TLR4 Mutant Research Articles

Cutting Edge: Heligmosomoides polygyrus Induces TLR4 on Murine Mucosal T Cells That Produce TGFβ 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.

Hepatic Ischemia/Reperfusion Injury Involves Functional TLR4 Signaling in Nonparenchymal Cells

Endogenous ligands from damaged cells, so-called damage-associated molecular pattern molecules, can activate innate immunity via TLR4 signaling. Hepatic warm ischemia and reperfusion (I/R) injury and inflammation is largely TLR4 dependent. We produced TLR4 chimeric mice to assess whether the TLR4-dependent injury required TLR4 expression on liver parenchymal or nonparenchymal cells. Chimeric mice were produced by adoptive transfer of donor bone marrow cells into irradiated recipient animals using reciprocal combinations of TLR4 wild-type (WT; C3H/HeOuj) and TLR4 mutant (C3H/HeJ) mouse bone marrow. Wild-type chimeric mice bearing TLR4 mutant hemopoietic cells and TLR4 mutant mice transplanted with their own bone marrow-derived cells were protected from hepatic I/R and exhibited decreased JNK and NF-kappaB activation compared with WT chimeric mice transplanted with their own bone marrow. In contrast, TLR4 mutant mice transplanted with TLR4 WT bone marrow were not protected from liver I/R and demonstrated pronounced increases in JNK and NF-kappaB activation when compared with autochthonous transplanted mutant mice. In addition, depletion of phagocytes taking up gadolinium chloride failed to provide any additional protection to TLR4 mutant mice, but substantially reduced damage in WT mice after hepatic I/R. Together, these results demonstrate that TLR4 engagement on actively phagocytic nonparenchymal cells such as Kupffer cells is required for warm I/R-induced injury and inflammation in the liver.

Protective Immunity to the Larval Stages ofOnchocerca volvulusIs Dependent on Toll-Like Receptor 4

Toll-like receptor 4 (TLR4) has been shown to be important for the induction of Th2-dependent immune responses in mice. Protective immunity against larval Onchocerca volvulus in mice depends on the development of a Th2 immune response mediated by both interleukin-4 (IL-4) and IL-5. In addition, O. volvulus contains the rickettsial endosymbiont Wolbachia, which has molecules with lipopolysaccharide-like activities that also signal through TLR4. We therefore hypothesized that protective immunity to O. volvulus would not develop in C3H/HeJ mice which have a mutation in the Tlr4 gene (TLR4 mutant), either because of a decreased Th2 response to the larvae or because of the absence of a response to Wolbachia. TLR4-mutant mice were immunized against O. volvulus with irradiated third-stage larvae, and it was observed that Th2 responses were elevated based on increased IL-5 production, total immunoglobulin E (IgE) levels, antigen-specific IgG1 response, and eosinophil recruitment. Protective immunity, however, did not develop in the TLR4-mutant mice. The Th1 response, as measured by gamma interferon production from spleen cells, was comparable in both wild-type and TLR4-mutant mice. Furthermore, antibody responses to Wolbachia were absent in both wild-type and TLR4-mutant mice. Therefore, the defect in the development of a protective immune response against O. volvulus in TLR4-mutant mice is not due to loss of Th2 immunity or the response to Wolbachia but is due to an unidentified TLR4-dependent larval killing mechanism.

Multiple Toll-like receptor ligands induce an IL-6 transcriptional response in skeletal myocytes

Toll-like receptors (TLRs) comprise a critical sentinel that monitors body compartments for the presence of pathogens. Skeletal muscle expresses TLRs and responds to pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), by mounting an innate immune response. In the present study, we used C2C12 myocytes as a model system for skeletal muscle during infection. C2C12 cells responded to LPS in a time frame and with a pattern of gene expression that faithfully mimicked the response of skeletal muscle to LPS in vivo. LPS from a variety of Escherichia coli serotypes stimulated IL-6 synthesis. C2C12 cells expressed TLR1-7, but not TLR8 or TLR9, mRNA by RT-PCR. A synthetic tripalmitoylated cysteine-, serine-, and lysine-containing peptide (Pam) and LPS from Porphyromonas gingivalis, two TLR2 ligands, also stimulated IL-6 expression. LPS and Pam stimulated luciferase activity driven from NF-kappaB and IL-6 promoter-containing plasmids, and this response was blunted when the NF-kappaB binding site was mutated. LPS- and Pam-stimulated IL-6 expression was inhibited by the proteasome inhibitor MG-132 and the IkappaB kinase-2 (IKK2) inhibitor 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1). Pam-stimulated NF-kappaB and IL-6 promoter activities were disrupted by a dominant-negative form of TLR2, but not TLR4. Local injection of LPS or Pam into the gastrocnemius muscle stimulated IL-6 mRNA expression in the injected, but not the contralateral, muscle. The LPS- but not Pam-stimulated expression of IL-6 mRNA was blunted in skeletal muscle of mice carrying an inactivating mutation in TLR4. The data suggest that skeletal muscle and muscle cells recognize pathogen-associated molecules with specific TLRs to initiate an IL-6 transcriptional response.

