Involvement of the platelet-activating factor receptor in host defense against Streptococcus pneumoniae during postinfluenza pneumonia

Although influenza infection alone may lead to pneumonia, secondary bacterial infections are a much more common cause of pneumonia. Streptococcus pneumoniae is the most frequently isolated causative pathogen during postinfluenza pneumonia. Considering that S. pneumoniae utilizes the platelet-activating factor receptor (PAFR) to invade the respiratory epithelium and that the PAFR is upregulated during viral infection, we here used PAFR gene-deficient (PAFR-/-) mice to determine the role of this receptor during postinfluenza pneumococcal pneumonia. Viral clearance was similar in wild-type and PAFR-/- mice, and influenza virus was completely removed from the lungs at the time mice were inoculated with S. pneumoniae (day 14 after influenza infection). PAFR-/- mice displayed a significantly reduced bacterial outgrowth in their lungs, a diminished dissemination of the infection, and a prolonged survival. Pulmonary levels of IL-10 and KC were significantly lower in PAFR-/- mice, whereas IL-6 and TNF-alpha were only trendwise lower. These data indicate that the pneumococcus uses the PAFR leading to severe pneumonia in a host previously exposed to influenza A.     

Pulmonary lipopolysaccharide (LPS)-binding protein inhibits the LPS-induced lung inflammation in vivo

LPS-binding protein (LBP) facilitates the interaction of the Gram-negative cell wall component LPS with CD14, thereby enhancing the immune response to LPS. Although lung epithelial cells have been reported to produce LBP in vitro, knowledge of the in vivo role of pulmonary LBP is limited. Therefore, in the present study we sought to determine the function of pulmonary LBP in lung inflammation induced by intranasal administration of LPS in vivo. Using LBP-deficient (LBP-/-) and normal wild-type mice, we show that the contribution of LBP to pulmonary LPS responsiveness depended entirely on the LPS dose. Although the inflammatory response to low dose (1 ng) LPS was attenuated in LBP-/- mice, neutrophil influx and cytokine/chemokine concentrations in the bronchoalveolar compartment were enhanced in LBP-/- mice treated with higher (>10 ng) LPS doses. This finding was specific for LBP, because the exogenous administration of LBP to LBP-/- mice reversed this phenotype and reduced the local inflammatory response to higher LPS doses. Our results indicate that pulmonary LBP acts as an important modulator of the LPS response in the respiratory tract in vivo. This newly identified function of pulmonary LBP might prove beneficial by enabling a protective immune response to low LPS doses while preventing an overwhelming, potentially harmful immune response to higher doses of LPS.     

Platelet-activating factor receptor contributes to host defense against Pseudomonas aeruginosa pneumonia but is not essential for the accompanying inflammatory and procoagulant response

Pseudomonas aeruginosa is a major cause of nosocomial pneumonia, which is associated with high morbidity and mortality. Because of its ubiquitous nature and its ability to develop resistance to antibiotics, it is a problematic pathogen from a treatment perspective. Platelet-activating factor receptor (PAFR) is involved in phagocytosis of several pathogens. To determine the role of PAFR in the innate immune response to P. aeruginosa pneumonia, pafr gene-deficient (PAFR-/-) mice and normal wild-type (Wt) mice were intranasally inoculated with P. aeruginosa. PAFR deficiency impaired host defense as reflected by increased bacterial outgrowth and dissemination in mice with a targeted deletion of the PAFR gene. PAFR-/- neutrophils showed a diminished phagocytosing capacity of P. aeruginosa in vitro. Relative to Wt mice, PAFR-/- mice demonstrated increased lung inflammation and injury as reflected by histopathology, relative lung weights and total protein concentrations in bronchoalveolar lavage fluid, which was accompanied by higher levels of proinflammatory cytokines in lung homogenates and plasma. In addition, PAFR deficiency was associated with exaggerated local and systemic activation of coagulation as determined by fibrin staining of lung tissue and pulmonary and plasma concentrations of thrombin-antithrombin complexes and D-dimer. These data suggest that PAFR is an essential component of an effective host response to P. aeruginosa pneumonia, at least partly via its contribution to the phagocytic properties of professional granulocytes. Additionally, our results indicate that PAFR signaling is not essential for the induction of a local and systemic inflammatory and procoagulant response to Pseudomonas pneumonia.     

Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii

GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-alpha production by macrophages exposed to T. gondii GPIs. Importantly, TNF-alpha response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88(-/-) mice were more susceptible to death than wild-type (WT), TLR2(-/-), TLR4(-/-), TLR2/4(-/-), and CD14(-/-) mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4(-/-), but not TLR2(-/-), TLR4(-/-), and CD14(-/-), mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-gamma production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2(-/-) and TLR2/4(-/-) mice produced more IFN-gamma than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.     

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.     

Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved

Lipoteichoic acid (LTA) derived from Streptococcus pneumoniae, purified employing a chloroform/methanol protocol, and from Staphylococcus aureus, prepared by the recently described butanol extraction procedure, was investigated regarding its interaction with lipopolysaccharide (LPS)-binding protein (LBP), CD14, Toll-like receptors (TLRs)-2 and -4, and MD-2. LTA from both organisms induced cytokine synthesis in human mononuclear phagocytes. Activation was LBP- and CD14-dependent, and formation of complexes of LTA with LBP and soluble CD14 as well as catalytic transfer of LTA to CD14 by LBP was verified by PhastGel(TM) native gel electrophoresis. Human embryonic kidney (HEK) 293/CD14 cells and Chinese hamster ovary (CHO) cells were responsive to LTA only after transfection with TLR-2. Additional transfection with MD-2 did not affect stimulation of these cells by LTA. Our data suggest that innate immune recognition of LTA via LBP, CD14, and TLR-2 represents an important mechanism in the pathogenesis of systemic complications in the course of infectious diseases brought about by the clinically most important Gram-positive pathogens. However, the involvement of TLR-4 and MD-2 in this process was ruled out.     

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

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

Expression of functional TLR4 confers proinflammatory responsiveness to Trypanosoma cruzi glycoinositolphospholipids and higher resistance to infection with T. cruzi

TLRs function as pattern recognition receptors in mammals and play an essential role in the recognition of microbial components. We found that the injection of glycoinositolphospholipids (GIPLs) from Trypanosoma cruzi into the peritoneal cavity of mice induced neutrophil recruitment in a TLR4-dependent manner: the injection of GIPL in the TLR4-deficient strain of mice (C57BL/10ScCr) caused no inflammatory response. In contrast, in TLR2 knockout mice, neutrophil chemoattraction did not differ significantly from that seen in wild-type controls. GIPL-induced neutrophil attraction and MIP-2 production were also severely affected in TLR4-mutant C3H/HeJ mice. The role of TLR4 was confirmed in vitro by testing genetically engineered mutants derived from TLR2-deficient Chinese hamster ovary (CHO)-K1 fibroblasts that were transfected with CD14 (CHO/CD14). Wild-type CHO/CD14 cells express the hamster TLR4 molecule and the mutant line, in addition, expresses a nonfunctional form of MD-2. In comparison to wild-type cells, mutant CHO/CD14 cells failed to respond to GIPLs, indicating a necessity for a functional TLR4/MD-2 complex in GIPL-induced NF-kappaB activation. Finally, we found that TLR4-mutant mice were hypersusceptible to T. cruzi infection, as evidenced by a higher parasitemia and earlier mortality. These results demonstrate that natural resistance to T. cruzi is TLR4 dependent, most likely due to TLR4 recognition of their GIPLs.     

Platelet-activating factor receptor-deficient mice are protected from experimental sleep apnea-induced learning deficits

Intermittent hypoxia (IH) during sleep, a hallmark of sleep apnea, is associated with neurobehavioral impairments, regional neurodegeneration and increased oxidative stress and inflammation in rodents. Platelet-activating factor (PAF) is an important mediator of both normal neural plasticity and brain injury. We report that mice deficient in the cell surface receptor for PAF (PAFR-/-), a bioactive mediator of oxidative stress and inflammation, are protected from the spatial reference learning deficits associated with IH. Furthermore, PAFR-/- exhibit attenuated elevations in inflammatory signaling (cyclo-oxygenase-2 and inducible nitric oxide synthase activities), degradation of the ubiquitin-proteasome pathway and apoptosis observed in wild-type littermates (PAFR+/+) exposed to IH. Collectively, these findings indicate that inflammatory signaling and neurobehavioral impairments induced by IH are mediated through PAF receptors.     

