Distinct roles for MyD88 and Toll-like receptors 2, 5, and 9 in phagocytosis of Borrelia burgdorferi and cytokine induction

The contribution of Toll-like receptors (TLRs) to phagocytosis of Borrelia burgdorferi has not been extensively studied. We show that bone marrow-derived macrophages (BMDM) from MyD88(-/-) mice or Raw cells transfected with a dominant-negative MyD88 were unable to efficiently internalize B. burgdorferi. Knockouts of TLR2 and TLR9 or knockdown of TLR5 by small interfering RNA produced no defects in phagocytosis of B. burgdorferi. Production of inflammatory cytokines was greatly diminished in MyD88(-/-) BMDM but only partially affected in TLR2(-/-) BMDM or knockdown of TLR5 and unaffected in TLR9(-/-) BMDM. Cytochalasin D reduced cytokine induction, but not to the level of the MyD88(-/-) BMDM. Addition of cytochalasin D to TLR2(-/-) BMDM inhibited inflammatory responses to B. burgdorferi to the level of MyD88(-/-) BMDM, consistent with a role for TLR2 in both recognition of extracellular products and lysosomal sampling by TLR2 after processing of the organism. Cytochalasin D had no impact on cytokine production in cells undergoing TLR5 knockdown. These results suggest that MyD88, but not TLR2, TLR5, and TLR9, is important for the uptake of B. burgdorferi and that MyD88 affects inflammatory responses through both its effects on phagocytosis and its role in transducing signals from TLR2 and TLR5.     

Schistosoma mansoni egg antigen-mediated modulation of Toll-like receptor (TLR)-induced activation occurs independently of TLR2, TLR4, and MyD88

Unlike most pathogens, helminth parasites and their products induce strong Th2 responses, and dendritic cells (DCs) and macrophages exposed to helminth antigens generally fail to produce interleukin-12. Rather, it has been shown that helminth products such as soluble egg antigens (SEA; a soluble extract from Schistosoma mansoni eggs) inhibit the activation of DCs in response to classical Toll-like receptor (TLR) ligands such as lipopolysaccharide or CpG. Nevertheless, recent work has suggested that TLR4 and/or TLR2 plays an important role in the recognition of helminth products by DCs and macrophages and in the development of Th2 responses. Using DCs derived from TLR4^−/−, TLR2^−/−, or MyD88^−/− mice, we have demonstrated that the ability of SEA to modulate DC activation is MyD88 independent and requires neither TLR4 nor TLR2. Moreover, TLR2 and TLR4 are not required for SEA-pulsed DCs to induce Th2 responses in naïve mice.Helminth parasites, which colonize organ systems as diverse as the lymphatics, gastrointestinal tract, and vasculature, have evolved multiple immunomodulatory mechanisms to evade host immune responses (20). A delicate balance is required in these chronic infections to establish parasite survival without eliciting lethal immunopathology. This balance is illustrated in schistosomiasis, which is caused by the trematode Schistosoma mansoni and causes chronic morbidity in more than 200 million people (24). Following infection, worms migrate to the portal vasculature, where they mature and pair. This early phase of infection is characterized by a Th1 response. After worm pairing, females lay eggs that cross the intestinal barrier to be excreted in the feces. However, some eggs become lodged in the intestinal wall and liver sinusoids, where soluble egg antigens (SEA) induce a polarized Th2 response (23). The Th2 response correlates with the downmodulation of the initial proinflammatory Th1 response to migrating immature worms and results in granuloma formation. Failure to switch to a Th2 response leads to hepatotoxic liver disease and host death (3, 6, 11).The mechanism by which host innate immune cells recognize SEA remains unclear. Pathogens such as bacteria, viruses, and intracellular parasites express conserved molecular signatures that are shared within classes of pathogens and their free-living relatives. These are recognized by highly conserved pattern recognition receptors (PRRs) expressed by innate defense cells, including dendritic cells (DCs) and macrophages. PRRs include C-type lectins and Toll-like receptors (TLRs) (10, 32). TLRs are the most well-described PRRs, and DC activation by TLR ligation is considered to play a major role in the coordination of innate and adaptive immune responses during infection (22). Typically, the ligation of TLRs initiates a proinflammatory program, which promotes innate defense mechanisms and adaptive Th1 or Th17 immune responses to the invasive pathogens (22). There is evidence, however, that lipopolysaccharide (LPS) activation of TLR4 can induce DCs to support the development of Th2 responses (5, 15).Emerging data have demonstrated that phospholipids or glycoproteins unique to extracellular helminths ligate TLRs to induce an anti-inflammatory and Th2-inducing antigen-presenting cell phenotype. A phosphorylcholine-containing glycoprotein, ES-62, from the nematode Acanthocheilonema viteae, has been shown to induce a polarized Th2 response and to work via TLR4 to modulate antigen-presenting cell activation by a variety of TLR ligands (7, 33). There is also evidence that S. mansoni products can stimulate antigen-presenting cells through TLRs. A lipid fraction from S. mansoni eggs containing lysophosphatidylserine has been shown, in a TLR2-dependent mechanism, to induce the activation of DCs that promote Th2 and regulatory T-cell development (28), and lacto-N-fucopentaose III (LNFPIII), a synthetic copy of a schistosome egg glycan, has been shown to promote Th2 differentiation by DCs via a TLR4-dependent pathway (26).Here, using gene-targeted mice, we demonstrate conclusively that the anti-inflammatory and Th2-inducing characteristics of SEA are MyD88 independent and require neither TLR2 nor TLR4.     