Lipopolysaccharide-induced elevation and secretion of interleukin-1beta in the submandibular gland of male mice.

The intraperitoneal injection of lipopolysaccharide (LPS) (400 microg/kg body weight) induced the expression of mRNAs of inflammatory cytokines such as interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF)-alpha in the submandibular gland (SMG) of C3H/HeN mice but not that of C3H/HeJ mice, a mutant strain for Toll-like receptor-4 (TLR-4(-) mutant). The mRNA levels of these cytokines in the SMG of the wild-type mice increased as early as 3 hr after injection, peaked at 3-6 hr, and had decreased again by 24 hr. In this study, we particularly focused on IL-1beta, and induction by this endotoxin was investigated in detail. Denervation of the superior cervical trunk and chorda tympani nerve did not diminish the LPS-induced elevation of IL-1beta mRNA in the SMG, indicating the irrelevance of the central nervous system in this induction. TLR-4 mRNA and protein were shown to be strongly expressed in the SMG, suggesting the direct action of LPS on this gland. IL-1beta proteins were localized in the secretory granules of granular convoluted tubular (GCT) cells, and their molecular weights in the gland were 17.5 and 20 kDa. IL-1beta of the same size appeared in the saliva 6 hr after LPS injection in C3H/HeN but not in C3H/HeJ mice. The present study thus suggests that IL-1beta, an inflammation cytokine, is induced and secreted into the saliva in response to endotoxin injected intraperitoneally.

LightTyper Assay with Locked-Nucleic-Acid–Modified Oligomers for Genotyping of the Toll-Like Receptor 4 Polymorphisms A896G and C1196T

The toll-like receptor-4 (TLR4; OMIM*603030), a member of a large family of transmembrane proteins, is predominantly expressed on monocytes and macrophages (1). TLR4 is involved in various diseases and in innate and adaptive immunity (2), and it is thought to be crucial in mediating lipopolysaccharide effects (3). The common co-segregating variants, Asp299Gly and Thr399Ile, affect the extracellular domain of the TLR4 receptor. These variants are associated in humans with a blunted response to inhaled lipopolysaccharide in humans and lead to an altered host immune response to pathogens (4). The corresponding TLR4 genetic variations A896G (D299G; OMIM*603030.001) and C1196T (T399I, OMIM*603030.002) have been analyzed by use of DNA sequencing (4), restriction fragment length polymorphism analysis (5), MGB-TaqMan probes, matrix-assisted laser desorption/ionization analysis (6), and LightCycler hybridization probes (7). We developed a new homogeneous assay for genotyping of A896G and C1196T by use of locked-nucleic-acid (LNA)–modified SimpleProbe oligomers (8) on the LightTyper instrument (Roche). This assay provides a specific and sensitive method for high-throughput genotyping of these TLR4 mutations and may be useful for evaluating the presence of TLR4 polymorphisms in patients and to predict susceptibility to bacterial infection. Genotyping of 96 samples in a short time period using DNA from various sources is possible with this method. Furthermore, it has a wide dynamic range and therefore is applicable for DNA sets with varying DNA yields and quality. DNA was extracted from whole blood (n = 120) by use of the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s guidelines. DNA from serum (n = 377) was extracted by use of the Magna Pure LC DNA Isolation Kit I (Roche). Mutation detection for the LightTyper was based on a reported protocol (7), but we used SimpleProbe oligomers (Table 1⇓ ) instead of pairs of hybridization probes. These singular …

TLR-4 pathway mediates the inflammatory response but not bacterial elimination inE. colipneumonia

