Differential recognition of TLR-dependent microbial ligands in human bronchial epithelial cells

Bronchial epithelial cells represent the first line of defense against invading airborne pathogens. They are important contributors to innate mucosal immunity and provide a variety of antimicrobial effectors. However, mucosal surfaces are prone to contact with pathogenic, as well as nonpathogenic microbes, and therefore, immune recognition principles have to be tightly controlled to avoid uncontrolled permanent activation. TLRs have been shown to recognize conserved microbial patterns and to mediate inducible activation of innate immunity. Our experiments demonstrate that bronchial epithelial cells express functional TLR1-6 and TLR9 and thus make use of a common principle of professional innate immune cells. Although it was observed that TLR2 ligands dependent on heterodimeric signaling either with TLR1 or TLR6 were functional, other ligands like lipoteichoic acid were not. Additionally, it was found that bronchial epithelial cells could be stimulated only marginally by Gram-positive bacteria bearing known TLR2 ligands while Gram-negative bacteria were easily recognized. This correlated with low expression of TLR2 and the missing expression of the coreceptor CD36. Transgenic expression of both receptors restored responsiveness to the complete set of TLR2 ligands and Staphylococcus aureus. Additional gene-array experiments confirmed hyporesponsiveness to this bacterium while Pseudomonas aeruginosa and respiratory syncytial virus induced common, as well as pathogen-specific, sets of genes. The findings indicate that bronchial epithelium regulates its sensitivity to recognize microbes by managing receptor expression levels. This could serve the special needs of controlled microbial recognition in mucosal compartments.     

Toll-like receptor 4-mediated signaling by epithelial surfaces: necessity or threat?

Recent data suggest that the lipopolysaccharide receptor Toll-like receptor (TLR) 4 is expressed by epithelial cells and might play a role in the mucosal host defense against Gram-negative bacteria. However, since many body surfaces are colonized by the physiological microflora, activation of epithelial TLRs must be tightly controlled to avoid unintended stimulation and mucosal inflammation. The present review summarizes the current understanding of TLR4-mediated recognition and addresses specific questions on microbial recognition on mucosal surfaces, with particular emphasis on the gastrointestinal and urinary tract.     

The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable haemophilus influenzae from the mouse lung

TLRs are important for the recognition of conserved motifs expressed by invading bacteria. TLR4 is the signaling receptor for LPS, the major proinflammatory component of the Gram-negative cell wall, whereas CD14 serves as the ligand-binding part of the LPS receptor complex. Triggering of TLR4 results in the activation of two distinct intracellular pathways, one that relies on the common TLR adaptor MyD88 and one that is mediated by Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF). Nontypeable Haemophilus influenzae (NTHi) is a common Gram-negative respiratory pathogen that expresses both TLR4 (LPS and lipooligosaccharide) and TLR2 (lipoproteins) ligands. To determine the roles of CD14, TLR4, and TLR2 during NTHi pneumonia, the following studies were performed: 1) Alveolar macrophages from CD14 and TLR4 knockout (KO) mice were virtually unresponsive to NTHi in vitro, whereas TLR2 KO macrophages displayed a reduced NTHi responsiveness. 2) After intranasal infection with NTHi, CD14 and TLR4 KO mice showed an attenuated early inflammatory response in their lungs, which was associated with a strongly reduced clearance of NTHi from the respiratory tract; in contrast, in TLR2 KO mice, lung inflammation was unchanged, and the number of NTHi CFU was only modestly increased at the end of the 10-day observation period. 3) MyD88 KO, but not TRIF mutant mice showed an increased bacterial load in their lungs upon infection with NTHi. These data suggest that the MyD88-dependent pathway of TLR4 is important for an effective innate immune response to respiratory tract infection caused by NTHi.     

Toll-Like Receptor 4 Mediates the Response of Epithelial and Stromal Cells to Lipopolysaccharide in the Endometrium

Background

Ascending infections of the female genital tract with bacteria causes pelvic inflammatory disease (PID), preterm labour and infertility. Lipopolysaccharide (LPS) is the main component of the cell wall of Gram-negative bacteria. Innate immunity relies on the detection of LPS by Toll-like receptor 4 (TLR4) on host cells. Binding of LPS to TLR4 on immune cells stimulates secretion of pro-inflammatory cytokines such as IL-6, chemokines such as CXCL1 and CCL20, and prostaglandin E₂. The present study tested the hypothesis that TLR4 on endometrial epithelial and stromal cells is essential for the innate immune response to LPS in the female genital tract.

