted in an inflammatory reaction characterized by a dose-dependent up-regulation of cytokines (IL-6, IL-8) and human beta-defensin 2. Up-regulation of NF-kappaB in epithelial cells in response to the CpG motif containing DNA was inhibited by overexpression of a dominant negative form of MyD88. These results provide clear evidence that the human respiratory epithelium is capable of detecting microbial DNA by TLR9. The respiratory epithelium has an important function in triggering innate immune responses and therefore represents an interesting target for anti-inflammatory therapy.     

The induction of a type 1 immune response following a Trypanosoma brucei infection is MyD88 dependent

The initial host response toward the extracellular parasite Trypanosoma brucei is characterized by the early release of inflammatory mediators associated with a type 1 immune response. In this study, we show that this inflammatory response is dependent on activation of the innate immune system mediated by the adaptor molecule MyD88. In the present study, MyD88-deficient macrophages are nonresponsive toward both soluble variant-specific surface glycoprotein (VSG), as well as membrane-bound VSG purified from T. brucei. Infection of MyD88-deficient mice with either clonal or nonclonal stocks of T. brucei resulted in elevated levels of parasitemia. This was accompanied by reduced plasma IFN-gamma and TNF levels during the initial stage of infection, followed by moderately lower VSG-specific IgG2a Ab titers during the chronic stages of infection. Analysis of several TLR-deficient mice revealed a partial requirement for TLR9 in the production of IFN-gamma and VSG-specific IgG2a Ab levels during T. brucei infections. These results implicate the mammalian TLR family and MyD88 signaling in the innate immune recognition of T. brucei.     

Mice lacking myeloid differentiation factor 88 display profound defects in host resistance and immune responses to Mycobacterium avium infection not exhibited by Toll-like receptor 2 (TLR2)- and TLR4-deficient animals

To assess the role of Toll-like receptor (TLR) signaling in host resistance to Mycobacterium avium infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88), as well as TLR2(-/-) and TLR4(-/-) animals, were infected with a virulent strain of M. avium, and bacterial burdens and immune responses were compared with those in wild-type (WT) animals. MyD88(-/-) mice failed to control acute and chronic M. avium growth and succumbed 9-14 wk postinfection. Infected TLR2(-/-) mice also showed increased susceptibility, but displayed longer survival and lower bacterial burdens than MyD88(-/-) animals, while TLR4(-/-) mice were indistinguishable from their WT counterparts. Histopathological examination of MyD88(-/-) mice revealed massive destruction of lung tissue not present in WT, TLR2(-/-), or TLR4(-/-) mice. In addition, MyD88(-/-) and TLR2(-/-), but not TLR4(-/-), mice displayed marked reductions in hepatic neutrophil infiltration during the first 2 h of infection. Although both MyD88(-/-) and TLR2(-/-) macrophages showed profound defects in IL-6, TNF, and IL-12p40 responses to M. avium stimulation in vitro, in vivo TNF and IL-12p40 mRNA induction was impaired only in infected MyD88(-/-) mice. Similarly, MyD88(-/-) mice displayed a profound defect in IFN-gamma response that was not evident in TLR2(-/-) or TLR4(-/-) mice or in animals deficient in IL-18. These findings indicate that resistance to mycobacterial infection is regulated by multiple MyD88-dependent signals in addition to those previously attributed to TLR2 or TLR4, and that these undefined elements play a major role in determining bacterial induced proinflammatory as well as IFN-gamma responses.     

Role of TLR2, TLR4, and MyD88 in murine ozone-induced airway hyperresponsiveness and neutrophilia.

Exposure to air pollutants such as ozone (O(3)) induces airway hyperresponsiveness (AHR) and airway inflammation. Toll-like receptors (TLR) are first-line effector molecules in innate immunity to infections and signal via adapter proteins, including myeloid differentiation factor-88 (MyD88). We investigated the sensing of ozone by TLR2, TLR4, and MyD88. Ozone induced AHR in wild-type (WT) C57BL/6 mice, but AHR was absent in TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice. Bronchoalveolar lavage neutrophilia induced by ozone was inhibited at 3 h but not at 24 h in TLR2(-/-) and TLR4(-/-) mice, while in MyD88(-/-) mice, this was inhibited at 24 h. We investigated the expression of inflammatory cytokines and TLR2, TLR4, and MyD88 in these mice. Ozone induced time-dependent increases in inflammatory gene expression of keratinocyte chemoattractant (KC) and IL-6 and of TLR2, TLR4, and MyD88 in WT mice. IL-6 and KC expression induced by ozone was inhibited in TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice. Expression of MyD88 was increased in TLR2(-/-) and TLR4(-/-) mice, while induction of TLR2 or TLR4 was reduced in TLR2(-/-) and TLR4(-/-) mice, respectively. TLR2 and TLR4 mediate AHR induced by oxidative stress such as ozone, while the adapter protein MyD88, but not TLR2 or TLR4, is important in mediating ozone-induced neutrophilia. TLR2 and TLR4 may also be important in regulating the speed of neutrophilic response. Therefore, ozone may induce murine AHR and neutrophilic inflammation through the activation of the Toll-like receptor pathway that may sense noninfectious stimuli such as oxidative stress.     