We examined the role of Toll-like receptor (TLR)-4 in modifying the lung inflammatory response and its effects on the bacterial recovery from the lungs following inhaled Escherichia coli in two different strains of TLR-4 mutant mice that are hyporesponsive to LPS. The C57BL/10ScN(tlr4(lps-del)) mice containing a deletion mutation in the TLR-4 gene showed lower proinflammatory cytokine levels, lower lung MPO activity, and less parenchymal and peribronchial inflammation compared with the C57BL/10ScSn mice, a related TLR-4 wild-type substrain. However, the C57BL/10ScN(tlr4(lps-del)) mutant showed lower bacterial recovery in the lungs following inhaled E. coli associated with a rapid but transient increase in air space neutrophil counts at 6 h. In comparison, the C3H/HeJ(tlr4(Lps-d)) mutant mice containing a Pro712His substitution in TLR-4 demonstrated lower proinflammatory cytokine levels, lower lung MPO activity, and lower neutrophil accumulation in the air spaces but showed no differences in the bacterial burden of inhaled E. coli at 6 h, when compared with the TLR-4 wild-type C3H/HeSnJ mice. Thus two different TLR-4 mutants showed attenuated inflammatory responses in the lungs, but the reduced inflammatory responses were not consistently associated with either improved or impaired bacterial elimination from the lungs. Our findings indicate that the inflammatory response to inhaled E. coli is TLR-4 dependent, but bacterial elimination depends on other factors in addition to TLR-4.

The matrix component biglycan is proinflammatory and signals through Toll-like receptors 4 and 2 in macrophages

Biglycan, a small leucine-rich proteoglycan, is a ubiquitous ECM component; however, its biological role has not been elucidated in detail. Here we show that biglycan acts in macrophages as an endogenous ligand of TLR4 and TLR2, which mediate innate immunity, leading to rapid activation of p38, ERK, and NF-kappaB and thereby stimulating the expression of TNF-alpha and macrophage inflammatory protein-2 (MIP-2). In agreement, the stimulatory effects of biglycan are significantly reduced in TLR4-mutant (TLR4-M), TLR2-/-, and myeloid differentiation factor 88-/- (MyD88-/-) macrophages and completely abolished in TLR2-/-/TLR4-M macrophages. Biglycan-null mice have a considerable survival benefit in LPS- or zymosan-induced sepsis due to lower levels of circulating TNF-alpha and reduced infiltration of mononuclear cells in the lung, which cause less end-organ damage. Importantly, when stimulated by LPS-induced proinflammatory factors, macrophages themselves are able to synthesize biglycan. Thus, biglycan, upon release from the ECM or from macrophages, can boost inflammation by signaling through TLR4 and TLR2, thereby enhancing the synthesis of TNF-alpha and MIP-2. Our results provide evidence for what is, to our knowledge, a novel role of the matrix component biglycan as a signaling molecule and a crucial proinflammatory factor. These findings are potentially relevant for the development of new strategies in the treatment of sepsis.

Consequence of functional Nod2 and Tlr4 mutations on gene transcription in Crohn’s disease patients

The concept that mutations in germ-line encoded pattern recognition receptors with immune activating functions are associated with an increased incidence in Crohn's disease (CD) is gaining acceptance. Whether these mutations have similar or distinct effects on cellular physiology remains obscure. The incidence of three single nucleotide polymorphisms (SNPs) within the Nod2 gene and one functional SNP within both the Tlr4 and Tlr5 gene in a Dutch cohort of 637 patients with inflammatory bowel disease and 127 controls was investigated. The functional consequence of mutant NOD2 and TLR4 was investigated by comparing gene expression profiles after stimulation of monocyte-derived dendritic cells (DCs) from homozygous TLR4- and NOD2-mutant patients with lipopolysaccharides and peptidoglycan, respectively. We observed that the R702W and 1007fs Nod2 alleles and the A299G Tlr4 alleles were significantly more prevalent in patients with CD as compared to healthy controls or patients with ulcerative colitis. The phenotype of TLR4- and NOD2-mutant DCs is distinct, but a large number of genes are up- or down-regulated concordantly. These data provide a concept for the genetic basis of CD; mutations in innate immunity cause similar effects on gene transcription and finally result in comparable clinical disease presentation.