MyD88, but not toll-like receptors 4 and 2, is required for efficient clearance of Brucella abortus

It is not clear how the host initially recognizes and responds to infection by gram-negative pathogenic Brucella spp. It was previously shown (D. S. Weiss, B. Raupach, K. Takeda, S. Akira, and A. Zychlinsky, J. Immunol. 172:4463-4469, 2004) that the early macrophage response against gram-negative bacteria is mediated by Toll-like receptor 4 (TLR4), which signals in response to lipopolysaccharide (LPS). Brucella, however, has a noncanonical LPS which does not have potent immunostimulatory activity. We evaluated the kinetics of TLR4 activation and the cytokine response in murine macrophages after Brucella infection. We found that during infection of macrophages, Brucella avoids activation of TLR4 at 6 h but activates TLR4, TLR2, and myeloid differentiation factor 88 (MyD88) at 24 h postinfection. Interestingly, even though its activation is delayed, MyD88 is important for host defense against Brucella infection in vivo, since MyD88(-/-) mice do not clear the bacteria as efficiently as wild-type, TLR4(-/-), TLR2(-/-), or TLR4/TLR2(-/-) mice.     

MyD88 and TLR2, but not TLR4, are required for host defense against Cryptococcus neoformans

We investigated here the potential role of Toll-like receptors (TLR) and the adaptor protein MyD88 in innate immunity responses to Cryptococcus neoformans, a pathogenic encapsulated yeast. Peritoneal macrophages from MyD88(-/-) or TLR2(-/-) mice released significantly less TNF-alpha, compared with wild-type controls, after in vitro stimulation with whole yeasts. In contrast, no differences in TNF-alpha release were noted between macrophages from C3H/HeJ mice, which have a loss of function mutation in TLR4, relative to C3H/HeN controls. When MyD88- or TLR2-deficient mice were infected with low doses of the H99 serotype A strain, all of the control animals, but none of MyD88(-/-) and only 38% of the TLR2(-/-) animals survived, in association with higher fungal burden in the mutant mice. Both MyD88(-/-) and TLR2(-/-) animals showed decreased TNF-alpha, IL-12p40 and/or IFN-gamma expression in various organs during infection. No difference in susceptibility to experimental cryptococcosis was found between C3H/HeJ mice and C3H/HeN controls. In conclusion, our data indicate that TLR2 and MyD88, but not TLR4, critically contribute to anti-cryptococcal defenses through the induction of increased TNF-alpha, IL-12 and IFN-gamma expression.     