IFN-alphabeta-mediated inflammatory responses and antiviral defense in liver is TLR9-independent but MyD88-dependent during murine cytomegalovirus infection

Chemokine responses critical for inflammation and promotion of effective innate control of murine CMV (MCMV) in liver have been shown to be dependent on immunoregulatory functions elicited by IFN-alphabeta. However, it remains to be determined whether upstream factors that promote viral sensing resulting in the rapid secretion of IFN-alphabeta in liver differ from those described in other tissues. Because plasmacytoid dendritic cells (pDCs) are known producers of high levels of systemic IFN-alpha in response to MCMV, this study examines the in vivo contribution of pDCs to IFN-alpha production in the liver, and whether production of the cytokine and ensuing inflammatory events are dependent on TLR9, MyD88, or both. We demonstrate that whereas MyD88 deficiency markedly impaired secretion of IFN-alpha, production of the cytokine was largely independent of TLR9 signaling, in the liver. MyD88 and TLR9 were needed for IFN-alpha production in the spleen. Moreover, hepatic but not splenic pDCs produced significant amounts of intracellular IFN-alpha in the absence of TLR9 function during infection. Furthermore, production of CCL2, CCL3, and IFN-gamma, as well as the accumulation of macrophages and NK cells, was not affected in the absence of functional TLR9 in the liver. In contrast, these responses were dramatically reduced in MyD88(-/-) mice. Additionally, MyD88(-/-) but not TLR9(-/-) mice exhibited increased sensitivity to virus infection in liver. Collectively, our results define contrasting compartmental functions for TLR9 and MyD88, and suggest that the infected tissue site uniquely contributes to the process of virus sensing and regulation of localized antiviral responses.     

Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis

Inflammatory bowel disease (IBD) arises from a dysregulated mucosal immune response to luminal bacteria. Toll-like receptor (TLR)4 recognizes LPS and transduces a proinflammatory signal through the adapter molecule myeloid differentiation marker 88 (MyD88). We hypothesized that TLR4 participates in the innate immune response to luminal bacteria and the development of colitis. TLR4-/- and MyD88-/- mice and littermate controls were given 2.5% dextran sodium sulfate (DSS) for 5 or 7 days followed by a 7-day recovery. Colitis was assessed by weight loss, rectal bleeding, and histopathology. Immunostaining was performed for macrophage markers, chemokine expression, and cell proliferation markers. DSS treatment of TLR4-/- mice was associated with striking reduction in acute inflammatory cells compared with wild-type mice despite similar degrees of epithelial injury. TLR4-/- mice experienced earlier and more severe bleeding than control mice. Similar results were seen with MyD88-/- mice, suggesting that this is the dominant downstream pathway. Mesenteric lymph nodes from TLR4-/- and MyD88-/- mice more frequently grew gram-negative bacteria. Altered neutrophil recruitment was due to diminished macrophage inflammatory protein-2 expression by lamina propria macrophages in TLR4-/- and MyD88-/- mice. The similarity in crypt epithelial damage between TLR4-/- or MyD88-/- and wild-type mice was seen despite decreased epithelial proliferation in knockout mice. TLR4 through the adapter molecule MyD88 is important in intestinal response to injury and in limiting bacterial translocation. Despite the diversity of luminal bacteria, other TLRs do not substitute for the role of TLR4 in this acute colitis model. A defective innate immune response may result in diminished bacterial clearance and ultimately dysregulated response to normal flora.     

TLR9信号通路介导缺血性脑损伤的研究进展

炎症反应是缺血性脑损伤的重要机制之一,过度的炎症免疫反应会加剧脑损伤的程度。作为先天免疫系统的重要组成部分,Toll样受体9(Toll like receptor 9,TLR9)通过识别其特异性的配体Cp G-DNA,从而激活先天免疫系统,释放大量前炎症细胞因子,参与缺血性脑损伤的炎症反应过程。近年来,有学者提出将TLR9作为一个新的缺血预处理靶点,即通过启动TLR9信号转导通路增加体内炎症细胞因子的水平来对抗缺血损伤。本文通过对近年TLR9信号通路介导的炎症反应与缺血性脑损伤相关研究进展作一综述,为寻求临床安全高效的缺血性脑损伤防治措施提供理论依据。     

