Neisseria meningitidis lipid A mutant LPSs function as LPS antagonists in humans by inhibiting TLR 4-dependent cytokine production

Lipopolysaccharide is a major constituent of the outer membrane of Gram-negative bacteria and important in the induction of pro-inflammatory responses. Recently, novel LPS species derived from Neisseria meningitidis H44/76 by insertional inactivation of the lpxL1 and lpxL2 genes have been created with a lipid A portion consisting of five (penta-acylated lpxL1) or four (tetra-acylated lpxL2) fatty acids connected to the glucosamine backbone instead of six fatty acids in the wild-type LPS. We show that these mutant LPS-types are poor inducers of cytokines (tumor-necrosis factor-α, IL-1β, IL-10, IL-RA) in human mononuclear cells. Both penta- and tetra-acylated meningococcal LPSs were able to inhibit cytokine production by wild-type Escherichia coli or meningococcal LPS. Binding of FITC-labelled E. coli LPS TLR4 transfected Chinese hamster ovary (CHO) cells was inhibited by both mutant LPS-types. Experiments with CHO fibroblasts transfected with human CD14 and TLR4 showed that the antagonizing effect was dependent on the expression of human TLR4. In contrast to the situation in humans, lpxL1 LPS has agonistic activity for cytokine production in peritoneal macrophages of DBA mice, and exacerbated arthritis in murine collagen induced arthritis model. N. meningitidis lipid A mutant LPSs lpxL1 and lpxL2 function as LPS antagonists in humans by inhibiting TLR4-dependent cytokine production but have agonistic activity in mice.     

Differential inductions of TNF-alpha and IGTP, IIGP by structurally diverse classic and non-classic lipopolysaccharides

The innate immune system recognizes microbes by characteristic molecules like the Gram-negative lipopolysaccharide (LPS). Lipid A (the LPS bioactive moiety) signals through toll-like receptors (TLRs) to induce pro-inflammatory molecules and small GTPases of the p47 family involved in intracellular pathogen control. We tested TNF-alpha and p47-GTPase induction in macrophages using classical LPSs [lipid As with glucosamine backbones, ester- and amide-linked C14:0(3-OH) and C12 to C16 in acyloxyacyl groups] of wild type and mutant Escherichia coli and Yersinia species and non-classical LPSs [lipid As with diaminoglucose, ester-linked 3-OH-fatty acids and C28:0(27-OH) and C23:0(29-OH) in acyloxyacyl groups] of plant endosymbionts (Rhizobium), intracellular pathogens (Brucella and Legionella) and phylogenetically related opportunistic bacteria (Ochrobactrum). Classical but not non-classical LPSs efficiently induced TNF-alpha, IIGP and IGTP p47-GTPase expression. Remarkably, the acyloxyacyl groups in classical LPSs necessary to efficiently induce TNF-alpha were not necessary to induce p47-GTPases, suggesting that different aspects of lipid A are involved in this differential induction. This was confirmed by using PPDM2, a non-endotoxic lipid A-structurally related synthetic glycolipid. Despite their different bioactivity, all types of LPSs signalled through TLR-4 and not through TLR-2. However, whereas TNF-alpha induction was myeloid differentiation factor 88 (MyD88)-dependent, that of p47-GTPases occurred via a MyD88-independent pathway. These observations show that different aspects of the LPS pathogen-associated molecular pattern may be triggering different signalling pathways linked to the same TLR. They also reinforce the hypothesis that non-classical lipid As act as virulence factors by favouring the escape from the innate immune system.     