B Lymphocyte Activation by Human Papillomavirus-Like Particles Directly Induces Ig Class Switch Recombination via TLR4-MyD88

Vaccination with human papillomavirus type 16 (HPV16) L1 virus-like particles (VLP) induces both high titer neutralizing IgG and protective immunity. Because protection from experimental infection by papillomavirus is mediated by neutralizing IgG, we sought the mechanisms that trigger humoral immunity to HPV16 L1 VLP. We find that HPV16 L1 VLP bind to murine B lymphocytes thereby inducing activation-induced cytidine deaminase expression and Ig class switch recombination to cause the generation of IgG. HPV16 L1 VLP also activate production of proinflammatory factors IFN-alpha, IL-6, MIP-1alpha, RANTES, and KC, up-regulate the expression of costimulatory molecules by naive B cells, and increase the B1 B cell subpopulation. These B cell responses to HPV16 L1 VLP are dependent upon MyD88. Although MyD88(-/-) B cells produce only mu transcript after exposure to HPV16 L1 VLP, MyD88(+/+) B cells express alpha, gamma, and mu Ig H chain and activation-induced cytidine deaminase transcripts. Notably, TLR4 mutant C3H/HeJ mice exhibited significantly reduced HPV16 VLP-specific IgG1, IgG2a, IgG2b, and IgG3 titers after vaccination as compared with the control C3H/HeOuJ mice. HPV16 L1 VLP directly activated class switch recombination and costimulatory molecule expression by B cells of C3H/HeOuJ mice but not C3H/HeJ mice. Thus HPV16 L1 VLP directly activate B cells to induce CD4(+) T cell independent humoral immune responses via TLR4- and MyD88-dependent signaling.

TLR4, but Not TLR2, Signals Autoregulatory Apoptosis of Cultured Microglia: A Critical Role of IFN-β as a Decision Maker

TLRs mediate diverse signaling after recognition of evolutionary conserved pathogen-associated molecular patterns such as LPS and lipopeptides. Both TLR2 and TLR4 are known to trigger a protective immune response as well as cellular apoptosis. In this study, we present evidence that TLR4, but not TLR2, mediates an autoregulatory apoptosis of activated microglia. Brain microglia underwent apoptosis upon stimulation with TLR4 ligand (LPS), but not TLR2 ligands (Pam(3)Cys-Ser-Lys(4), peptidoglycan, and lipoteichoic acid). Based on studies using TLR2-deficient or TLR4 mutant mice and TLR dominant-negative mutants, we also demonstrated that TLR4, but not TLR2, is necessary for microglial apoptosis. The critical difference between TLR2 and TLR4 signalings in microglia was IFN regulatory factor-3 (IRF-3) activation, followed by IFN-beta expression: while TLR4 agonist induced the activation of IRF-3/IFN-beta pathway, TLR2 did not. Nevertheless, both TLR2 and TLR4 agonists strongly induced NF-kappaB activation and NO production in microglia. Neutralizing Ab against IFN-beta attenuated TLR4-mediated microglial apoptosis. IFN-beta alone, however, did not induce a significant cell death. Meanwhile, TLR2 activation induced microglial apoptosis with help of IFN-beta, indicating that IFN-beta production following IRF-3 activation determines the apoptogenic action of TLR signaling. TLR4-mediated microglial apoptosis was mediated by MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-beta, and was associated with caspase-11 and -3 activation rather than Fas-associated death domain protein/caspase-8 pathway. Taken together, TLR4 appears to signal a microglial apoptosis via autocrine/paracrine IFN-beta production, which may act as an apoptotic sensitizer.

Toll-like receptor 4 deficiency and acute pancreatitis act similarly in reducing host defense during murine Escherichia coli peritonitis

Acute pancreatitis is frequently complicated by Gram-negative sepsis. Mammalian cells recognize lipopolysaccharide from Gram-negative bacteria via Toll-like receptor (TLR) 4. The objective of this study was to determine the role of TLR4 in the defense against Gram-negative sepsis in previously healthy mice and in animals with preexisting pancreatitis. A controlled, in vivo laboratory study. Research laboratory of a health sciences university. Female C3H/HeJ (nonfunctional TLR4 mutant) and C3H/HeN (wild-type) mice. Abdominal sepsis was induced by the intraperitoneal injection of Escherichia coli. Pancreatitis was induced by 12 hourly intraperitoneal injections of cerulein. The following experiments were performed. First, healthy TLR4 mutant mice demonstrated an enhanced bacterial load and dissemination of the infection relative to wild-type mice after intraperitoneal injection with E. coli, associated with a reduced early release of proinflammatory cytokines and an attenuated influx of neutrophils into the peritoneal fluid. Second, wild-type mice in which acute pancreatitis was induced by repeated cerulein injections showed an increased bacterial load and dissemination of E. coli relative to wild-type mice without pancreatitis, which was accompanied by a blunted proinflammatory cytokine response by peritoneal macrophages ex vivo and a diminished early cytokine and neutrophil response in vivo. Third, whereas the severity of cerulein-induced pancreatitis was similar in TLR4 mutant and wild-type mice, the important contribution of TLR4 to an effective host defense against E. coli sepsis observed in previously healthy mice was no longer present in mice with preexisting pancreatitis. These data suggest that TLR4 deficiency and acute pancreatitis act similarly in reducing host defense against E. coli peritonitis and that the role of TLR4 in severe Gram-negative infection depends, at least in part, on the presence of preexisting critical illness.