TLR4, TLR9 and MyD88 are not required for the positive selection of autoreactive B cells into the primary repertoire

Toll-like receptors (TLR) have been shown to play an essential role in the generation of autoantibodies in mouse models of autoimmunity, but the timing and context of these effects are poorly understood. One hypothesis is that TLR ligands assist in the positive selection of self-reactive B cells into the primary repertoire and, in this way, distinguish between immunogenic and tolerogenic forms of self-antigen. To explore this idea we generated hen egg lysozyme-specific immunoglobulin (Ig(HEL)) and isotype class-switching anti-HEL mice deficient in MyD88, TLR4 or TLR9 signalling and studied B cell development and autoantibody secretion in the presence or absence of an intracellular form of self-antigen HEL that positively selects B1 cells. Our findings show that TLR4, TLR9 and MyD88 are not required for the positive selection of autoreactive B cells in the primary B cell repertoire, nor is MyD88 required for the generation of isotype-switched antibodies in the absence of antigen. These results suggest that the significant effects of TLR on autoimmunity occur in the established repertoire and not during B cell development.     

Susceptibility of prostate epithelial cells to Chlamydia muridarum infection and their role in innate immunity by recruitment of intracellular Toll-like receptors 4 and 2 and MyD88 to the inclusion

Although Chlamydia infections are widespread throughout the world, data about immunopathogenesis of genitourinary tract infections in males are very limited. In the present work we present an in vitro model of male genital tract-derived epithelial cells, more precisely prostate epithelial cells (PEC), to analyze if they are susceptible and able to respond to Chlamydia muridarum infection. Our results demonstrate that rat PEC are susceptible to C. muridarum infection and respond to this pathogen by up-regulating different proinflammatory cytokine and chemokine genes that could participate in the recruitment and local activation of immune cells, therefore influencing innate and adaptive immune responses during Chlamydia infection. Moreover, we analyzed the expression of Toll-like receptor 4 (TLR4), TLR2, and related molecules on PEC and the effect of C. muridarum infection on their expression. Our results demonstrate that PEC express significant levels of TLR4, CD14, TLR2, and the adaptor molecule MyD88 and up-regulate these proteins in response to C. muridarum infection. Indeed, TLR4, CD14, TLR2, and the adaptor MyD88 are specifically recruited to the vicinity of the bacterial inclusion, suggesting that these TLRs are actively engaged in signaling from this intracellular location in these cells. This is, to our knowledge, the first time that an in vitro model of infection with Chlamydia of male tract-derived epithelial cells has been achieved, and it provides the opportunity to determine how these cells respond and participate in modulating innate and adaptive immune response during Chlamydia infections.     

关节置换后外周血白细胞中Toll样受体4和9的表达

背景：目前对关节置换后机体内Toll样受体变化和相关机制研究甚少,有研究证实全髋关节置换后松动假体表面生物膜中表达Toll样受体。 目的：观察关节假体置入后Toll样受体4,9在关节置换者外周血白细胞上的表达。 方法：实验组选择11例行关节置换患者,对照组为10例行关节镜检查患者,于入院次日和术后第3天早晨空腹采血,流式细胞术分析外周血白细胞中Toll样受体4,9的阳性表达,同时送血样至检验科检查白细胞计数、血细胞沉降率、C-反应蛋白水平。 结果与结论：两组白细胞计数、血细胞沉降率、C-反应蛋白均在正常范围内。Toll样受体4,9均主要表达于外周血单核细胞,而在外周血淋巴细胞和中性粒细胞的表达率较低;两组患者干预后Toll样受体4,9在外周血单核细胞阳性表达率较置换前明显降低(P > 0.05)。实验组患者Toll样受体9置换前后外周血单核细胞阳性表达率的变化明显大于关节镜组(P < 0.05);而Toll样受体4变化差异无显著性意义(P > 0.05)。说明在无感染的情况下,手术本身的应激和局部损伤不影响Toll样受体4,9的表达,假体置入则下调外周血白细胞对Toll样受体9的表达。     