Genes other than TLR4 are involved in the response to inhaled LPS

For several decades, the mouse strains C3H/HeJ and C57BL/10ScNCr have been known to be hyporesponsive to endotoxin or lipopolysaccharide (LPS). Recently, mutations in Toll-like receptor (TLR) 4 have been shown to underlie this aberrant response to LPS. To further determine the relationship between TLR4 and responsiveness to LPS, we genotyped 18 strains of mice for TLR4 and evaluated the physiological and biological responses of these strains to inhaled LPS. Of the 18 strains tested, 6 were wild type for TLR4 and 12 had mutations in TLR4. Of those strains with TLR4 mutations, nine had mutations in highly conserved residues. Among the strains wild type for TLR4, the inflammatory response in the airway induced by inhalation of LPS showed a phenotype ranging from very sensitive (DBA/2) to hyporesponsive (C57BL/6). A broad spectrum of airway hyperreactivity after inhalation of LPS was also observed among strains wild type for TLR4. Although the TLR4 mutant strains C3H/HeJ and C57BL/10ScNCr were phenotypically distinct from the other strains with mutations in the TLR4 gene, the other strains with mutations for TLR4 demonstrated a broad distribution in their physiological and biological responses to inhaled LPS. The results of our study indicate that although certain TLR4 mutations can be linked to a change in the LPS response phenotype, additional genes are clearly involved in determining the physiological and biological responses to inhaled LPS in mammals.     

Penta-acylated lipopolisaccharide binds to murine MD-2 but does not induce the oligomerization of TLR4 required for signal transduction

A mutant lipopolysaccharide (LPS) lacking a myristate chain in lipid A was shown to be non-pathogenic both in humans and mice. The mutant penta-acylated LPS from the lpxM-strain did not induce TNF-alpha production in murine peritoneal macrophages, or activation of NF-kappaB in transfected cells expressing murine TLR4/MD-2. We prepared a recombinant murine MD-2 in Escherichia coli (E. coli), and examined the binding function. Unexpectedly, specific binding was detected to both wild type and mutant LPS. However, the mutant LPS did not induce conformation changes or oligomerization of TLR4, which have been shown to be required for signal transduction. Mutant LPS appears to fail to induce appropriate conformational changes, resulting in oligomerization of the murine complex for triggering cell responses.     

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.     

Hierarchical gene expression profiles of HUVEC stimulated by different lipid A structures obtained from Porphyromonas gingivalis and Escherichia coli

The ability of lipid A structural variants to elicit unique endothelial cell gene expression was examined by measuring global gene expression profiles in human umbilical cord vein endothelial cells (HUVEC) using Affymetrix full genome chips. Two lipid A structural variants obtained from Porphyromonas gingivalis designated PgLPS(1435/1449) and PgLPS(1690) as well as LPS obtained from Escherichia coli wild type and an E. coli msbB mutant (missing myristic acid in the lipid A) were examined. Each of these lipid A structures has been shown to interact with TLR4; however, PgLPS(1435/1449) and E. coli msbB LPS have been shown to be TLR4 antagonists while PgLPS(1690) and wild-type E. coli LPS are TLR4 agonists. It was found that PgLPS(1435/1449) and PgLPS(1690) as well as E. coli msbB LPS activated a subset of those genes significantly transcribed in response to E. coli wild-type LPS. Furthermore, the subset of genes expressed in response to the different lipid A structural forms were those most significantly activated by wild-type E. coli LPS demonstrating a hierarchy in TLR4-dependent endothelial cell gene activation. A unique gene expression profile for the weak TLR4 agonist PgLPS(1690) was observed and represents a TLR4 hierarchy in endothelial cell gene activation.     

The Lipopolysaccharide Core of Brucella abortus Acts as a Shield Against Innate Immunity Recognition

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.     

O-antigen modulates infection-induced pain States

The molecular initiators of infection-associated pain are not understood. We recently found that uropathogenic E. coli (UPEC) elicited acute pelvic pain in murine urinary tract infection (UTI). UTI pain was due to E. coli lipopolysaccharide (LPS) and its receptor, TLR4, but pain was not correlated with inflammation. LPS is known to drive inflammation by interactions between the acylated lipid A component and TLR4, but the function of the O-antigen polysaccharide in host responses is unknown. Here, we examined the role of O-antigen in pain using cutaneous hypersensitivity (allodynia) to quantify pelvic pain behavior and using sacral spinal cord excitability to quantify central nervous system manifestations in murine UTI. A UPEC mutant defective for O-antigen biosynthesis induced chronic allodynia that persisted long after clearance of transient infections, but wild type UPEC evoked only acute pain. E. coli strains lacking O-antigen gene clusters had a chronic pain phenotype, and expressing cloned O-antigen gene clusters altered the pain phenotype in a predictable manner. Chronic allodynia was abrogated in TLR4-deficient mice, but inflammatory responses in wild type mice were similar among E. coli strains spanning a wide range of pain phenotypes, suggesting that O-antigen modulates pain independent of inflammation. Spinal cords of mice with chronic allodynia exhibited increased spontaneous firing and compromised short-term depression, consistent with centralized pain. Taken together, these findings suggest that O-antigen functions as a rheostat to modulate LPS-associated pain. These observations have implications for an infectious etiology of chronic pain and evolutionary modification of pathogens to alter host behaviors.     