Innate Immunity to the Pathogenic Fungus Coccidioides posadasii Is Dependent on Toll-Like Receptor 2 and Dectin-1

Coccidioides posadasii is a pathogenic fungus that causes endemic and epidemic coccidioidomycosis in the deserts of North, Central, and South America. How the innate immune system responds to the organism is not well understood. Here we show that elicited mouse peritoneal macrophages respond to spherules (the tissue form of the fungus) by producing proinflammatory cytokines as measured by quantitative PCR of cellular transcripts and by enzyme-linked immunosorbent assay (ELISA) assays for secreted protein. We examined the contribution of Toll-like receptors (TLR) and MyD88 in macrophage responses to formalin-killed spherules (FKS) by comparing cytokine responses of elicited macrophages from different knockout mice. FKS were added to elicited mouse peritoneal macrophages from wild-type, TLR2-/-, and MyD88-/- cells, and wild-type cells made more tumor necrosis factor alpha, MIP-2, and interleukin 6 than did the mutant macrophages. In contrast, the C3H/HeJ mice, which have a point mutation in TLR4, and TLR4-/- B6 mice exhibited no defect in cytokine production compared to the control mice. We also investigated the role of the macrophage beta-glucan receptor, Dectin-1. RAW 264.7 macrophages overexpressing Dectin-1 produced more cytokines in respond to FKS, live spherules, and purified beta-glucan than did control RAW cells. Blockage of Dectin-1 with antibodies inhibited cytokine production in elicited mouse peritoneal macrophages. Taken together, these results show that cytokine responses in mouse peritoneal macrophages to C. posadasii spherules are dependent on TLR2, MyD88, and Dectin-1.

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.

Mutations Innate Immune System NOD/CARD15 Gene and Toll-Like-Receptor-4 Gene Are Associated with Increased Risk for Severe Acute Gvhd in Patients Who Underwent Allogeneic Transplantation.

The innate immune system detects invading pathogens through several pattern-recognition receptors and represents the first line of mucosal host defense. Mutations of the NOD2/CARD15 and Toll like receptor −2, −4, −9 genes have been associated with an increased incidence of inflammatory bowel disease due to a diminished response to bacterial cell wall products. Moreover, it was reported that mutations of NOD2 gene loci might be associated with an increased risk for TRM and severe GVHD in patients who underwent allogeneic blood stem cell transplantation. Here we analyzed 201 patients and their respective donors for NOD2, TLR-2, TLR-4 and TLR-9 mutations and correlated the results with the incidence of overall acute GVHD and intestinal GVHD. Further, we evaluated if the occurrence of NOD2/CARD15 and TLR allele mutations are accompanied with an increased risk for transplant-related mortality and overall survival.Mutated alleles of the NOD2 gene were observed in 17% of the patients and 16% of the donors. NOD2 gene mutations in patients and donors together were associated with an increased incidence of severe acute GVHD grade III-IV (6/11 patients; 55% versus 35/145 patients; 24%). If a mutated allele of the NOD2 gene was found either in patients or in donors only no correlation with increased incidence of severe acute GVHD was seen in this study.Patients with mutated alleles in TLR-4 genes (Asp299Gly and Thr399Ile) had a higher incidence of intestinal GVHD (p<0.02), whereas mutations of donor alleles only had no influence on the occurrence of intestinal GVHD in this analysis.No correlation with intestinal GVHD or overall acute GVHD was seen in patients with mutated alleles for TLR-2 and TLR-9. Overall survival, TRM and relapse risks was not influenced in none of the patients with mutated alleles of the innate immune system genes. Multivariate analysis including all potential factors, confirmed that that mutations of NOD2/CARD15 in patients and donors together influenced the occurrence of severe acute GVHD, whereas the occurrence of intestinal GVHD was influenced in patients by mutated alleles of TLR-4 gene. But mutations in alleles of the NOD2 or TLR-2,−4,−9 had no influence on the outcome of allogeneic transplant (TRM and overall survival) in the multivariate analysis.Since all patients in our institution had received an intestinal bacterial decontamination using metronidazole and ciprofloxacin after allogeneic stem cell transplantation, it might be speculative, if the reduction of concentrations of anaerobic bacteria in the intestinum might have protected patients from the occurrence of increased TRM. Decontamination of anaerobic bacteria is associated with a reduced risk for severe acute GVHD as reported earlier (Blood 1999; 93:3267–75).