Involvement of CD14, toll-like receptors 2 and 4, and MyD88 in the host response to the fungal pathogen Cryptococcus neoformans in vivo

The major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans. MyD88(-/-) mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with live C. neoformans. CD14(-/-) mice had reduced survival when infected i.v., while TLR2(-/-) mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge with C. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14(-/-), TLR2(-/-), and MyD88(-/-) mice. MyD88(-/-) mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs of C. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88(-/-) mice correlating with increased numbers of lung CFU and serum and lung GXM levels.     

An angiogenic switch in macrophages involving synergy between Toll-like receptors 2, 4, 7, and 9 and adenosine A(2A) receptors

Adenosine A(2A) receptor (A(2A)R) agonists synergize with Escherichia coli (E. coli) LPS [toll-like receptor (TLR)4 agonist] to up-regulate vascular endothelial growth factor (VEGF) expression in murine macrophages. Here, we demonstrate that TLR2, TLR7, and TLR9, but not TLR3 and TLR5 agonists, also synergize with A(2A)R agonists and adenosine to up-regulate VEGF, while simultaneously strongly down-regulating TNFalpha expression. In the absence of adenosine or A(2A)R agonists, Porphyromonas gingivalis (P. gingivalis) LPS and PAM(3)CAG (TLR2 agonists), resiquimod (R848) (TLR7 agonist), and non-methylated CpG DNA (TLR9 agonist) strongly up-regulate TNFalpha expression, with no effect on VEGF. In the presence of adenosine or A(2A)R agonists, but not A(1)R agonists, TLR2, 4, 7, and 9 agonists strongly up-regulate VEGF expression, while simultaneously down-regulating TNFalpha. C57BL/10ScN (TLR4 deletion mutant) macrophages produce TNFalpha in response to TLR2, 3, 7, and 9 agonists, but not the TLR4 agonist E. coli LPS. With adenosine or A(2A)R agonists, TLR2, 7, and 9, but not TLR4 agonists, also synergistically up-regulate VEGF, while down-regulating TNFalpha expression. Polyinosinic-polycytidilic acid (poly(I:C)) (TLR3 agonist) stimulates TNFalpha expression in macrophages from both C57BL/10ScSn and C57BL/10ScN mice, but has little effect on VEGF expression in the presence of adenosine or A(2A)R agonists. R-flagellins from Serratia marcescens (S. marcescens) and Salmonella muenchen (S. muenchen) do not stimulate TNFalpha expression in either C57BL/10ScSn or C57BL10/ScN mice, and have no effect on VEGF production in the presence of adenosine or A(2A)R agonists. While adenosine and A(2A)R agonists strongly down-regulate TNFalpha protein expression induced by TLR2, 3, 4, 7, and 9 agonists, TNFalpha mRNA and NF-kappaB activation are not reduced. We propose a novel signaling pathway in murine macrophages involving synergy between TLRs 2, 4, 7, and 9 and A(2A)Rs, that up-regulates VEGF and down-regulates TNFalpha expression, thus acting as an angiogenic switch. This angiogenic switch may play an important role in ischemia when TLR agonists are present, providing an interface between innate immunity and wound healing.     

Wound healing is impaired in MyD88-deficient mice: a role for MyD88 in the regulation of wound healing by adenosine A2A receptors

