Role of MyD88 and Toll-Like Receptors 2 and 4 in the Sensing of Parachlamydia acanthamoebae

Parachlamydia acanthamoebae is a Chlamydia-related organism whose pathogenic role in pneumonia is supported by serological and molecular clinical studies and an experimental mouse model of lung infection. Toll-like receptors (TLRs) play a seminal role in sensing microbial products and initiating innate immune responses. The aim of this study was to investigate the roles of MyD88, TLR2, and TLR4 in the interaction of Parachlamydia with macrophages. Here, we showed that Parachlamydia entered bone-marrow derived macrophages (BMDMs) in a TLR-independent manner but did not multiply intracellularly. Interestingly, compared to live bacteria, heat-inactivated Parachlamydia induced the production of substantial amounts of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and IL-12p40 by BMDMs and of TNF and IL-6 by peritoneal macrophages as well as RAW 264.7 and J774 macrophage cell lines. Cytokine production by BMDMs, which was partially inhibited upon trypsin treatment of Parachlamydia, was dependent on MyD88, TLR4, and, to a lesser extent, TLR2. Finally, MyD88^−/−, TLR4^−/−, and TLR2^−/− mice were as resistant as wild-type mice to lung infection following the intratracheal instillation of Parachlamydia. Thus, in contrast to Chlamydia pneumoniae, Parachlamydia acanthamoebae weakly stimulates macrophages, potentially compensating for its low replication capacity in macrophages by escaping the innate immune surveillance.Parachlamydia acanthamoebae is a strict intracellular bacterium which naturally infects free-living amoebae. Like other members of the Chlamydiales order, it exhibits a two-stage developmental cycle with infectious elementary bodies and metabolically active replicating reticulate bodies (25). Several pieces of evidence support the role of P. acanthamoebae as a new agent of lower respiratory tract infection (reviewed in references 18 and 32). The first hint was provided by the recovery of P. acanthamoebae strain Hall''s coccus from the water of a humidifier associated with an outbreak of fever and the presence of anti-Parachlamydia antibodies among exposed individuals (2). Additional serological studies demonstrated a higher seropositivity rate among patients with pneumonia than among controls (20, 33). Furthermore, parachlamydial DNA was detected by PCR in mononuclear cells of a patient with bronchitis and in sputa and bronchoalveolar lavage samples from patients with lower respiratory tract infections (11, 12, 19, 37). Moreover, Parachlamydia infects human pneumocytes and macrophages in vitro (7, 23, 24). Finally, we recently developed an experimental model in which mice injected with living Parachlamydia showed signs of severe pneumonia and bacterial localization in cells that were likely pneumocytes and macrophages (9, 10).The sensing of invasive pathogens by innate immune cells relies on their capacity to sense microbial molecular motifs through pattern recognition receptors. Toll-like receptors (TLRs) expressed on the surface or in the endosomes of immune cells allow the detection of microbially derived molecular structures such as lipids, proteins, and nucleic acids. TLR4 is an obligate partner for the host response to bacterial lipopolysaccharide (LPS) (endotoxin) and most Gram-negative bacteria (1). TLR2, which generates heterodimers in combination with either TLR1 or TLR6, has been reported to recognize a broad range of microbial compounds, among which are lipopeptides, lipoproteins, peptidoglycan subcomponents, and β-glucans (29). The activation of the intracellular signaling pathways upon microbial recognition by TLRs engages several adaptor molecules. Myeloid differentiation primary response gene 88 (MyD88) specifies most TLRs and the TLR4 MyD88-dependent signaling pathway, which is involved in the activation of mitogen-activated protein kinases and nuclear factor κB and in the generation of proinflammatory cytokines and immune-related genes (35).Although Parachlamydia may represent an emerging agent of pneumonia (18, 26), very little is known about its recognition by innate immune cells. Unfortunately, the mechanisms involved in the sensing of Chlamydia are most likely irrelevant for Parachlamydia given the specificities of this bacterium, such as its ability to replicate within amoebae (22) and its genome size, which is more than twice that of Chlamydia (21). In addition, the bioinformatics-based annotation of the genome of the Parachlamydia-related symbiont UWE25 indicates that it likely possesses a truncated LPS and lacks most immunogenic outer membrane proteins present in the Chlamydiaceae (27). Considering the central role played by TLRs in microbial sensing, we studied the contributions of MyD88, TLR2, and TLR4 in the recognition of P. acanthamoebae by macrophages in vitro and in the outcome of parachlamydial infection in an experimental model of pneumonia in mice. Our results show that living P. acanthamoebae enters but does not multiply in macrophages and that heat-inactivated P. acanthamoebae stimulates cytokine production by macrophages in a MyD88/TLR4-dependent manner and to a lesser extend through TLR2. Furthermore, MyD88^−/−, TLR4^−/−, and TLR2^−/− mice are resistant to P. acanthamoebae infection. Taken together, these data indicate that P. acanthamoebae weakly stimulates the innate immune system, which may allow the bacterium to survive in infected cells despite its low replication capacity.     

