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PubMed2002

Non-LPS components of Chlamydia pneumoniae stimulate cytokine production through Toll-like receptor 2-dependent pathways.

Recent studies suggest that infection with Chlamydia pneumoniae is associated with atherosclerosis, and that cytokines play an important role in the initiation and progression of Chlamydia-induced inflammation. When freshly isolated peripheral blood mononuclear cells (PBMC) were stimulated for 24 h with sonicated C. pneumoniae, significant amounts of the pro-inflammatory cytokines TNF-alpha and IL-1beta and of the anti-inflammatory cytokine IL-10 were released into the supernatant. The addition of serum increased cytokine release induced by C. pneumonia two- to fivefold (p < 0.01). This effect was not due to complement, mannose-binding lectin (MBL) or lipopolysaccharide-binding protein (LBP). Incubation of PBMC with either anti-Toll-like receptor 4 (TLR4) or anti-CD14 blocking antibodies did not influence the production of cytokines induced by Chlamydia. The induction of cytokines by C. pneumoniae in macrophages from C3H / HeJ mice, known to have a defective TLR4, was identical to that measured in control macrophages from C3H / HeN mice. In contrast, incubation of PBMC with an anti-TLR2 blocking antibody significantly inhibited the production of TNF by 67 % and of IL-1beta by 72 %. In conclusion, C. pneumoniae stimulates cytokine production in a serum-dependent manner, but independently of complement, MBL and LBP. C. pneumoniae induces the pro-inflammatory cytokines TNF and IL-1beta through TLR2, but not TLR4 and CD14.

Acute-Phase Proteins↗
PubMed2002

Role of chlamydial heat shock protein 60 in the stimulation of innate immune cells by Chlamydia pneumoniae.

Chlamydia pneumoniae stimulates potently maturation of and cytokine secretion by bone marrow-derived dendritic cells (BMDDC). BMDDC responses depend mainly on Toll-like receptor (TLR)2 and to a minor extent on TLR4. We demonstrate here using C. pneumoniae in an infectious model with the replication-permissive epithelial cell line HEp2 that HSP60 is produced in substantial amounts in chlamydial inclusions during infection. Electron microscopy of chlamydial inclusions revealed that HSP60 was mainly associated with reticulate bodies, but was also located in between the different chlamydial developmental forms. Supernatants of permissive HEp2 cells infected with C. pneumoniae contained soluble chlamydial HSP60 as demonstrated by Western blotting and were able to stimulate BMDDC of wild-type mice. The stimulatory capacity of culture supernatants correlated with the presence of chlamydial HSP60. In contrast, BMDDC from TLR4-mutant mice crossed to TLR2-deficient mice were not stimulated by the culture supernatant, indicating that chlamydial HSP60 but not cytokines, possibly secreted by infected HEp2 cells, are responsible for the observed stimulation of BMDDC. Purified recombinant HSP60 from C. pneumoniae stimulated BMDDC in a TLR2- and TLR4-dependent fashion similar to the whole microorganism. In summary, these data suggest chlamydial HSP60 as an important mediator of inflammatory responses during infection with C. pneumoniae.

Animals↗
PubMed2003

Caspase-dependent and -independent apoptosis of mast cells induced by withdrawal of IL-3 is prevented by Toll-like receptor 4-mediated lipopolysaccharide stimulation.

IL-3-dependent mucosal-like mast cells undergo apoptosis upon withdrawal of IL-3. Generally, the apoptosis is mediated by the activation of caspases and inhibited by addition of the pan-caspase inhibitors z-VAD-FMK or BOC-D-FMK. However, DNA fragmentation, a typical characteristic of apoptosis, is not inhibited by z-VAD-FMK or BOC-D-FMK in mast cell apoptosis. In this study, we demonstrate that the apoptosis of mast cells is mediated by both caspase-dependent and -independent mechanisms. The caspase-independent apoptosis is mediated by the translocation of endonuclease G from mitochondria into nuclei. Withdrawal of IL-3 caused down-regulation of Bcl-xL, resulting in a drop in mitochondrial membrane transition potential followed by the release of cytochrome c and endonuclease G from mitochondria. However, stimulation of mast cells through Toll-like receptor 4 (TLR4) by lipopolysaccharide prevented mast cell apoptosis by inducing expression of Bcl-xL. Moreover, the activation of mast cells by LPS is enhanced in the presence of IFN-gamma, which up-regulates the expression of cell surface TLR4. Taken together, these observations provide evidence that mast cells play important roles not only in allergic reactions but also in innate immunity recognizing enterobacteria through TLR4, and are regulated differently from allergic inflammation by Th1 cytokines.