Synergy between Toll-like receptor (TLR) and adenosine A2A receptor (A2AR) signaling switches macrophages from production of inflammatory cytokines such as tumor necrosis factor-alpha to production of the angiogenic growth factor vascular endothelial growth factor (VEGF). We show in this study that this switch critically requires signaling through MyD88, IRAK4, and TRAF6. Macrophages from mice lacking MyD88 (MyD88(-/-)) or IRAK4 (IRAK4(-/-)) lacked responsiveness to TLR agonists and did not respond to A2AR agonists by expressing VEGF. Suppression of TRAF6 expression with siRNA in RAW264.7 macrophages also blocked their response to TLR and A2AR agonists. Excisional skin wounds in MyD88(-/-) mice healed at a markedly slower rate than wounds in wild-type MyD88(+/+) mice, showing delayed contraction, decreased and delayed granulation tissue formation, and reduced new blood vessel density. Although macrophages accumulated to higher levels in MyD88(-/-) wounds than in controls, expression of VEGF and HIF1-alpha mRNAs was elevated in MyD88(+/+) wounds. CGS21680, an A2AR agonist, promoted repair in MyD88(+/+) wounds and stimulated angiogenesis but had no significant effect on healing of MyD88(-/-) wounds. These results suggest that the synergistic interaction between TLR and A(2A)R signaling observed in vitro that switches macrophages from an inflammatory to an angiogenic phenotype also plays a role in wound healing in vivo.     

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.     

Containment of aerogenic Mycobacterium tuberculosis infection in mice does not require MyD88 adaptor function for TLR2, -4 and -9

The role of Toll-like receptors (TLR) and MyD88 for immune responses to Mycobacterium tuberculosis (Mtb) infection remains controversial. To address the impact of TLR-mediated pathogen recognition and MyD88-dependent signaling events on anti-mycobacterial host responses, we analyzed the outcome of Mtb infection in TLR2/4/9 triple- and MyD88-deficient mice. After aerosol infection, both TLR2/4/9-deficient and wild-type mice expressed pro-inflammatory cytokines promoting antigen-specific T cells and the production of IFN-gamma to similar extents. Moreover, TLR2/4/9-deficient mice expressed IFN-gamma-dependent inducible nitric oxide synthase and LRG-47 in infected lungs. MyD88-deficient mice expressed pro-inflammatory cytokines and were shown to expand IFN-gamma-producing antigen-specific T cells, albeit in a delayed fashion. Only mice that were deficient for MyD88 rapidly succumbed to unrestrained mycobacterial growth, whereas TLR2/4/9-deficient mice controlled Mtb replication. IFN-gamma-dependent restriction of mycobacterial growth was severely impaired only in Mtb-infected MyD88, but not in TLR2/4/9-deficient bone marrow-derived macrophages. Our results demonstrate that after Mtb infection neither TLR2, -4, and -9, nor MyD88 are required for the induction of adaptive T cell responses. Rather, MyD88, but not TLR2, TLR4 and TLR9, is critical for triggering macrophage effector mechanisms central to anti-mycobacterial defense.     

MyD88 is essential for clearance of Leishmania major: possible role for lipophosphoglycan and Toll-like receptor 2 signaling

Leishmania major is an obligate intracellular eukaryotic pathogen of mononuclear phagocytes. Invasive promastigotes gain entry into target cells by receptor-mediated phagocytosis, transform into non-motile amastigotes and establish in the phagolysosome. Glycosylphosphatidylinositol-anchored lipophosphoglycan (LPG) is a virulence factor and a major parasite molecule involved in this process. We observed that mice lacking the Toll-like receptor (TLR) pathway adaptor protein MyD88 were more susceptible to infection with L. major than wild-type C57BL/6 mice, demonstrating a central role for this innate immune recognition pathway in control of infection, and suggesting that L. major possesses a ligand for TLR. We sought to identify parasite molecules capable of activating the protective Toll pathway, and found that purified Leishmania LPG, but not other surface glycolipids, activate innate immune signaling pathways via TLR2. Activation of cytokine synthesis by LPG required the presence of the lipid anchor and a functional MyD88 adaptor protein. LPG also induced the expression of negative regulatory pathways mediated by members of thesuppressors of cytokine signaling family SOCS-1 and SOCS-3. Thus, the Toll pathway is required for resistance to L. major and LPG is a defined TLR agonist from this important human pathogen.     

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.     