Toll-Like Receptor 2- and MyD88-Dependent Phosphatidylinositol 3-Kinase and Rac1 Activation Facilitates the Phagocytosis of Listeria monocytogenes by Murine Macrophages

Toll-like receptors (TLRs) play a key role in the innate immune response by sensing bacterial ligands. The mechanisms involved in the TLR-mediated cytokine response are well established; however, the possible contribution of TLR-dependent recognition of bacteria to macrophage phagocytosis remains unclear. Listeria monocytogenes is an intracellular, parasitic, Gram-positive bacterium recognized mainly by TLR2. In this study, we investigated whether TLR2-dependent signaling is involved in the phagocytosis of L. monocytogenes by macrophages. We found no difference in the number of L. monocytogenes cells associating with wild-type (WT) and TLR2^−/− macrophages 1 h after infection. However, the number of L. monocytogenes cells phagocytosed in TLR2^−/− and MyD88^−/− macrophages was significantly lower than that of WT macrophages. In addition, lipopolysaccharide (LPS) treatment restored impaired phagocytic activity of TLR2^−/− macrophages but did not enhance the activity of MyD88^−/− macrophages. The efficiency of phagocytosis was suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K) and the small Rho GTPases but not by cycloheximide. Moreover, functional activation of PI3K and Rac1 was impaired in TLR2^−/− and MyD88^−/− macrophages. In an in vivo infection model, we found significantly lower numbers of L. monocytogenes cells phagocytosed in peritoneal macrophages of TLR2^−/− and MyD88^−/− mice after intraperitoneal infection. Moreover, a lower number of bacteria were detected in the spleens of TLR2^−/− mice 1 day after intravenous infection than in WT mice. These results clearly indicated that TLR2-MyD88-dependent signaling enhances the basal level of phagocytosis of L. monocytogenes by macrophages through activation of PI3K and Rac1, not by synthesis of proinflammatory cytokines or expression of phagocytic receptors.Listeria monocytogenes is a Gram-positive, facultative, intracellular bacterium that causes severe disease (listeriosis) in humans and various animal species, with a mortality rate of approximately 30% (14, 38). L. monocytogenes can invade various types of cells, including epithelial cells, hepatocytes, endothelial cells, fibroblasts, and macrophages. After entry into host cells, L. monocytogenes is trapped temporarily in an endosome. However, the bacterium quickly escapes from the endosome into the cell cytoplasm, where it undergoes rapid replication by means of expressing various virulence genes, including prfA, plcA, hlyA, actA, mpl, and plcB, all of which are located in a locus called Listeria pathogenicity island 1 (14).Accumulating evidence suggests that L. monocytogenes gains entry into nonprofessional phagocytes by utilizing bacterial invasion factors called internalins (11, 20, 24, 34, 42, 45). Indeed, internalin A mediates the entry of L. monocytogenes into human intestinal cells through binding to E-cadherin and the interaction of internalin B with c-Met (hepatocyte growth factor receptor) induces endocytosis of the bacterium into various types of cells (8, 25, 37). In comparison, professional phagocytes such as macrophages are able to phagocytose and subsequently kill various pathogens through phagolysosome fusion. Macrophages are known to express various receptors that recognize bacterial components or bind to opsonins attached to bacteria (54); a series of intracellular signaling pathways are then activated that lead to the dynamic and rapid reorganization of the actin cytoskeleton for phagocytic engulfment. Of these receptors, Fcγ receptor and complement receptor serve as opsonin receptors for IgG and C3b, respectively, that bind to the surface of bacteria (19, 33). Mannose receptor and CD14 can contribute to phagocytosis of bacteria through direct binding to mannosylated components (15, 17, 44). Scavenger receptor is also implicated in phagocytosis (43). These different cell surface receptors likely operate alone or in combination in the recognition and efficient internalization of bacteria into macrophages.Toll-like receptors (TLRs) are type I integral membrane glycoproteins that are expressed in all lymphoid tissues, with the highest level of expression in peripheral blood leukocytes. TLRs are pattern recognition receptors (PRR) characterized by the presence of a Toll/IL-1 receptor (TIR) domain homologous to interleukin-1 receptor (IL-1R) in their cytoplasmic portion and a variable number of leucine-rich repeats (LRR) in their extracellular portion (31). There are more than 10 TLRs identified to date in mammals, and each TLR exhibits a distinct function in innate immune recognition (1, 5, 52). For example, TLR2 recognizes lipoteichoic acid, peptidoglycan, and lipoproteins of Gram-positive bacteria. TLR4 recognizes lipopolysaccharide (LPS) from Gram-negative bacteria, and TLR9 recognizes bacterial hypomethylated CpG DNA motifs. TLR-dependent recognition of bacteria induces a signal through myeloid differentiation factor 88 (MyD88) and is accompanied by inflammatory responses in macrophages (51). TLR2-MyD88 signaling is known to be critical for the proinflammatory cytokine response during L. monocytogenes infection. Several studies in vivo have revealed that TLR2 is required for optimum control of L. monocytogenes infection (29, 53). The increased susceptibility of MyD88^−/− mice to L. monocytogenes infection may support the idea that TLR2 plays a role in resistance to L. monocytogenes (46). On the other hand, Edelson and Unanue showed that MyD88 was necessary for resistance to L. monocytogenes infection but TLR2 deficiency did not influence the propagation of L. monocytogenes in vivo (16). These conflicting findings appeared to indicate that though TLR2 participates in host resistance to L. monocytogenes through the induction of cytokine production in vivo, there are some signaling pathways that compensate for the lack of TLR2 to control bacterial infection.In addition to this established function of TLRs in the host cytokine response, recent studies suggested that TLR signaling modulates the phagocytosis of pathogens (27, 41). Indeed, Blander and Medzhitov (6) reported that TLR-mediated signaling regulates phagolysosomal maturation in bone-marrow-derived macrophages after infection with Escherichia coli, heat-killed Salmonella enterica serovar Typhimurium, and Staphylococcus aureus. Letiembre et al. (35) reported that TLR2 promotes the phagocytosis of Streptococcus pneumoniae and killing of bacteria by polymorphonuclear leukocytes. Moreover, Luther et al. (39) have shown that TLR2, MyD88, and dectin-1 are required for efficient phagocytosis of Aspergillus fumigatus conidia. Doyle et al. (13) also reported that TLR-mediated enhancement of phagocytosis is due to the upregulation of scavenger receptors, and another recent report on MyD88-mediated phagocytosis of Borrelia burgdorferi has emphasized the importance of downstream signaling through phosphatidylinositol 3-kinase (PI3K) (48). From these findings it is likely that TLR signaling contributes to the phagocytosis and phagolysosome formation in professional phagocytes upon infection with extracellular parasitic bacteria or killed bacteria. On the other hand, little is known about whether the phagocytosis of L. monocytogenes, a representative Gram-positive intracellular parasitic bacterium, is dependent on the TLR signaling pathway. In this study, we analyze the contribution of TLR2-MyD88 signaling to phagocytosis of L. monocytogenes both in vitro and in vivo.     