Lipopolysaccharide increases alveolar type II cell number in fetal mouse lungs through Toll-like receptor 4 and NF-kappaB.

Chorioamnionitis is a major cause of preterm delivery. Infants exposed to inflammation in utero and then born preterm may have improved lung function in the immediate postnatal period. We developed a mouse model of chorioamnionitis to study the inflammatory signaling mechanisms that might influence fetal lung maturation. With this in vivo model, we found that Escherichia coli lipopolysaccharide (LPS) increased the number of alveolar type II cells in the fetal mouse lung. LPS also increased type II cell number in cultured fetal lung explants, suggesting that LPS could directly signal the fetal lung in the absence of maternal influences. Using immunostaining, we localized cells within the fetal mouse lung expressing the LPS receptor molecule Toll-like receptor 4 (TLR4). Similar to the signaling pathways in inflammatory cells, LPS activated NF-kappaB in fetal lung explants. Activation of the TLR4/NF-kappaB pathway appeared to be required, as LPS did not increase the number of type II cells in C.C3H-Tlr4(Lps-d) mice, a congenic strain containing a loss of function mutation in tlr4. In addition, the sesquiterpene lactone parthenolide inhibited NF-kappaB activation following LPS exposure and blocked the LPS-induced increase in type II cells. On the basis of these data from our mouse model of chorioamnionitis, it appears that LPS specifically activated the TLR4/NF-kappaB pathway, leading to increased type II cell maturation. These data implicate an important signaling mechanism in chorioamnionitis and suggest the TLR4/NF-kappaB pathway can influence lung development.

The Development of Early Host Response to Pseudomonas aeruginosa Lung Infection Is Critically Dependent on Myeloid Differentiation Factor 88 in Mice

Toll-like receptors (TLR) induce distinct patterns of host responses through myeloid differentiation factor 88 (MyD88)-dependent and/or -independent pathways, depending on the nature of the pathogen. Pseudomonas aeruginosa is a cause of serious lung infection in immunocompromised individuals and cystic fibrosis patients. The role of the TLR-MyD88 pathway in P. aeruginosa-induced lung infection in vivo was examined in this study. MyD88-/- mice demonstrated an impaired clearance of P. aeruginosa from the lung. Little or no neutrophil recruitment was observed in the airways of MyD88-/- mice following P. aeruginosa lung infection. This observation was associated with a reduced production of inflammatory mediators that affect neutrophil recruitment, including macrophage-inflammatory protein-2, tumor necrosis factor, and interleukin-1beta in the airways of MyD88-/- mice. Similarly, MyD88-/- mice showed inhibited NF-kappaB activation in the lung following P. aeruginosa infection. Interestingly, P. aeruginosa infection induced a 7.5-fold increase of TLR2 mRNA expression in the lungs of MyD88+/+ mice. Furthermore, host responses to P. aeruginosa lung infection in TLR2-/- and TLR4 mutant mice were partially inhibited compared with the responses of respective control mice. Taken together, our results indicate that the MyD88-dependent pathway is essential for the development of early host responses to P. aeruginosa infection, leading to the clearance of this bacterium, and that TLR2 and TLR4 are involved in this process.