Chemokine production and pattern recognition receptor (PRR) expression in whole blood stimulated with pathogen-associated molecular patterns (PAMPs)

Recognition of conserved bacterial structures called pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), may lead to induction of a variety of "early immediate genes" such as chemokines. In the current study, we have in an ex vivo whole blood model studied the induction of the chemokines MIP-1alpha, MCP-1 and IL-8 by various PAMPs. The rate of appearance of Escherichia coli-Lipopolysaccharide (LPS) induced chemokines differed. The production of MIP-1alpha and IL-8 was after 1 h of stimulation significantly higher when compared to unstimulated whole blood, whereas MCP-1 was not significantly elevated until after 3 h. At peak levels the MIP-1alpha concentration induced by E. coli-LPS was 3-5-fold higher than MCP-1 and IL-8. By specific cell depletion, we demonstrated that all three chemokines were mainly produced by monocytes. However, the mRNA results showed that IL-8 was induced in both monocytes and granulocytes. The production of all three chemokines, induced by the E. coli-LPS and Neisseria meningitidis-LPS, was significantly inhibited by antibodies against CD14 and TLR4, implying these receptors to be of importance for the effects of LPS in whole blood. The chemokine production induced by lipoteichoic acid (LTA) and non-mannose-capped lipoarabinomannan (AraLAM) was, however, less efficiently blocked by antibodies against CD14 and TLR2. E. coli-LPS and LTA induced a dose-dependent increase of CD14, TLR2 and TLR4 expression on monocytes in whole blood. These data show that PAMPs may induce chemokine production in whole blood and that antibodies against PRRs inhibit the production to different extent.     

TLR4 is the signaling but not the lipopolysaccharide uptake receptor

TLR4 is the primary recognition molecule for inflammatory responses initiated by bacterial LPS (endotoxin). Internalization of endotoxin by various cell types is an important step for its removal and detoxification. Because of its role as an LPS-signaling receptor, TLR4 has been suggested to be involved in cellular LPS uptake as well. LPS uptake was investigated in primary monocytes and endothelial cells derived from TLR4 and CD14 knockout C57BL/6 mice using tritiated and fluorescein-labeled LPS. Intracellular LPS distribution was investigated by deconvolution confocal microscopy. We could not observe any difference in LPS uptake and intracellular LPS distribution in either monocytes or endothelial cells between TLR4(-/-) and wild-type cells. As expected, CD14(-/-) monocytes showed a highly impaired LPS uptake, confirming CD14-dependent uptake in monocytes. Upon longer incubation periods, the CD14-deficient monocytes mimicked the LPS uptake pattern of endothelial cells. Endothelial cell LPS uptake is slower than monocyte uptake, LBP rather than CD14 dependent, and sensitive to polyanionic polymers, which have been shown to block scavenger receptor-dependent uptake mechanisms. We conclude that TLR4 is not involved in cellular LPS uptake mechanisms. In membrane CD14-positive cells, LPS is predominantly taken up via CD14-mediated pathways, whereas in the CD14-negative endothelial cells, there is a role for scavenger receptor-dependent pathways.     

A requirement for microglial TLR4 in leukocyte recruitment into brain in response to lipopolysaccharide

To study the mechanisms involved in leukocyte recruitment induced by local bacterial infection within the CNS, we used intravital microscopy to visualize the interaction between leukocytes and the microvasculature in the brain. First, we showed that intracerebroventricular injection of LPS could cause significant rolling and adhesion of leukocytes in the brain postcapillary venules of wild-type mice, while negligible recruitment was observed in TLR4-deficient C57BL/10ScCr mice and CD14 knockout mice, suggesting recruitment is mediated by TLR4/CD14-bearing cells. Moreover, we observed reduced but not complete inhibition of recruitment in MyD88 knockout mice, indicating both MyD88-dependent and -independent pathways are involved. The leukocyte recruitment responses in chimeric mice with TLR4-positive microglia and endothelium, but TLR4-negative leukocytes, were comparable to normal wild-type mice, suggesting either endothelium or microglia play a crucial role in the induction of leukocyte recruitment. LPS injection induced both microglial and endothelial activation in the CNS. Furthermore, minocycline, an effective inhibitor of microglial activation, completely blocked the rolling and adhesion of leukocytes in the brain and blocked TNF-alpha production in response to LPS in vivo. Minocycline did not affect activation of endothelium by LPS in vitro. TNFR p55/p75 double knockout mice also exhibited significant reductions in both rolling and adhesion in response to LPS, indicating TNF-alpha signaling is critical for the leukocyte recruitment. Our results identify a TLR4 detection system within the blood-brain barrier. The microglia play the role of sentinel cells detecting LPS thereby inducing endothelial activation and leading to efficient leukocyte recruitment to the CNS.     