Group B Streptococcus and Streptococcus suis Capsular Polysaccharides Induce Chemokine Production by Dendritic Cells via Toll-Like Receptor 2- and MyD88-Dependent and -Independent Pathways

Streptococcus agalactiae (also known as group B Streptococcus [GBS]) and Streptococcus suis are encapsulated streptococci causing severe septicemia and meningitis. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. The mechanisms underlying anti-CPS antibody responses are not fully elucidated, but the biochemistry of CPSs, particularly the presence of sialic acid, may have an immunosuppressive effect. We investigated the ability of highly purified S. suis and GBS native (sialylated) CPSs to activate dendritic cells (DCs), which are crucial actors in the initiation of humoral immunity. The influence of CPS biochemistry was studied using CPSs extracted from different serotypes within these two streptococcal species, as well as desialylated CPSs. No interleukin-1β (IL-1β), IL-6, IL-12p70, tumor necrosis factor alpha (TNF-α), or IL-10 production was observed in S. suis or GBS CPS-stimulated DCs. Moreover, these CPSs exerted immunosuppressive effects on DC activation, as a diminution of gamma interferon (IFN-γ)-induced B cell-activating factor of the tumor necrosis factor family (BAFF) expression was observed in CPS-pretreated cells. However, S. suis and GBS CPSs induced significant production of CCL3, via partially Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88 (MyD88)-dependent pathways, and CCL2, via TLR-independent mechanisms. No major influence of CPS biochemistry was observed on the capacity to induce chemokine production by DCs, indicating that DCs respond to these CPSs in a patterned way rather than a structure-dedicated manner.     

Distinct Roles for MyD88 and Toll-Like Receptor 2 during Leishmania braziliensis Infection in Mice