Heligmosomoides polygyrus induces TLR4 on murine mucosal T cells that produce TGFbeta after lipopolysaccharide stimulation

Helminths are immune modulators that down-regulate colitis in inflammatory bowel disease. In animal models, intestinal bacteria drive colitis and in humans certain alleles of the LPS receptor protein TLR4 increase inflammatory bowel disease susceptibility. To understand helminthic immune modulation in the gut, we studied the influence of intestinal Heligmosomoides polygyrus colonization on LPS-induced lamina propria mononuclear cell (LPMC) cytokine responses in mice. LPS did not stimulate TGFbeta production from LPMC of uninfected mice. LPS strongly induced LPMC from worm-infected animals to secrete TGFbeta, but not TNF-alpha or IL-12. The TGFbeta derived from mucosal T cells. Helminth infection up-regulated TLR4 expression only in lamina propria T cells. LPMC from worm-infected TLR4 mutant animals did not respond to LPS, suggesting that LPS required TLR4 to stimulate TGFbeta secretion. Thus, during helminth infection, LPS challenge induces mucosal T cells to make TGFbeta through a TLR4-dependent process without promoting synthesis of proinflammatory cytokines.     

TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells

Whole cell pertussis vaccines (Pw) induce Th1 responses and protect against Bordetella pertussis infection, whereas pertussis acellular vaccines (Pa) induce Ab and Th2-biased responses and also protect against severe disease. In this study, we show that Pw failed to generate protective immunity in TLR4-defective C3H/HeJ mice. In contrast, protection induced with Pa was compromised, but not completely abrogated, in C3H/HeJ mice. Immunization with Pw, but not Pa, induced a population of IL-17-producing T cells (Th-17), as well as Th1 cells. Ag-specific IL-17 and IFN-gamma production was significantly lower in Pw-immunized TLR4-defective mice. Furthermore, treatment with neutralizing anti-IL-17 Ab immediately before and after B. pertussis challenge significantly reduced the protective efficacy of Pw. Stimulation of dendritic cells (DC) with Pw promoted IL-23, IL-12, IL-1beta, and TNF-alpha production, which was impaired in DC from TLR4-defective mice. B. pertussis LPS, which is present in high concentrations in Pw, induced IL-23 production by DC, which enhanced IL-17 secretion by T cells, but the induction of Th-17 cells was also dependent on IL-1. In addition, we identified a new effector function for IL-17, activating macrophage killing of B. pertussis, and this bactericidal activity was less efficient in macrophages from TLR4-defective mice. These data provide the first definitive evidence of a role for TLRs in protective immunity induced by a human vaccine. Our findings also demonstrate that activation of innate immune cells through TLR4 helps to direct the induction of Th1 and Th-17 cells, which mediate protective cellular immunity to B. pertussis.     

Stimulation of toll-like receptor 2 by Coxiella burnetii is required for macrophage production of pro-inflammatory cytokines and resistance to infection