Methodology/Principal Findings

Wild type (WT) mice expressed TLR4 in the endometrium. Intrauterine infusion of purified LPS caused pelvic inflammatory disease, with accumulation of granulocytes throughout the endometrium of WT but not Tlr4^−/− mice. Intra-peritoneal infusion of LPS did not cause PID in WT or Tlr4^−/− mice, indicating the importance of TLR4 in the endometrium for the detection of LPS in the female genital tract. Stromal and epithelial cells isolated from the endometrium of WT but not Tlr4^−/− mice, secreted IL-6, CXCL1, CCL20 and prostaglandin E₂ in response to LPS, in a concentration and time dependent manner. Co-culture of combinations of stromal and epithelial cells from WT and Tlr4^−/− mice provided little evidence of stromal-epithelial interactions in the response to LPS.

Conclusions/Significance

The innate immune response to LPS in the female genital tract is dependent on TLR4 on the epithelial and stromal cells of the endometrium.     

TLR4-dependent recognition of lipopolysaccharide by epithelial cells requires sCD14

Epithelial cells lining the urinary bladder mucosa are engaged in numerous functions that act in concert to prevent exposure of the sensitive upper urinary tract to bacteria. This protective effect was recently suggested to be achieved mainly by compartmentalized, organ-specific expression of the lipopolysaccharide (LPS) receptor Toll-like receptor (TLR) 4 within epithelial cells of the urogenital tract. Here, we show that bladder epithelial cells recognize similarly low amounts of LPS as macrophages. LPS responsiveness measured as secretion of the chemoattractant interleukin 8 demonstrates a dependency on TLR4 in epithelial cells, which is similar to the situation in macrophages. The TLR4-mediated LPS response in bladder epithelial cells also uses the co-receptor CD14 for efficient LPS signalling. However, bladder epithelial cells do not express endogenous CD14 and are therefore dependent on the soluble form of CD14 that is present in body fluids. Furthermore, we demonstrate that epithelial chemokine production is augmented by type 1-mediated attachment of uropathogenic Escherichia coli in the absence, but not in the presence, of CD14. Collectively, our findings strengthen the role for bladder epithelial cells as important players in the innate immune system within the urinary tract.     

Human toll-like receptors mediate cellular activation by Mycobacterium tuberculosis.

Recent studies have implicated a family of mammalian Toll-like receptors (TLR) in the activation of macrophages by Gram-negative and Gram-positive bacterial products. We have previously shown that different TLR proteins mediate cellular activation by the distinct CD14 ligands Gram-negative bacterial LPS and mycobacterial glycolipid lipoarabinomannan (LAM). Here we show that viable Mycobacterium tuberculosis bacilli activated both Chinese hamster ovary cells and murine macrophages that overexpressed either TLR2 or TLR4. This contrasted with Gram-positive bacteria and Mycobacterium avium, which activated cells via TLR2 but not TLR4. Both virulent and attenuated strains of M. tuberculosis could activate the cells in a TLR-dependent manner. Neither membrane-bound nor soluble CD14 was required for bacilli to activate cells in a TLR-dependent manner. We also assessed whether LAM was the mycobacterial cell wall component responsible for TLR-dependent cellular activation by M. tuberculosis. We found that TLR2, but not TLR4, could confer responsiveness to LAM isolated from rapidly growing mycobacteria. In contrast, LAM isolated from M. tuberculosis or Mycobacterium bovis bacillus Calmette-Guérin failed to induce TLR-dependent activation. Lastly, both soluble and cell wall-associated mycobacterial factors were capable of mediating activation via distinct TLR proteins. A soluble heat-stable and protease-resistant factor was found to mediate TLR2-dependent activation, whereas a heat-sensitive cell-associated mycobacterial factor mediated TLR4-dependent activation. Together, our data demonstrate that Toll-like receptors can mediate cellular activation by M. tuberculosis via CD14-independent ligands that are distinct from the mycobacterial cell wall glycolipid LAM.     