We have previously reported that Leishmania braziliensis infection can activate murine dendritic cells (DCs) and upregulate signaling pathways that are essential for the initiation of innate immunity. However, it remains unclear whether Toll-like receptors (TLRs) are involved in L. braziliensis-mediated DC activation. To address this issue, we generated bone marrow-derived DCs from MyD88^−/− and TLR2^−/− mice and examined their responsiveness to parasite infection. While wild-type DCs were efficiently activated to produce cytokines and prime naïve CD4⁺ T cells, L. braziliensis-infected MyD88^−/− DCs exhibited less activation and decreased production of interleukin-12 (IL-12) p40. Furthermore, MyD88^−/− mice were more susceptible to infection in that they developed larger and prolonged lesions compared to those in control mice. In sharp contrast, the lack of TLR2 resulted in an enhanced DC activation and increased IL-12 p40 production after infection. As such, L. braziliensis-infected TLR2^−/− DCs were more competent in priming naïve CD4⁺ T cells in vitro than were their controls, findings which correlated with an increased gamma interferon production in vivo and enhanced resistance to infection. Our results suggest that while MyD88 is indispensable for the generation of protective immunity to L. braziliensis, TLR2 seems to have a regulatory role during infection.Leishmaniasis is a vector-borne disease that has a great socioeconomic impact in many tropical and neotropical countries (40). Leishmania parasites multiply as flagellated promastigotes in the midguts of sand flies and are transmitted to the vertebrate host via the bites of parasite-carrying female flies (3, 22). The insult at the bite site initiates a strong neutrophil influx and parasite capture by these cells (38). Interestingly, some of the captured parasites remain viable, and these infected neutrophils actually facilitate the silent entry of parasites into macrophages (Mφs) (29), where parasites survive and replicate as intracellular amastigotes (3). The magnitude and nature of inflammatory responses at the infection site and the profile of subsequent T-cell responses determine the outcome of the infection. In South America, Leishmania braziliensis infection causes cutaneous leishmaniasis in most cases and mucocutanous leishmaniasis in some individuals. The latter is a severe and disfiguring form of the disease. At present, it remains unclear why the infection is controlled in some individuals but progressive in others (40).Dendritic cell (DC)-pathogen interactions are initiated by interaction between receptors on DCs and pathogen-associated molecular patterns, including lipopolysaccharide (LPS), glycolipids, and nucleic acids. Signals through Toll-like receptors (TLRs) can induce DC maturation and the production of proinflammatory cytokines (20, 39), thereby bridging the innate and adaptive immune responses (9). Upon ligand binding, downstream signaling of all TLRs (with the exception of TLR3) uses the adaptor protein MyD88 (32). Gene knockout studies in mice have suggested that TLR signaling is essential for the immune responses against Leishmania parasites (52). For example, MyD88 and TLR4 contribute to the control of Leishmania major infection in C57BL/6 mice (27, 33). TLR9 is involved in NK cell activation in animal models of visceral (Leishmania donovani) and cutaneous (L. major and L. braziliensis) leishmaniasis (30, 45), while TLR2 and TLR3 are required for the intracellular killing of L. donovani in gamma interferon (IFN-γ)-primed Mφs (15). Leishmania lypophosphoglycan (LPG), an abundant molecule in the surfaces of promastigotes, not only is a virulence factor for some Leishmania species (e.g., L. major and L. donovani) (49) but also acts as a ligand for TLR2-mediated signaling (5). However, different species of Leishmania display relatively high variations (biochemical modifications) in LPG molecules (7). In the case of L. braziliensis, the procyclic form of the parasite lacks side chain sugar substitutions on its LPG, whereas the metacyclic form appears to contain decreased amounts of LPG compared to other Leishmania species (47). On the DC surface, TLR2 is present as preexisting heterodimers of TLR2/1 and/or TLR2/6, recognizing triacylated and diacylated lipoproteins, respectively (51). TLR2 has been shown to be important for NK cell activation in vitro by purified L. major LPG (5); however, the functional roles of TLR2 remain largely unclear during both parasite-DC interactions and the course of Leishmania infection in vivo.Most inbred strains of mice are genetically resistant to L. braziliensis infection, due to the capacity of mice to establish a strong Th1 response (43). This self control of infection is accompanied by the selective expansion of IFN-γ-producing CD4⁺ T cells, which induce nitric oxide production in infected Mφs to promote parasite killing (3, 12). We have previously revealed that several key molecules in the innate immunity pathways (e.g., STAT1, STAT3, and ISG15) were upregulated in L. braziliensis-infected DCs and that such DCs were highly efficient in priming CD4⁺ T cells in vitro and in vivo (53). However, it remains unclear whether DC-Leishmania cell interactions in the absence of MyD88 and TLR2 impact T-cell functions and in vivo containment of infection. In the present study, we generated bone marrow-derived DCs (BMDCs) from MyD88^−/− and TLR2^−/− mice and examined their responsiveness to L. braziliensis infection. We found that infected MyD88^−/− DCs showed low levels of cell activation and interleukin-12 (IL-12) p40 production, which correlated with increased susceptibilities of these mice to L. braziliensis infection and decreased expansion of IFN-γ-producing and IL-17-producing CD4⁺ T cells during the course of infection. Given that most TLR pathways share MyD88 and that TLR2 is involved in LPG recognition, we then examined the role of TLR2 in L. braziliensis recognition. Contrary to MyD88^−/− DCs, the lack of TLR2 enhanced DC activation, IL-12 p40 production, and T-cell priming in vitro. Consequently, TLR2^−/− mice were more resistant to infection than were the control mice, a finding that was associated with enhanced IFN-γ production in the draining lymph nodes (dLN). Collectively, our results show that while MyD88 is critical for L. braziliensis recognition in vitro and in vivo, TLR2 appears to have a regulatory role in modulating immune responses to the parasite.     