Innate and adaptive immune responses are initiated upon recognition of microbial molecules by Toll-like receptors (TLRs). We have investigated the importance of these receptors in the induction of pro-inflammatory cytokines and macrophage resistance to infection with Coxiella burnetii, an obligate intracellular bacterium and the etiological agent of Q fever. By using a Chinese hamster ovary/CD14 cell line expressing either functional TLR2 or TLR4, we determined that C. burnetii phase II activates TLR2 but not TLR4. Macrophages deficient for TLR2, but not TLR4, produced less tumor necrosis factor-alpha and interleukin-12 upon C. burnetii infection. Furthermore, it was found that TLR2 activation interfered with C. burnetii intracellular replication, as macrophages from TLR2-deficient mice were highly permissive for C. burnetii growth compared with macrophages from wild type mice or TLR4-deficient mice. Although LPS modifications distinguish virulent C. burnetii phase I bacteria from avirulent phase II organisms, electrospray ionization-mass spectrometry analysis showed that the lipid A moieties isolated from these two phase variants are identical. Purified lipid A derived from either phase I or phase II LPS failed to activate TLR2 and TLR4. Indeed, the lipid A molecules were able to interfere with TLR4 signaling in response to purified Escherichia coli LPS. These studies indicate that TLR2 is an important host determinant that mediates recognition of C. burnetii and a response that limits growth of this intracellular pathogen.     

Bone Marrow and Nonbone Marrow Toll Like Receptor 4 Regulate Acute Hepatic Injury Induced by Endotoxemia

Background

Toll-like receptors (TLRs) are expressed in immune cells and hepatocytes. We examined whether hepatic Toll-like receptor 4 (TLR4) is involved in the acute hepatic injury caused by the administration of lipopolysaccharide (LPS) (septic shock model).

Methods

Wild type (WT), TLR4-deficient and chimera mice underwent myeloablative bone marrow transplantation to dissociate between TLR4 expression in the liver or in the immune-hematopoietic system. Mice were injected with LPS and sacrificed 4 hours later.

Results

Compared to TLR4 deficient mice, WT mice challenged with LPS displayed increased serum liver enzymes and hepatic cellular inflammatory infiltrate together with increased serum and hepatic levels of interleukin 1β (IL-1β), tumor necrosis factor α (TNFα) ,Up-regulation of hepatic mRNA encoding TLR4, IκB and c-jun expressions. TLR4 mutant mice transplanted with WT bone marrow were more protected than WT chimeric mice bearing TLR4 mutant hemopoietic cells from LPS, as seen by IL-1β and TNFα levels. We then used hepatocytes (Huh7) and macrophages from monocytic cell lines to detect TLR mRNA expression. Macrophages expressed a significantly higher level of TLR4 mRNA and TLR2 (more than 3000- and 8000-fold respectively) compared with the hepatocyte cell line. LPS administration induced TLR4 activation in a hepatocyte cell line in a dose dependent manner while TLR2 mRNA hardly changed.

Conclusions

These results suggest that TLR4 activation of hepatocytes participate in the immediate response to LPS induced hepatic injury. However, in this response, the contribution of TLR4 on bone marrow derived cells is more significant than those of the hepatocytes. The absence of the TLR4 gene plays a pivotal role in reducing hepatic LPS induced injury.     

Highly-purified Helicobacter pylori LPS preparations induce weak inflammatory reactions and utilize Toll-like receptor 2 complex but not Toll-like receptor 4 complex

Helicobacter pylori is recognized as an etiologic agent of gastroduodenal diseases. Among toxic substances produced by H. pylori, LPS exhibits extremely low endotoxic activity as compared to the typical LPSs, such as that produced by Escherichia coli. We found that the LPS-low-responder stomach cancer cell line MKN28, which expresses Toll-like receptor 4 (TLR4) at extremely low levels, showed similar levels of interleukin-8 (IL-8) induction by H. pylori or E. coli LPS preparations. Weak IL-8 induction by H. pylori LPS preparations was suppressed by expression of a dominant negative mutant of TLR2 but not of TLR4. Data from luciferase reporter analysis indicated that cotransfection of TLR2-TLR1 or TLR2-TLR6 was required for the activation induced by H. pylori LPS preparations. In conclusion, the H. pylori LPS preparations significantly induce an inflammatory reaction via the receptor complex containing TLR2-TLR1 or TLR2-TLR6 but not that containing TLR4. The TLR2-TLR1 complex was preferentially recognized by the H. pylori LPS preparations over the TLR2-TLR6 complex. Whereas the magnitude of response to H. pylori LPS preparation was markedly less than that to E. coli LPS preparation in LPS-high-responder cells strongly expressing TLR4, it was comparable to that of E. coli LPS in low-responder cells expressing negligible amount of TLR4.     