Role of TLR4 in ethanol effects on innate and adaptive immune responses in peritoneal macrophages

Toll-like receptors (TLRs) play an important role in the innate immune response and these receptors link innate and adaptive responses. We have reported that ethanol modulates TLR4 receptors by activating or inhibiting its response. However, the role of TLRs in the effects of ethanol on the innate and adaptive responses during acute or chronic treatment is presently unknown. Peritoneal macrophages of wild-type and TLR4-deficient mice treated with acute ethanol (4?g?kg(-1), intraperitoneally) or chronic ethanol consumption (5 months) were used. Here we report how acute ethanol dose induces inflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-1β, macrophage inflammatory protein 1α (MIP-1α), interferon β1 and IL-12β) and chemokines (monocyte chemoattractant protein -1α and MIP-1α), and upregulates major histocompatibility complexes class I and II (MHC-I and -II), but inhibits the activation of the costimulatory molecules (CD86 and CD40), leading to the suppression of the CD4(+) T-cell proliferation in the macrophages of wild-type mice. Chronic ethanol consumption downregulates the number of F4/80(+) cells expressing MHC-I and -II and decreases CD4(+) T-cell activation in wild-type mice. Interestingly, elimination of TLR4 abolishes the effects of ethanol on the innate and the adaptive inflammatory response induced by both ethanol treatments in macrophages. Taken together, our findings support the role of TLR4 in the effects of ethanol on the immune system, and suggest that alterations in the function of this receptor might modulate the immune response induced by alcohol abuse.     

In vivo humoral immune responses to isolated pneumococcal polysaccharides are dependent on the presence of associated TLR ligands

We determined whether T cell-independent Ig isotype responses to isolated pneumococcal polysaccharides (PPS) required TLR signaling in vivo. IgG anti-PPS responses to PPS3, PPS14, and C-polysaccharide (C-PS) were virtually undetectable in TLR2(-/-) mice, whereas specific IgM induction was variably reduced compared with wild-type mice. All PPS-containing preparations induced IL-6 and TNF-alpha from wild-type, but not TLR2-/-, macrophages. TLR2 activity was distinct from that of PPS, in that it was phenol extractable. Immunization of wild-type mice with phenol-extracted PPS14 also resulted in a marked reduction in the IgG, although not the IgM-anti-PPS14, response compared with untreated PPS14. The commercial 23-valent PPS vaccine, Pneumovax-23 also contained TLR ligands (TLR2 and TLR4), which were absolutely critical for the IgG-inducing activity of the vaccine in mice. Finally, the commercial pneumococcal conjugate vaccine, Prevnar, contained a TLR2 ligand(s) that substantially enhanced both the primary and secondary anti-PPS responses in mice, especially the type 1 IgG isotypes. These data strongly suggest the absolute need for a distinct, TLR-dependent second signal for inducing in vivo IgG T cell-independent humoral immune responses to isolated pneumococcal polysaccharide Ags and highlight the potential importance of previously unappreciated copurified and/or contaminating TLR ligands in PPS vaccine preparations.     

TLR2- and TLR4-mediated signals determine attenuation or augmentation of inflammation by acute alcohol in monocytes

Most pathogens express ligands for multiple TLRs that share common downstream signaling. In this study, we investigated the effects of acute alcohol on inflammatory pathways induced by TLR2 or TLR4 ligands and their combination. In human monocytes, alcohol attenuated TLR4- but not TLR2-induced TNF-alpha protein and mRNA levels and NF-kappaB activation. In contrast, acute alcohol augmented TNF-alpha production when both TLR2 and TLR4 ligands were present. IL-1R-associated kinase (IRAK)-1 activity was reduced by alcohol in TLR4, but it was augmented in TLR2- plus TLR4-stimulated cells. IRAK-monocyte, an inhibitor of IRAK-1, was induced in TLR4, but it was reduced in TLR2- plus TLR4-stimulated monocytes by alcohol. This was supported by decreased IRAK-1:TRAF6 association in TLR4 induced but sustained presence of IRAK-1:TRAF6 complexes in TLR2- plus TLR4-stimulated monocytes after alcohol treatment. Phosphorylation of MAPKs such as ERK1/2 was selectively inhibited by acute alcohol in TLR4-stimulated cells. In contrast, JNK phosphorylation as well as AP-1 nuclear binding were augmented by acute alcohol in the presence of combined TLR4 and TLR2 stimulation. Consistent with this result, the JNK inhibitor prevented alcohol-induced augmentation of TNF-alpha production. These results suggest that acute alcohol attenuates TLR4-induced inflammation via inhibition of IRAK-1 and ERK1/2 kinases and increases in IRAK-monocyte levels in monocytes. Conversely, in the presence of TLR2 and TLR4 ligands, acute alcohol augments inflammatory responses via IRAK-1 activation and JNK phosphorylation. Thus, the complexity of TLR-mediated signals may determine attenuation or augmentation of inflammatory responses by acute alcohol.     

Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2

NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-alpha and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-alpha. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-alpha. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-beta expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-alpha production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-beta, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.     

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.     

Renal collecting duct epithelial cells react to pyelonephritis-associated Escherichia coli by activating distinct TLR4-dependent and -independent inflammatory pathways

TLR4 plays a central role in resistance to pyelonephritis caused by uropathogenic Escherichia coli (UPEC). It has been suggested that renal tubule epithelial cells expressing TLRs may play a key role in inflammatory disorders and in initiating host defenses. In this study we used an experimental mouse model of ascending urinary tract infection to show that UPEC isolates preferentially adhered to the apical surface of medullary collecting duct (MCD) intercalated cells. UPEC-infected C3H/HeJ (Lps(d)) mice carrying an inactivating mutation of tlr4 failed to clear renal bacteria and exhibited a dramatic slump in proinflammatory mediators as compared with infected wild-type C3H/HeOuJ (Lps(n)) mice. However, the level of expression of the leukocyte chemoattractants MIP-2 and TNF-alpha still remained greater in UPEC-infected than in naive C3H/HeJ (Lps(d)) mice. Using primary cultures of microdissected Lps(n) MCDs that expressed TLR4 and its accessory molecules MD2, MyD88, and CD14, we also show that UPECs stimulated both a TLR4-mediated, MyD88-dependent, TIR domain-containing adaptor-inducing IFN-beta-independent pathway and a TLR4-independent pathway, leading to bipolarized secretion of MIP-2. Stimulation by UPECs of the TLR4-mediated pathway in Lps(n) MCDs leads to the activation of NF-kappaB, and MAPK p38, ERK1/2, and JNK. In addition, UPECs stimulated TLR4-independent signaling by activating a TNF receptor-associated factor 2-apoptosis signal-regulatory kinase 1-JNK pathway. These findings demonstrate that epithelial collecting duct cells are actively involved in the initiation of an immune response via several distinct signaling pathways and suggest that intercalated cells play an active role in the recognition of UPECs colonizing the kidneys.     

Involvement of lipopolysaccharide binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Treponema glycolipids

Culture supernatants from Treponema maltophilum associated with periodontitis in humans and Treponema brennaborense found in a bovine cattle disease accompanied with cachexia caused a dose-dependent TNF-alpha synthesis in human monocytes increasing with culture time. This activity could be reduced significantly by blocking the CD14-part of the LPS receptor using the My 4 mAb and by polymyxin B. In the murine macrophage cell line RAW 264.7, Treponema culture supernatants induced TNF-alpha secretion in a LPS binding protein (LBP)-dependent fashion. To enrich for active compounds, supernatants were extracted with butanol, while whole cells were extracted using a phenol/water method resulting in recovery of material exhibiting a similar activity profile. An LPS-LBP binding competition assay revealed an interaction of the treponeme phenol/water extracts with LBP, while precipitation studies implied an affinity to polymyxin B and endotoxin neutralizing protein. Macrophages obtained from C3H/HeJ mice carrying a Toll-like receptor (TLR)-4 mutation were stimulated with treponeme extracts for NO release to assess the role of TLRs in cell activation. Furthermore, NF-kappaB translocation in TLR-2-negative Chinese hamster ovary (CHO) cells was studied. We found that phenol/water-extracts of the two strains use TLRs differently with T. brennaborense-stimulating cells in a TLR-4-dependent fashion, while T. maltophilum-mediated activation apparently involved TLR-2. These results indicate the presence of a novel class of glycolipids in Treponema initiating inflammatory responses involving LBP, CD14, and TLRs.