Chlamydial Heat Shock Protein 60 Induces Acute Pulmonary Inflammation in Mice via the Toll-Like Receptor 4- and MyD88-Dependent Pathway

Heat shock protein 60 derived from Chlamydia pneumoniae (cHSP60) activates Toll-like receptor 4 (TLR4) signaling through the MyD88 pathway in vitro, but it is not known how cHSP60 contributes to C. pneumoniae-induced lung inflammation. We treated wild-type (WT), TLR2^−/−, TLR4^−/−, or MyD88^−/− mice intratracheally (i.t.) with recombinant cHSP60 (50 μg), UV-killed C. pneumoniae (UVCP; 5 × 10⁶ inclusion-forming units/mouse), lipopolysaccharide (2 μg), or phosphate-buffered saline (PBS) and sacrificed mice 24 h later. Bronchoalveolar lavage (BAL) was obtained to measure cell counts and cytokine levels, lungs were analyzed for histopathology, and lung homogenate chemokine concentrations were determined. Bone marrow-derived dendritic cells (BMDDCs) were generated and stimulated with live C. pneumoniae (multiplicity of infection [MOI], 5), UVCP (MOI, 5), or cHSP60 for 24 h, and the expression of costimulatory molecules (CD80 and CD86) was measured by fluorescence-activated cell sorting. cHSP60 induced acute lung inflammation with the same intensity as that of UVCP-induced inflammation in WT mice but not in TLR4^−/− or MyD88^−/− mice. cHSP60- and UVCP-induced lung inflammation was associated with increased numbers of cells in BAL, increased neutrophil recruitment, and elevated BAL interleukin-6 (IL-6) levels. Both cHSP60 and UVCP induced IL-6 release and CD80 and CD86 expression in WT cells but not in MyD88^−/− BMDDCs. cHSP60 stimulated DC activation in a TLR4- and MyD88-dependent manner with an intensity similar to that induced by UVCP. These data suggest that cHSP60 promotes lung inflammation and DC activation via TLR4 and MyD88 and therefore may play a significant role in the pathogenesis of C. pneumoniae-induced chronic inflammatory lung diseases.Chlamydia pneumoniae is an obligate intracellular gram-negative bacterium that causes upper and lower respiratory tract infections throughout the world; it is responsible for 10% of community-acquired pneumonia (17). The estimated number of cases of C. pneumoniae-induced pneumonia is 300,000 cases per year. Approximately 50% of young adults and 75% of the elderly population have serological evidence of previous infection (8, 17). In addition to acute infection, there is increasing evidence that implicates C. pneumoniae and heat shock protein 60 derived from C. pneumoniae (cHSP60) in the pathogenesis of atherosclerosis and chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and bronchitis (7, 9, 10, 29).The three major effector antigens of Chlamydia are lipopolysaccharide (LPS), the major outer membrane protein (MOMP), and cHSP60 (16). Chlamydia pneumoniae has a unique biphasic life cycle. The metabolically inactive infectious elementary bodies (EBs) attach and enter the host cell, where they differentiate into the metabolically active reticulate bodies. The reticulate bodies replicate within the expanding endosome, resulting in the development of characteristic cytoplasmic inclusions. These persistent intracellular inclusions contain increased quantities of cHSP60, a highly immunogenic protein that has been implicated in the stimulation of the innate immune system and the pathogenesis of chronic inflammatory lung diseases (9). Chlamydia can achieve a state of chronic intracellular infection in which they remain viable but quiescent and do not replicate. During such persistent infections, cHSP60 is abundantly produced and might stimulate the innate immune and inflammatory responses, thus contributing to chronic inflammatory lung diseases (9, 14, 16).The involvement of the cHSP60 of C. trachomatis in the immunopathogenesis of trachoma, pelvic inflammatory disease, and tubal infertility is well established, but the role of cHSP60 in the pathogenesis of C. pneumoniae-induced infections and chronic lung disease has not been discerned (25, 26, 32). Evidence that cHSP60 indeed plays a role comes from studies showing the presence of anti-cHSP60 antibodies (Abs) in adult patients with asthma who developed symptoms after an acute respiratory illness (11). However, the cHSP60 responses might simply represent a marker for exposure to chlamydial infection. Conversely, Huittinen et al. showed an independent association between cHSP60 immunoglobulin A (IgA) Abs and the severity of allergic asthma, even after controlling for the effects of migration inhibition factor IgA Abs to C. pneumoniae (12). Importantly, these authors demonstrated that only Abs against cHSP60, and not those against C. pneumoniae EBs, were associated with chronic airway disease (31). These results clearly suggest the possibility that the persistent presence of cHSP60 after C. pneumoniae infection in the lungs participates in the immunopathology of chronic airway disease. Furthermore, several studies have linked cHSP60 with asthma and decreased pulmonary functions (11, 12, 34).Previous reports indicate that C. pneumoniae infection and cHSP60 alone can activate the innate immune system through Toll-like receptors (TLRs), one of the sensors of innate immunity (5, 24, 28, 30, 33). Live Chlamydia pneumoniae infection can activate the innate immune response by both TLR2 and TLR4, and we have demonstrated a critical role of MyD88 in host defense against C. pneumoniae (22, 28). Some studies with dendritic cells (DCs) indicate that the immune recognition of live Chlamydia pneumoniae was dependent largely on TLR2 and only to a lesser extent on TLR4 (24, 27). However, we reported that cHSP60 is a potent inducer of vascular endothelial cells (EC) and macrophage inflammatory responses, and these inflammatory effects are mediated through the innate immune receptor complex TLR4-MD2 (4). These responses proceed via the MyD88-dependent signaling pathway.In this study, we show that the i.t. administration of cHSP60 induces acute lung inflammation in a TLR4- and MyD88-dependent manner with severity comparable to that seen after the inoculation of UVCP in mice. We also demonstrate that cHSP60 induces lung inflammation and stimulates DC activation and maturation in a MyD88-dependent manner and with intensity similar to that induced by UVCP. These data suggest that cHSP60 is a key inflammatory component of Chlamydia pneumoniae that induces lung inflammation and DC activation, and therefore it may play a significant role in the pathogenesis of C. pneumoniae-induced acute and chronic inflammatory lung diseases.     