Dioscorin isolated from Dioscorea alata activates TLR4-signaling pathways and induces cytokine expression in macrophages

The Toll-like receptor 4 (TLR4)-signaling pathway is crucial for activating both innate and adaptive immunity. TLR4 is a promising molecular target for immune-modulating drugs, and TLR4 agonists are of therapeutic potential for treating immune diseases and cancers. Several medicinal herb-derived components have recently been reported to act via TLR4-dependent pathways, suggesting that medicinal plants are potential resources for identifying TLR4 activators. We have applied a screening procedure to systematically identify herbal constituents that activate TLR4. To exclude possible LPS contamination in these plant-derived components, a LPS inhibitor, polymyxin B, was added during screening. One of the plant components we identified from the screening was dioscorin, the glycoprotein isolated from Dioscorea alata. It induced TLR4-downstream cytokine expression in bone marrow cells isolated from TLR4-functional C3H/HeN mice but not from TLR4-defective C3H/HeJ mice. Dioscorin also stimulated multiple signaling molecules (NF-kappaB, ERK, JNK, and p38) and induced the expression of cytokines (TNF-alpha, IL-1beta, and IL-6) in murine RAW 264.7 macrophages. Furthermore, the ERK, p38, JNK, and NF-kappaB-mediated pathways are all involved in dioscorin-mediated TNF-alpha production. In summary, our results demonstrate that dioscorin is a novel TLR4 activator and induces macrophage activation via typical TLR4-signaling pathways.     

Toll-like receptor 4-mediated innate IL-10 activates antigen-specific regulatory T cells and confers resistance to Bordetella pertussis by inhibiting inflammatory pathology

Signaling through Toll-like receptors (TLR) activates dendritic cell (DC) maturation and IL-12 production, which directs the induction of Th1 cells. We found that the production of IL-10, in addition to inflammatory cytokines and chemokines, was significantly reduced in DCs from TLR4-defective C3H/HeJ mice in response to Bordetella pertussis. TLR4 was also required for B. pertussis LPS-induced maturation of DCs, but other B. pertussis components stimulated DC maturation independently of TLR4. The course of B. pertussis infection was more severe in C3H/HeJ than in C3H/HeN mice. Surprisingly, Ab- and Ag-specific IFN-gamma responses were enhanced at the peak of infection, whereas Ag-specific IL-10-producing T cells were significantly reduced in C3H/HeJ mice. This was associated with enhanced inflammatory cytokine production, cellular infiltration, and severe pathological changes in the lungs of TLR4-defective mice. Our findings suggest that TLR-4 signaling activates innate IL-10 production in response to B. pertussis, which both directly, and by promoting the induction of IL-10-secreting type 1 regulatory T cells, may inhibit Th1 responses and limit inflammatory pathology in the lungs during infection with B. pertussis.     

Reduced hepatic injury in Toll-like receptor 4-deficient mice following D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure

Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), D-galactosamine (GalN)-induced FHF is a well established model of liver injury in mice. Toll-Like Receptor 4 (TLR4) has been identified as a receptor for LPS. The aim of this study was to investigate the role of TLR4 in FHF induced by D-GalN/LPS administration in mice. Wild type (WT) and TLR4 deficient (TLR4ko) mice were studied in vivo in a fulminant model induced by GalN/LPS. Hepatic TLR4 expression, serum liver enzymes, hepatic and serum TNF-α and interleukin-1β levels were determined. Apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor-kappaβ (NF-κ β) and phosphorylated c-Jun hepatic expression were studied using Western blot analysis. All WT mice died within 24 hours after administration of GalN/LPS while all TLR4ko mice survived. Serum liver enzymes, interleukin-1β, TNF-α level, TLR4 mRNA expression, hepatic injury and hepatocyte apoptosis all significantly decreased in TLR4ko mice compared with WT mice. A significant decrease in hepatic c-Jun and IκB signaling pathway was noted in TLR4ko mice compared with WT mice. In conclusion, following induction of FHF, the inflammatory response and the liver injury in TLR4ko mice was significantly attenuated through decreased hepatic c-Jun and NF-κB expression and thus decreased TNF-α level. Down-regulation of TLR4 expression plays a pivotal role in GalN/LPS induced FHF. These findings might have important implications for the use of the anti TLR4 protein signaling as a potential target for therapeutic intervention in FHF.     