Multiple MyD88-dependent responses contribute to pulmonary clearance of Legionella pneumophila

MyD88-dependent signalling is important for secretion of early inflammatory cytokines and host protection in response to Legionella pneumophila infection. Although toll-like receptor (TLR)2 contributes to MyD88-dependent clearance of L. pneumophila , TLR-independent functions of MyD88 could also be important. To determine why MyD88 is critical for host protection to L. pneumophila , the contribution of multiple TLRs and IL-18 receptor (IL-18R)-dependent interferon-gamma (IFN-γ) production in a mouse was examined. Mice deficient for TLR5 or TLR9, or deficient for TLR2 along with either TLR5 or TLR9, were competent for controlling bacterial replication and had no apparent defects in cytokine production compared with control mice. MyD88-dependent production of IFN-γ in the lung was mediated primarily by natural killer cells and required IL-18R signalling. Reducing IFN-γ levels did not greatly affect the kinetics of L. pneumophila replication or clearance in infected mice. Additionally, IFN-γ-deficient mice did not have a susceptibility phenotype as severe as the MyD88-deficient mice and were able to control a pulmonary infection by L. pneumophila . Thus, MyD88-dependent innate immune responses induced by L. pneumophila involve both TLR-dependent responses and IL-18R-dependent production of IFN-γ by natural killer cells, and these MyD88-dependent pathways can function independently to provide host protection against an intracellular pathogen.     

Molecular chaperoning by glucose-regulated protein 170 in the extracellular milieu promotes macrophage-mediated pathogen sensing and innate immunity

Recognition of pathogen-associated molecular patterns by innate immune receptors is essential for host defense responses. Although extracellular stress proteins are considered as indicators of the stressful conditions (e.g., infection or cell injury), the exact roles of these molecules in the extracellular milieu remain less defined. We found that glucose-regulated protein 170 (Grp170), the largest stress protein and molecular chaperone, is highly efficient in binding CpG oligodeoxynucleotides (CpG-ODN), the microbial DNA mimetic sensed by toll-like receptor 9 (TLR9). Extracellular Grp170 markedly potentiates the endocytosis and internalization of CpG-ODN by mouse bone marrow-derived macrophages and directly interacts with endosomal TLR9 on cell entry. These molecular collaborations result in the synergistic activation of the MyD88-dependent signaling and enhanced production of proinflammatory cytokines and nitric oxide in mouse primary macrophages as well as human THP-1 monocyte-derived macrophages, suggesting that Grp170 released from injured cells facilitates the sensing of pathogen-associated "danger" signals by intracellular receptors. This CpG-ODN chaperone complex-promoted innate immunity confers increased resistance in mice to infection of Listeria monocytogenes compared with CpG-ODN treatment alone. Our studies reveal a previously unrecognized attribute of Grp170 as a superior DNA-binding chaperone capable of amplifying TLR9 activation on pathogen recognition, which provides a conceptual advance in understanding the dynamics of ancient chaperoning functions inside and outside the cell.     

MyD88-dependent and -independent murine cytomegalovirus sensing for IFN-alpha release and initiation of immune responses in vivo

Antiviral immunity requires early and late mechanisms in which IFN-alpha and IL-12 play major roles. However, the initial events leading to their production remain largely unclear. Given the crucial role of TLR in innate recognition, we investigated their role in antiviral immunity in vivo. Upon murine CMV (MCMV) infection, both MyD88-/- and TLR9-/- mice were more susceptible and presented increased viral loads compared with C57BL/6, TLR2-/-, TLR3-/-, or TLR4-/- mice. However, in terms of resistance to infection, IFN-alpha production and in many other parameters of early inflammatory responses, the MyD88-/- mice showed a more defective response than TLR9-/- mice. In the absence of the TLR9/MyD88 signaling pathway, cytokine production was dramatically impaired with a complete abolition of bioactive IL-12p70 serum release contrasting with a high flexibility for IFN-alpha release, which is initially (36 h) plasmacytoid dendritic cell- and MyD88-dependent, and subsequently (44 h) PDC-, MyD88-independent and, most likely, TLR-independent. NK cells from MCMV-infected MyD88-/- and TLR9-/- mice displayed a severely impaired IFN-gamma production, yet retained enhanced cytotoxic activity. In addition, dendritic cell activation and critical inflammatory cell trafficking toward the liver were still effective. In the long term, except for isotype switching to MCMV-specific IgG1, the establishment of Ab responses was not significantly altered. Thus, our results demonstrate a critical requirement of TLR9 in the process of MCMV sensing to assure rapid antiviral responses, coordinated with other TLR-dependent and -independent events that are sufficient to establish adaptive immunity.