Activation and regulation of Toll-Like Receptors (TLRs) by helminth parasites

Helminth (worm) infections are major public health problems that have important socioeconomic consequences for the more than 2 billion infected individuals. Chronicity (their hallmark) can lead to anemia (in hookworm infection), river blindness (onchcerciasis), cirrhosis (schistosomiasis), and elephantiasis (lymphatic filariasis). Although there have been many studies examining innate immune responses (including TLR expression and function) in response to intracellular pathogens, fewer have examined the interaction of the multicellular helminth parasites and the innate immune system. This review will focus on two "systemic" helminth parasitic infections (lymphatic filariasis and schistosomiasis) and the regulation of TLRs that may contribute to infection outcome.     

Antagonism between MyD88- and TRIF-dependent signals in B7RP-1 up-regulation

Type I interferons (IFN) play a critical role in the Toll-like receptor (TLR)-mediated expression of B7 costimulatory family members. For example, LPS-induced up-regulation of CD80 (B7.1) and CD86 (B7.2) is abrogated in antigen-presenting cells (APC) deficient in TRIF or TRAM, two adaptors that are responsible for TLR4-mediated production of Type I IFN. In this report, we demonstrate that LPS-induced up-regulation of B7-related protein 1 (B7RP-1), a ligand for ICOS, is dependent primarily upon the MyD88-dependent signaling pathway. Signaling via the TRIF pathway sharply limits MyD88-dependent B7RP-1 up-regulation. Hence, LPS induces significantly higher B7RP-1 expression on TRIF- or TRAM-deficient mouse peritoneal macrophages and on TRIF-deficient mouse splenic B cells as compared to wild-type cells. Further studies reveal that Type I IFN are general suppressors of TLR-mediated up-regulation of B7RP-1. These data indicate that Type I IFN play a dual role in the TLR-mediated expression of B7 costimulatory family members and suggest that they may act to limit B7RP-1 expression and thus limit signals derived from B7RP-1-ICOS interaction.     

TRAF6 distinctively mediates MyD88- and IRAK-1-induced activation of NF-kappaB

MyD88 and IL-1R-associated kinase 1 (IRAK-1) play crucial roles as adaptor molecules in signal transduction of the TLR/IL-1R superfamily, and it is known that expression of these proteins leads to the activation of NF-kappaB in a TNFR-associated factor 6 (TRAF6)-dependent manner. We found in this study, however, that a dominant-negative mutant of TRAF6, lacking the N-terminal RING and zinc-finger domain, did not inhibit IRAK-1-induced activation of NF-kappaB in human embryonic kidney 293 cells, although the TRAF6 mutant strongly suppressed the MyD88-induced activation. The dominant-negative mutant of TRAF6 did not affect the IRAK-1-induced activation, regardless of the expression level of IRAK-1. In contrast, small interfering RNA silencing of TRAF6 expression inhibited MyD88-induced and IRAK-1-induced activation, and supplementation with the TRAF6 dominant-negative mutant did not restore the IRAK-1-induced activation. Expression of IRAK-1, but not MyD88, induced the oligomerization of TRAF6, and IRAK-1 and the TRAF6 dominant-negative mutant were associated with TRAF6. These results indicate that TRAF6 is involved but with different mechanisms in MyD88-induced and IRAK-induced activation of NF-kappaB and suggest that TRAF6 uses a distinctive mechanism to activate NF-kappaB depending on signals.     