Toll-like receptor 4 mutation impairs the macrophage TNFα response to peptidoglycan

Macrophages produce TNFα when infected by bacteria, a response that follows recognition of microbial components by members of the Toll-like receptor (TLR) family. Cells that lack functional TLR4 are known to have markedly diminished responses to Gram-negative lipopolysaccharide. We demonstrate in the present work that peritoneal macrophages derived from strains of mice that carry a spontaneous, inactivating mutation in TLR4 also have impaired production of TNFα in response to peptidoglycan, a ligand for TLR2. This impairment is at a step of biosynthesis subsequent to the generation of mRNA. TLR4-activated signals act at this step to enhance peptidoglycan-induced TNFα production in wild-type mice. Based on these observations, we conclude that macrophages from wild-type mice are primed by chronically acting TLR4 signals, probably resulting from exposure to environmental lipopolysaccharide. These signals are required for optimal production of TNFα in response to TLR2 stimulation, and are absent in macrophages from TLR4 mutant animals.

Non-mannose-capped lipoarabinomannan induces lung inflammation via toll-like receptor 2.

Non-mannose-capped lipoarabinomannan (AraLAM) is part of the cell membrane of atypical mycobacteria. To determine the capacity of AraLAM to induce lung inflammation in vivo and to determine the signaling receptors involved herein, wild-type (WT) mice, lipopolysaccharide binding protein knockout mice, CD14-deficient (CD14 KO) mice, Toll-like receptor (TLR) 4 mutant mice, or TLR2 KO mice were intranasally inoculated with purified AraLAM. AraLAM induced high lung levels of tumor necrosis factor, interleukin-1beta, interleukin-6, and cytokine-induced neutrophil chemoattractant (KC) and an influx of neutrophils into the pulmonary compartment of WT mice. Lipopolysaccharide binding protein knockout, CD14 KO, and TLR4 mutant mice displayed similar inflammatory responses as WT mice, whereas in TLR2 KO mice, AraLAM-induced lung inflammation was strongly diminished. In addition, TLR2 KO mice, but not CD14 KO or TLR4 mutant mice, displayed a delayed clearance of pulmonary infection with the atypical AraLAM expressing Mycobacterium smegmatis. These data indicate that TLR2 is the signaling receptor for purified AraLAM in the lung in vivo and that this receptor contributes to an effective clearance of M. smegmatis from the pulmonary compartment.

LPS-induced decrease of neutrophil apoptosis is regulated through TLR2, not through TLR4

Abstract Introduction: Decreased neutrophil apoptosis during sepsis may amplify or prolong systemic inflammation. Toll-like receptor (TLR) 4 is thought to recognize LPS from gram-negative bacteria and TLR2 is thought to recognize gram-positive bacterial products. The purpose of this study was to investigate the role of TLRs in LPS-induced modulation of neutrophil apoptosis in mice. Methods: Neutrophils were isolated from C3H/HeJ mice which have a point mutation in TLR4 or from TLR2KO mice. C3H/HeN mice and C57BL6 mice were used as controls. Blood-derived neutrophils from the mice were cultured with or without LPS or synthetic bacterial lipopeptide (sBLP) in FBS-containing medium. Polymyxyn B (PMB) was used to block the effect of LPS-induced activity. After 24 hours, apoptosis was assessed by two-color flowcytometry using a neutrophil-specific antibody and propidium iodide. LPS-induced mRNA expression for A1, Mcl-1, Bcl-XL, and Bak was determined by RT followed by real-time PCR. Results: LPS and sBLP significantly decreased the apoptosis of neutrophils from C3H/HeN mice as well as from C3H/HeJ mice, and the LPS-induced decrease in apoptosis was blocked by PMB in these mice. However, neither LPS nor sBLP decreased apoptosis in neutrophils from TLR2KO mice. Finally there were no significant differences in LPS-induced mRNA expression for the apoptosis-related factors examined in this study. ∗ . C3H/HeN C3H/HeJ WT TLR2KO VEHICLE 41 ± 8 36 ± 5 53 ± 6 39 ± 8 LPS (1 μg/ml) 22 ± 4 ∗ 18 ± 7 ∗ 16 ± 4 ∗ 39 ± 11 ∗ LPS (1 μg/ml)/PMB (50 μg/ml) 47 ± 7 32 ± 2 ND ∗∗ ND BLP (1 μg/ml) 15 ± 0 ∗ 15 ± 1 ∗ 16 ± 9 ∗ 39 ± 9 ∗ Data depict mean ± SD percent neutrophil apoptosis; ∗ p ∗∗ ND = not done. Conclusions: These results suggest that LPS decreases neutrophil apoptosis mainly through TLR2, not through TLR4, and the transcriptional activity of A1, Mcl-1, Bcl-XL, and Bak apoptosis-associated factors do not appear to play a major role in this process.