Active Transport, Cell Nucleus↗
PubMed2006

Endotoxin modulates the capacity of CpG-activated liver myeloid DC to direct Th1-type responses.

DC are believed to play important roles in the induction and regulation of immune responses in the liver, an organ implicated in peripheral tolerance. Since the liver is located downstream of the gut, it is constantly exposed to bacterial LPS. Our recent observations indicate that prior exposure to endotoxin modulates subsequent liver DC responses to this TLR4 ligand. In this study, we demonstrate that endotoxin modifies the capacity of mouse liver myeloid DC (MDC) activated by CpG (TLR9 ligand) to direct Th1-type responses. IL-12 production by liver MDC was significantly lower than that of spleen MDC following CpG or Imiquimod (R837; TLR7 ligand) activation in vitro. In addition, allogeneic T cells stimulated by CpG-activated liver MDC secreted significantly lower levels of IFN-gamma than T cells stimulated with CpG-activated spleen MDC. A similar effect on liver DC was observed in response to in vivo CpG administration. This effect may be explained by exposure of the DC to endotoxin, because LPS attenuated IL-12 production by CpG-stimulated liver MDC, both in vitro and in vivo. Moreover, attenuation of the response to CpG was not observed in liver MDC from TLR4-mutant (C3H/HeJ) mice, in which TLR4 signaling is impaired. These data suggest that endotoxin-induced 'cross-tolerance' to TLR ligands in liver DC may contribute to down-regulation of hepatic immune responses.

Adjuvants, Immunologic↗
PubMed2006

Pathogen-specific TLR signaling in mucosa: mutual contribution of microbial TLR agonists and virulence factors.

Detection of microorganisms through microbe-associated molecular patterns (MAMP) by Toll-like receptors (TLR) is crucial to trigger protective immunity. In the mucosa, sentinel cells are exposed to MAMP from both pathogens and commensals; however, the TLR response is tightly controlled to avoid inflammation in response to commensals. Uropathogenic Escherichia coli (UPEC) trigger innate responses during urinary tract infection in a TLR4-dependent and CD14-independent manner. UPEC express virulence factors, such as type 1 fimbriae and/or P fimbriae, allowing bacterial attachment to the epithelium. In this issue of the European Journal of Immunology, Fisher et al. show that fimbriae are required to induce a TLR4-specific epithelial response. Depending on the fimbriae expressed by UPEC, different adaptor molecules are involved in TLR4 signaling. These data add to the recent body of evidence suggesting that TLR responses are regulated by co-receptors, such as receptors for virulence factors. In conclusion, the "pathogenic" TLR stimulation provides a novel way for the host to ignore commensal bacteria.

Animals↗
PubMed2007

Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.

UNLABELLED: Toll-like receptors (TLRs) play a key role in the innate immune response. The aim of this study was to examine the expression of TLR2 and TLR4 in chronic hepatitis B (CHB). The TLR2 and TLR4 expression on hepatocytes and Kupffer cells from fresh liver biopsies was measured from 21 patients with untreated hepatitis B e antigen (HBeAg)-positive and HBeAg-negative CHB. Parallel studies were also undertaken on monocytes from their peripheral blood. Expression of TLR2 on hepatocytes, Kupffer cells, and peripheral monocytes was significantly reduced in patients with HBeAg-positive CHB in comparison with HBeAg-negative CHB and controls, whereas it was significantly increased in HBeAg-negative CHB compared with controls. The level of TLR4 expression did not differ significantly between the groups. These results were confirmed in vitro using hepatic cell lines transduced with recombinant HBV baculovirus expressing wild-type HBV (HBeAg-positive), precore stop codon (G1896A) mutant HBV (HBeAg-negative). The functional relevance of these findings was established by the demonstration of significantly reduced cytokine production (TNF-alpha) and phospho-p38 kinase expression in the presence of the HBeAg. In the absence of HBeAg, HBV replication was associated with up-regulation of the TLR2 pathway leading to increased TNF-alpha production. CONCLUSION: This study demonstrates a potentially important interaction between HBeAg, HBV, and the innate immune response.

Adult↗
PubMed2004

The expression of toll-like receptor 2 and 4 mRNA in local tissues of model of oropharyngeal candidiasis in mice.