Interleukin-1 Receptor but Not Toll-Like Receptor 2 Is Essential for MyD88-Dependent Th17 Immunity to Coccidioides Infection

Interleukin-17A (IL-17A)-producing CD4⁺ T helper (Th17) cells have been shown to be essential for defense against pulmonary infection with Coccidioides species. However, we have just begun to identify the required pattern recognition receptors and understand the signal pathways that lead to Th17 cell activation after fungal infection. We previously reported that Card9^−/− mice vaccinated with formalin-killed spherules failed to acquire resistance to Coccidioides infection. Here, we report that both MyD88^−/− and Card9^−/− mice immunized with a live, attenuated vaccine also fail to acquire protective immunity to this respiratory disease. Like Card9^−/− mice, vaccinated MyD88^−/− mice revealed a significant reduction in numbers of both Th17 and Th1 cells in their lungs after Coccidioides infection. Both Toll-like receptor 2 (TLR2) and IL-1 receptor type 1 (IL-1r1) upstream of MyD88 have been implicated in Th17 cell differentiation. Surprisingly, vaccinated TLR2^−/− and wild-type (WT) mice showed similar outcomes after pulmonary infection with Coccidioides, while vaccinated IL-1r1^−/− mice revealed a significant reduction in the number of Th17 cells in their infected lungs compared to WT mice. Thus, activation of both IL-1r1/MyD88- and Card9-mediated Th17 immunity is essential for protection against Coccidioides infection. Our data also reveal that the numbers of Th17 cells were reduced in IL-1r1^−/− mice to a lesser extent than in MyD88^−/− mice, raising the possibility that other TLRs are involved in MyD88-dependent Th17 immunity to coccidioidomycosis. An antimicrobial action of Th17 cells is to promote early recruitment of neutrophils to infection sites. Our data revealed that neutrophils are required for vaccine immunity to this respiratory disease.     

Lack of antigen-specific Th1 response alters granuloma formation and composition in Schistosoma mansoni-infected MyD88-/- mice

To evaluate the role of the innate immune system during schistosomiasis in vivo, we infected myeloid differentiation factor 88 (MyD88)-deficient mice with Schistosoma mansoni and analyzed their pathognomonic formation of hepatic granulomas and T cell responses. Even though the differences between knockout and wild-type mice in terms of mortality, liver damage, serum IgE and parasite burden were insignificant, the liver granulomas in the MyD88-deficient mice were significantly smaller, less cellular and contained a reduced percentage of eosinophils. Histologically, these granulomas revealed stronger fibrosis, confirmed also by increased levels of soluble collagen and IL-13, implying a Th2 bias. Spleen cells from infected MyD88-deficient mice also produced significantly less IFN-gamma than their wild-type controls upon restimulation with Schistosoma-egg-antigen (SEA). Furthermore, SEA-loaded APC from naive wild-type or MyD88-deficient mice induced equal amounts of proliferation and cytokine secretion by T cells from wild-type infected mice. In contrast, Ag-specific T cells from infected MyD88-deficient mice produced hardly any IFN-gamma but considerably more IL-10, again regardless of the APC type. These findings indicate that the loss of IFN-gamma production is not due to impaired antigen presentation but may perhaps is due to suppression by IL-10-producing T cells. Thus, MyD88 plays an important role in cellular infiltration, granuloma composition and T cell responses during schistosomiasis.     

Roles of Toll-Like Receptor 2 (TLR2), TLR4, and MyD88 during Pulmonary Coxiella burnetii Infection

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular, primarily pulmonary, bacterial pathogen. Although much is known about adaptive immune responses against this bacterium, our understanding of innate immune responses against C. burnetii is not well defined, particularly within the target tissue for infection, the lung. Previous studies examined the roles of the innate immune system receptors Toll-like receptor 2 (TLR2) and TLR4 in peripheral infection models and described minimal phenotypes in specific gene deletion animals compared to those of their wild-type controls (S. Meghari et al., Ann N Y Acad Sci 1063:161–166, 2005, http://dx.doi.org/10.1196/annals.1355.025; A. Honstettre et al., J Immunol 172:3695–3703, 2004, http://dx.doi.org/10.4049/jimmunol.172.6.3695) . Here, we assessed the roles for TLR2, TLR4, and MyD88 in pulmonary C. burnetii infection and compared responses to those that occurred in TLR2- and TLR4-deficient animals following peripheral infection. As observed previously, neither TLR2 nor TLR4 was needed for limiting bacterial growth after peripheral infection. In contrast, TLR2 and, to a lesser extent, TLR4 limited growth (or dissemination) of the bacterium in the lung and spleen after pulmonary infection. TLR2, TLR4, and MyD88 were not required for the general inflammatory response in the lungs after pulmonary infection. However, MyD88 signaling was important for infection-induced morbidity. Finally, TLR2 expression on hematopoietic cells was most important for limiting bacterial growth in the lung. These results expand on our knowledge of the roles for TLR2 and TLR4 in C. burnetii infection and suggest various roles for these receptors that are dictated by the site of infection.     