Local delivery of CpG oligodeoxynucleotides induces rapid changes in the genital mucosa and inhibits replication,but not entry,of herpes simplex virus type 2

Mucosal surfaces are the entry sites for the vast majority of infectious pathogens and provide the first line of defense against infection. In addition to the epithelial barrier, the innate immune system plays a key role in recognizing and rapidly responding to invading pathogens via innate receptors, such as Toll-like receptors (TLR). Bacterial CpG DNA, a potent activator of innate immunity, is recognized by TLR9. Here, we confirm that local mucosal, but not systemic, delivery of CpG oligodeoxynucleotides (ODN) to the genital tract protects mice from a subsequent lethal vaginal herpes simplex virus type 2 (HSV-2) challenge. Since these effects were so local in action, we examined the genital mucosa. Local delivery of CpG ODN induced rapid proliferation and thickening of the genital epithelium and caused significant recruitment of inflammatory cells to the submucosa. Local CpG ODN treatment also resulted in inhibition of HSV-2 replication but had no effect on HSV-2 entry into the genital mucosa. CpG ODN-induced protection against HSV-2 was not associated with early increases in gamma interferon (IFN-gamma) secretion in the genital tract, and CpG ODN-treated IFN-gamma(-/-) mice were protected from subsequent challenge with a lethal dose of HSV-2. Treatment of human HEK-293 cells transfected with murine TLR9 showed that the antiviral activity of CpG ODN was mediated through TLR9. These studies suggest that local induction of mucosal innate immunity can provide protection against sexually transmitted infections, such as HSV-2 or possibly human immunodeficiency virus, at the mucosal surfaces.     

Innate immunity at the mucosal surface: role of toll-like receptor 3 and toll-like receptor 9 in cervical epithelial cell responses to microbial pathogens

Toll-like receptors (TLRs) are a family of pattern recognition receptors that recognize distinct molecular patterns shared by a broad range of pathogens, including nucleic acids. TLR9, for example, recognizes unmethylated deoxycytidyl-phosphate-deoxyguanosine (CpG) dinucleotides that are common in bacterial and some viral nucleic acids, whereas TLR3 recognizes double-stranded RNA and TLR7/TLR8 recognize single-stranded RNA, which would be found during viral replication. We were interested in whether TLR3, TLR9, and the related TLR9 family members TLR7/TLR8 might play a role in antiviral immune defense at the mucosal epithelial surface of the lower female reproductive tract. We studied cervical epithelial cells and found that they expressed mRNA for TLR3, TLR9, and TLR7, but had only a weak signal for TLR8. For TLR3 and TLR9, protein expression was confirmed to be intracellular. When epithelial cells were incubated with polyinosine-polycytidylic acid and CpG oligodinucleotides, we observed dose-dependent upregulation of interleukin-8 secretion. However, cells failed to respond to a variety of TLR7/TLR8 ligands. Polyinosine-polycytidylic acid also induced production of interferon-beta and chemokine C-C motif ligand 5, whereas CpG DNA did not. Cell activation by synthetic oligodinucleotides occurred only in response to the B class sequences, and required the presence of human-specific CpG motifs. In addition, responses to CpG oligodinucleotides could be inhibited by chloroquine, demonstrating the requirement for endosomal maturation. These data demonstrate that mucosal epithelial cells express functional TLR3 and TLR9, and suggest that these receptors play a role in regulating the proinflammatory cytokine and antiviral environment of the lower female reproductive tract during infection with viral and bacterial pathogens.     

Type 1 fimbriae deliver an LPS- and TLR4-dependent activation signal to CD14-negative cells

Fimbriae target bacteria to different mucosal surfaces and enhance the inflammatory response at these sites. Inflammation may be triggered by the fimbriae themselves or by fimbriae-dependent delivery of other host activating molecules such as lipopolysaccharide (LPS). Although LPS activates systemic inflammation through the CD14 and Toll-like receptor 4 (TLR4) pathways, mechanisms of epithelial cell activation by LPS are not well understood. These cells lack CD14 receptors and are unresponsive to pure LPS, but fimbriated Escherichia coli overcome this refractoriness and trigger epithelial cytokine responses. We now show that type 1 fimbriae can present an LPS- and TLR4-dependent signal to the CD14-negative epithelial cells. Human uroepithelial cells were shown to express TLR4, and type 1 fimbriated E. coli strains triggered an LPS-dependent response in those cells. A similar LPS- and fimbriae-dependent response was observed in the urinary tract of TLR4-proficient mice, but not in TLR4-defective mice. The moderate inflammatory response in the TLR4-defective mice was fimbriae dependent but LPS independent. The results demonstrate that type 1 fimbriae present LPS to CD14-negative cells and that the TLR4 genotype determines this response despite the absence of CD14 on the target cells. The results illustrate how the host "sees" LPS and other microbial products not as purified molecules but as complexes, and that fimbriae determine the molecular context in which LPS is presented to host cells.     

Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2.

Invasive infection with Gram-positive and Gram-negative bacteria often results in septic shock and death. The basis for the earliest steps in innate immune response to Gram-positive bacterial infection is poorly understood. The LPS component of the Gram-negative bacterial cell wall appears to activate cells via CD14 and Toll-like receptor (TLR) 2 and TLR4. We hypothesized that Gram-positive bacteria might also be recognized by TLRs. Heterologous expression of human TLR2, but not TLR4, in fibroblasts conferred responsiveness to Staphylococcus aureus and Streptococcus pneumoniae as evidenced by inducible translocation of NF-kappaB. CD14 coexpression synergistically enhanced TLR2-mediated activation. To determine which components of Gram-positive cell walls activate Toll proteins, we tested a soluble preparation of peptidoglycan prepared from S. aureus. Soluble peptidoglycan substituted for whole organisms. These data suggest that the similarity of clinical response to invasive infection by Gram-positive and Gram-negative bacteria is due to bacterial recognition via similar TLRs.     

Induction of innate immune responses by Escherichia coli and purified lipopolysaccharide correlate with organ- and cell-specific expression of Toll-like receptors within the human urinary tract

Mucosal epithelial linings function as physical barriers against microbes. In addition, they participate in the first line of host defence by production of a variety of proinflammatory mediators when exposed to microbes and microbial agents. Here, we use a human urinary tract infection model to demonstrate that organ- and cell-specific innate responses induced by lipopolysaccharides (LPS) present on Gram-negative bacteria correlates with the expression of Toll-like receptor 4 (TLR4). The presence of TLR4 on human bladder epithelial cells enables them to rapidly respond to bacterial infections in vitro and in vivo . In contrast, TLR4 is not expressed on human proximal tubule cells isolated from the renal cortex, which may explain the cortical localization of bacteria in pyelonephritis. TLR4-negative renal epithelial cells, A498, are non-responsive to purified LPS, however, they respond to viable bacteria via a mannose-sensitive attachment-mediated pathway. To identify LPS components recognised by bladder epithelial cells, a bacterial lipid A mutant and LPS of different chemotypes were tested. Full interleukin 8 induction required hexa-acylated lipid A and was decreased by between 50% and 70% in the presence of O-antigen. Taken together, we propose that multiple independent pathways, which are organ- and cell-specifically expressed, mediate bacterial recognition and determine the outcome of innate responses to infection.     

Phase variation of lipopolysaccharide directs interconversion of invasive and immuno-resistant phenotypes of Neisseria gonorrhoeae.

Phase variation of Neisseria gonorrhoeae lipopolysaccharide (LPS) controls both bacterial entry into human mucosal cells, and bacterial susceptibility to killing by antibodies and complement. The basis for this function is a differential sialylation of the variable oligosaccharide moiety of the LPS. LPS variants that incorporate low amounts of sialic acid enter human mucosal epithelial cells very efficiently, but are susceptible to complement-mediated killing. Phase transition to a highly sialylated LPS phenotype results in equally adhesive but entry deficient bacteria which, however, resist killing by antibodies and complement because of dysfunctional complement activation. Phase variation of N. gonorrhoeae LPS thus functions as an adaptive mechanism enabling bacterial translocation across the mucosal barrier, and, at a later stage of infection, escape from the host immune defence.     