High levels of susceptibility and T helper 2 response in MyD88-deficient mice infected with Leishmania major are interleukin-4 dependent

Myeloid differentiation protein 88 (MyD88) is a general adaptor for the signaling cascade through receptors of the Toll/IL-1R family. When infected with Leishmania major parasites, MyD88-deficient mice displayed a dramatically enhanced parasite burden in their tissues similar to that found in susceptible BALB/c mice. In contrast, MyD88 knockout mice did not develop ulcerating lesions despite a lack of interleukin-12 (IL-12) production and a predominant T helper 2 cell response. Blockade of IL-4 produced early (day 1) after infection restored a protective T helper 1 response in MyD88 knockout mice.     

Epigenetic regulation of Toll-like receptors 2 and 4 in kidney disease

Journal of Molecular Medicine - Kidney disease affects more than 10% of the worldwide population and causes significant morbidity and mortality. Epigenetic mechanisms such as DNA methylation,...     

Toll-like receptors 3, 7, 8, and 9 in gastric cancer

Toll-like receptors (TLRs) have been shown to have anti-tumor, pro-tumor, or even dual effects in cancer, and are thus potential prognostic biomarkers and immunotherapeutic targets. The present study aimed to evaluate associations between endosomal TLRs, namely TLR3, TLR7, TLR8, and TLR9, expression and clinicopathological variables and survival in gastric cancer. A total of 564 gastric adenocarcinoma patients were included in this retrospective cohort study. Samples and clinicopathological data were retrieved and organized into tissue microarray blocks. Protein expressions were detected by immunohistochemical staining. The patients were divided into low expression and high expression groups by median values of expression. Cox regression provided hazard ratios (HR) with 95% confidence intervals (CI), adjusted for confounders. Patients with high nuclear TLR3 expression had significantly poorer 5-year survival than the low nuclear TLR3 expression group in the univariable analysis (crude HR 1.31, 95% CI 1.07–1.60). With radically resected patients, poor prognosis was also seen in the multivariable analysis (adjusted HR 1.38, 95% CI 1.08–1.77). Cytoplasmic TLR3, TLR7, TLR8, and TLR9 were not associated with 5-year survival. In conclusion, high nuclear TLR3 expression seems to have prognostic impact in gastric cancer, while TLR7, TLR8, and TLR9 do not.     

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.     

Expression and function of Toll-like receptors 2 and 4 in human keratinocytes

Keratinocytes have the ability to kill pathogenic fungi and bacteria by producing antimicrobial substances. Recent studies suggest that microbial components use signaling molecules of the human Toll-like receptor (TLR) family to transduce signals in various cells. Here we provide evidence that keratinocytes express both TLR2 and TLR4 at the mRNA and protein levels, and show that TLR2 and TLR4 are present in the normal human epidermis in vivo and that their expression is regulated by microbial components. The expression of myeloid differentiation protein gene (MyD88), which is involved in the signaling pathway of many TLR, was also demonstrated in keratinocytes. LPS + IFN-gamma increased the expression of TLR2 and TLR4 50- and 5-fold respectively. Treatment of keratinocytes with Candida albicans, mannan, Mycobacterium tuberculosis or LPS with IFN-gamma resulted in the activation and nuclear translocation of NF-kappaB. Inhibition of NF-kappaB blocked the Candida-killing activity of keratinocytes, suggesting that the antimicrobial effect of keratinocytes requires NF-kappaB activation. LPS + IFN-gamma, C. albicans (4 Candida/KC), peptidoglycan (1 micro g/ml) or M. tuberculosis extract significantly increased IL-8 gene expression after 3 h of treatment (P < 0.05). The increases over the 0-h level were 15-, 8-, 10.8- and 7-fold, respectively. The microbial compound-induced increase in IL-8 gene expression could be inhibited by anti-TLR2 and anti-TLR4 neutralizing antibodies, suggesting that TLRs are involved in the pathogen-induced expression of this pro-inflammatory cytokine. Our findings stress the importance of the role of keratinocytes as a component of innate immunity.