The Alveolar Epithelial Cell Chemokine Response to Pneumocystis Requires Adaptor Molecule MyD88 and Interleukin-1 Receptor but Not Toll-Like Receptor 2 or 4

Pneumocystis is an opportunistic fungal pathogen that causes pneumonia in a variety of clinical settings. An early step in Pneumocystis infection involves the attachment of organisms to alveolar epithelial cells (AECs). AECs produce chemokines in response to Pneumocystis stimulation, but the upstream host-pathogen interactions that activate AEC signaling cascades are not well-defined. MyD88 is an adaptor molecule required for activation of proinflammatory signaling cascades following Toll-like receptor (TLR)-dependent recognition of conserved molecular patterns on pathogens. To determine whether the TLR/MyD88 pathway is required for the AEC chemokine response to Pneumocystis, wild-type (WT) and MyD88-deficient AECs were incubated with Pneumocystis. As expected, WT AECs produced CCL2 and CXCL2 following Pneumocystis stimulation. In contrast, MyD88-deficient AECs were severely impaired in their ability to respond to Pneumocystis. MyD88-deficient AECs did not display Pneumocystis-induced Jun N-terminal protein kinase activation and produced much less chemokine than Pneumocystis-stimulated WT AECs. Using a panel of TLR agonists, primary murine AECs were found to respond vigorously to TLR2 and TLR4 agonists. However, the AEC chemokine response to Pneumocystis did not require TLR2 or TLR4. Surprisingly, the interleukin-1 receptor (IL-1R) was required for an AEC chemokine response to Pneumocystis. The role of MyD88 in early responses during Pneumocystis infection was supported by in vivo studies demonstrating that MyD88-deficient mice showed impaired Pneumocystis-stimulated chemokine production and impaired inflammatory cell recruitment. These data indicate an important role for MyD88 in the AEC inflammatory response to Pneumocystis.     

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。     

1-[4-Fluoro-2-(2-nitrovinyl)phenyl]pyrrolidine Suppresses Toll-Like Receptor 4 Dimerization Induced by Lipopolysaccharide

Toll-like receptor 4 (TLR4) recognizes LPS and triggers the activation of the myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter, inducing interferon-β (TRIF)-dependent major downstream signaling pathways. Previously, we presented biochemical evidence that 1-[4-Fluoro-2-(2-nitrovinyl)phenyl]pyrrolidine (FPP), which was synthesized in our laboratory, inhibits NF-κB activation induced by LPS. Here, we investigated whether FPP modulates the TLR4 downstream signaling pathways and what anti-inflammatory target in TLR4 signaling is regulated by FPP. FPP inhibited LPS-induced NF-κB activation by targeting TLR4 dimerization. These results suggest that FPP can modulate the TLR4 signaling pathway at the receptor level to decrease inflammatory gene expression.     

Menin 对 MyD88表达调控的初步研究

目的：探讨 menin 蛋白对髓样分化因子88(myeloid differentiation factor 88, MyD88)基因表达的调控作用。方法在 Men1基因敲除的小鼠 mef (Men1-/- mef)细胞中,转染可以过表达 menin 蛋白的 me-nin-PCI-NEO 质粒,运用 Western 印迹技术检测 menin 对 MyD88蛋白表达的影响。构建包含 MyD88启动子的报告基因质粒,在 Men1-/- mef 细胞中,将 MyD88启动子质粒和 menin-PCI-NEO 质粒或其阴性对照 PCI-NEO 共转染,利用荧光报告基因系统分析 menin 对 MyD88基因启动子活性的影响。结果转染 menin-PCI-NEO 质粒后, menin 蛋白表达升高的同时, MyD88蛋白的表达减少。在荧光报告基因系统中,转染 menin-PCI-NEO 质粒后 MyD88基因启动子活性被抑制。结论 menin 蛋白可以下调 MyD88蛋白的表达,这种作用可能通过抑制 MyD88基因的转录而实现。     

The Significance of Toll-Like Receptors in the Development of Ischemic Damage

Current views on the involvement of immune processes in the development of ischemic lesions are presented, with a special focus on the role of the key receptors of innate immunity, i.e., Toll-like receptors (TLR). The main types of these receptors and their ligands are characterized, as are the key mechanisms of signal transduction from TLR. Data from experimental and clinical studies of the factors of innate immunity in the development of ischemic stroke and myocardial infarction are summarized. The reasons for the similarities in the results of these studies are discussed; the main reason may be the evolutionary conservation of these receptors, and the occurrence of a single pathological process – arterial atherosclerosis – in different vascular basins may be a common cause for the development of cerebral and myocardial ischemia.