The CD14 ligands lipoarabinomannan and lipopolysaccharide differ in their requirement for Toll-like receptors

Mammalian Toll-like receptor (TLR) proteins are new members of the IL-1 receptor family that participate in activation of cells by bacteria and bacterial products. Several recent reports indicate that TLR proteins mediate cellular activation by bacterial LPS via a signaling pathway that is largely shared by the type I IL-1 receptor. We previously showed that Chinese hamster ovary (CHO) fibroblasts engineered to express CD14 (CHO/CD14) were responsive to LPS, but not to a distinct CD14 ligand, mycobacterial lipoarabinomannan (LAM). These CHO/CD14 cells were subsequently found to possess a frame-shift mutation within the TLR2 gene which resulted in their inability to express functional TLR2 protein. Thus, we hypothesized that TLR2, but not TLR4, was necessary for LAM signaling. In this paper we show that CHO/CD14 cells engineered to express functional TLR2 protein acquired the ability to be activated by LAM. Similarly, overexpression of TLR2 in murine macrophages conferred enhanced LAM responsiveness. Together, our data demonstrate that the distinct CD14 ligands LAM and LPS utilize different TLR proteins to initiate intracellular signals. These findings suggest a novel receptor signaling paradigm in which the binding of distinct ligands is mediated by a common receptor chain, but cellular activation is initiated via distinct signal-transducing chains that confer ligand specificity. This paradigm contrasts with many cytokine receptor complexes in which receptor specificity is conferred by a unique ligand-binding chain but cellular activation is initiated via shared signal-transducing chains.     

Toll-like receptor 4 and MYD88-dependent signaling mechanisms of the innate immune system are essential for the response to lipopolysaccharide by epithelial and stromal cells of the bovine endometrium

Infection of the bovine endometrium with Gram-negative bacteria commonly causes uterine disease. Toll-like receptor 4 (TLR4) on cells of the immune system bind Gram-negative bacterial lipopolysaccharide (LPS), stimulating the secretion of the proinflammatory cytokines interleukin 1B (IL1B) and IL6, and the chemokine IL8. Because the endometrium is the first barrier to infection of the uterus, the signaling cascade triggered by LPS and the subsequent expression of inflammatory mediators were investigated in endometrial epithelial and stromal cells, and the key pathways identified using short interfering RNA (siRNA) and biochemical inhibitors. Treatment of endometrial cells with ultrapure LPS stimulated an inflammatory response characterized by increased IL1B, IL6, and IL8 mRNA expression, and IL6 protein accumulation in epithelial cells, and by increased IL1B and IL8 mRNA expression, and IL6 and IL8 protein accumulation in stromal cells. Treatment of endometrial cells with LPS also induced the degradation of IKB and the nuclear translocation of NFKB, as well as rapid phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) and MAPK14. Knockdown of TLR4 or its signaling adaptor molecule, myeloid differentiation factor 88 (MYD88), using siRNA reduced the inflammatory response to LPS in epithelial and stromal cells. Biochemical inhibition of MAPK3/1, but not JNK or MAPK14, reduced LPS-induced IL1B, IL6, and IL8 expression in endometrial cells. In conclusion, epithelial and stromal cells have an intrinsic role in innate immune surveillance in the endometrium, and in the case of LPS this recognition occurs via TLR4- and MYD88-dependent cell signaling pathways.     

Oxidized phospholipid inhibition of toll-like receptor (TLR) signaling is restricted to TLR2 and TLR4: roles for CD14, LPS-binding protein, and MD2 as targets for specificity of inhibition

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysaccharide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor-alpha production, IkappaBalpha degradation, p38 MAPK phosphorylation, and NF-kappaB-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I.C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from approximately 30 microm. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.     

Cutting edge: TLR2-deficient and MyD88-deficient mice are highly susceptible to Staphylococcus aureus infection

Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns. We previously showed that TLR2 recognizes Gram-positive bacterial components whereas TLR4 recognizes LPS, a component of Gram-negative bacteria. MyD88 is shown to be an adaptor molecule essential for TLR family signaling. To investigate the role of TLR family in host defense against Gram-positive bacteria, we infected TLR2- and MyD88-deficient mice with Staphylococcus aureus. Both TLR2- and MyD88-deficient mice were highly susceptible to S. aureus infection, with more enhanced susceptibility in MyD88-deficient mice. Peritoneal macrophages from MyD88-deficient mice did not produce any detectable levels of cytokines in response to S. aureus. In contrast, TLR2-deficient macrophages produced reduced, but significant, levels of the cytokines, and TLR4-deficient macrophages produced the same amounts as wild-type cells, indicating that S. aureus is recognized not only by TLR2, but also by other TLR family members except for TLR4.