脂代谢相关三基因突变小鼠肝脏差异表达蛋白组研究

应用双向电泳及质谱技术对5周龄三基因(apoE^-1- / LDLR^-1-/Lepr^db/db)联合突变小鼠和野生型小鼠肝组织的差异蛋白质进行比较研究,借此分析脂代谢相关三基因联合突变小鼠肝脏蛋白质表达特点,研究差异表达蛋白与血脂代谢紊乱和动脉粥样硬化的关系.在实验中检测到三基因联合突变小鼠和野生型小鼠肝脏中分别平均有（841±57）个和（1 017±50）个蛋白点（n=3）,两者的平均匹配率分别为71.9%,83.2%.三基因联合突变小鼠有140个蛋白点未能与野生型小鼠匹配,其中相差5倍以上的上调点和下调点分别为7个和39个.选取其中的6个点做质谱分析,鉴定为endoplasmin precursor(Grp-94)、酸性富亮氨酸核磷蛋白32家族成员A(acidic leucin-rich nuclear phosphoprotein 32 family member A)、转铁蛋白前体、果糖二磷酸酶1、纤维连接蛋白前体、补体C3前体,纤维蛋白原B β多肽7种蛋白. 该结果提示,差异表达的蛋白对三基因联合突变小鼠的血脂代谢紊乱和动脉粥样硬化发生发展过程起一定作用.     

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.     

Cutting edge: Toll-like receptor (TLR)2- and TLR4-mediated pathogen recognition in resistance to airborne infection with Mycobacterium tuberculosis

Innate resistance against Mycobacterium tuberculosis is thought to depend critically on engagement of pattern recognition receptors on macrophages. However, the relative contribution of these receptors for containing M. tuberculosis infection has remained unexplored in vivo. To address this issue, we infected mice defective in CD14, TLR2, or TLR4 with M. tuberculosis by aerosol. Following infection with 100 mycobacteria, either mutant strain was as resistant as congenic control mice. Granuloma formation, macrophage activation, and secretion of proinflammatory cytokines in response to low-dose aerosol infection were identical in mutant and control mice. However, high-dose aerosol challenge with 2000 CFU M. tuberculosis revealed TLR2-, but not TLR4-defective mice to be more susceptible than control mice. In conclusion, while TLR2 signaling contributes to innate resistance against M. tuberculosis in borderline situations, its function, and that of CD14 and TLR4, in initiating protective responses against naturally low-dose airborne infection is redundant.     

Regulation of toll-like receptor 2 expression by macrophages following Mycobacterium avium infection

Recent studies have implicated Toll-like receptors (TLR), especially TLR2 and TLR4, as sentinel receptors that signal the interaction of macrophages with bacterial pathogens via a NF-kappaB-mediated pathway. The regulation of TLR gene expression, however, has not been intensively studied. Here, we report that TLR2 mRNA was induced following infection of murine macrophages with Mycobacterium avium. The changes in TLR2 mRNA correlated with an increase in TLR2 surface expression. Infection with M. avium resulted in a concomitant decrease in TLR4 mRNA. The effect of M. avium infection on TLR2 mRNA appeared to be mediated, in part, by TLR2 because the induction of the mRNA was partially blocked by preincubation of the macrophages with an anti-human TLR2 Ab. In contrast, the effect of LPS stimulation was mediated via TLR4 because infection of macrophages from LPS(d) mice, which do not express active TLR4, resulted in an increase in TLR2 mRNA, while treatment of macrophages from these mice with LPS failed to induce TLR2 mRNA. Several cytokines, including TNF-alpha, IL-1alpha, and GM-CSF, but not IFN-gamma, induced TLR2 mRNA. M. avium infection resulted in the induction of TLR2 mRNA by macrophages from both TNFRI knockout and NF-kappaB p50 knockout mice.