To investigate the expression of Toll-like receptor (TLR) 2 and 4 mRNA in local tissues of model of oropharyngeal candidiasis in mice and to explore the potential role of TLR2 and TLR4 in earlier period of immune response, a murine model of oropharyngeal candidiasis inoculated by cotton wool balls saturated with Candida albicans was established. Mice were sacrificed at the indicated time points and the oropharyngeal tissues were excised. The expression of TLR2 and TLR4 mRNA was detected by RT-PCR. The results showed that low level of TLR2/4 mRNA could be detected in oropharyngeal tissues, but they were markedly up-regulated 6 h after inoculation, peaking after 12-24 h. Tissue TLR4 mRNA was gradually down-regulated 24-48 h, while TLR2 mRNA levels remained high up to the 72nd h. These data suggested that oropharyngeal infection of Candida albicans could result in up-regulation of TLR2/4 mRNA expression in local tissues, which might play important roles in earlier period of immune response.

Animals↗
PubMed2005

Dialyzable leukocyte extract differentially regulates the production of TNFalpha, IL-6, and IL-8 in bacterial component-activated leukocytes and endothelial cells.

OBJECTIVE: To investigate i) whether the Dialyzable Leukocyte Extract (DLE) modulates the production of proinflammatory cytokines in leukocytes activated by the bacterial cell wall components lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN); ii) the effect of DLE on LPS-stimulated endothelial cells; and iii) whether the regulatory effect of DLE on inflammatory mediators is related to the modulation of Toll-like receptors (TLRs), NF-kappaB and cAMP signaling pathways. METHODS: Leukocytes were stimulated with LPS, LTA, and PGN in the presence of DLE. Endothelial cells were stimulated with LPS and treated with DLE. The levels of Tumor Necrosis Factor-alpha(TNFalpha), Interleukin-6 (IL-6), and IL-8 in culture supernatants were evaluated by ELISA. The expression of Toll-like receptor 2 (TLR2) and 4 (TLR4), NF-kappaB activity and cAMP levels were evaluated by flow cytometry, EMSA, and EIA, respectively. RESULTS: The addition of DLE to leukocytes stimulated with cell wall constituents suppressed the production of TNFalpha. However, DLE induced IL-8 release in monocytes and enhanced IL-6 and IL-8 production by activated monocytes and endothelial cells. Also, DLE induced TLR2 and TLR4 expression, and increased cAMP levels, whereas NF-kappaB activity was inhibited. CONCLUSIONS: The present data indicate the differential regulation by DLE of the production of TNFalpha, IL-6, and IL-8 cytokines, associated with effects on TLR2 and TLR4 expression and NF-kappaB and cAMP activities. We suggest a putative mechanism for the biological effects of DLE in activated leukocytes and endothelial cells.

Cells, Cultured↗
PubMed2002

In vivo response of neutrophils and epithelial cells to lipopolysaccharide injected into the monkey ileum.

Since few previous studies have investigated the in vivo response of intestinal mucosa to the luminally administered lipopolysaccharide (LPS), we examined the cellular localization of exogenously applied LPS in the intestinal mucosa and the expression of Toll-like receptor (TLR) and IL-1 receptor-associated kinase (IRAK) in the epithelial cells of monkey ileum. FITC-labeled LPS was injected into the lumen of monkey ileum. Thirty minutes after the LPS injection, the ileal tissue was fixed and localization of FITC fluorescence in the ileal mucosa was examined. We applied Factor C immunohistochemistry to demonstrate the bioactivity of LPS taken up by the mucosal tissue. The expression of TLR4 and IRAK-1 in the epithelial cells was also examined by immunohistochemistry. FITC fluorescence was detected in the cells migrated into the epithelium and those in the lamina propria. The FITC-labeling cells were completely overlapped with the Factor C immunoreactive cells. These FITC-labeling/Factor C-positive cells were identified as neutrophils by the immunoelectron microscopic analysis. TLR4 and IRAK-1 were expressed at the apical membrane of the epithelial cells in the ileum of both control and FITC-LPS injected animals. These results suggest that intraluminal injection of LPS stimulates the transmigration of neutrophils into the epithelium and these neutrophils may uptake luminally applied LPS and possibly inactivate the enterotoxin. Expression of TLR4 and IRAK-1 in the epithelial cells suggests that epithelial cells may react to LPS and produce chemoattractant mediator to induce the neutrophil chemotaxis.

Animals↗
PubMed2006

The lack of genetic association of the Toll-like receptor 2 (TLR2) Arg753Gln and Arg677Trp polymorphisms with rheumatic heart disease.

UNLABELLED: Acute rheumatic fever (ARF) is a non-suppurative inflammatory disease after group A, beta haemolytic streptococcal pharyngitis. Certain individuals can develop ARF. This finding implies variability in host predisposition to ARF. A variety of studies have linked specific genetic markers with ARF or rheumatic heart disease (RHD) as a sequelae of ARF. For this purpose, we aimed to search the role of polymorphisms in Toll-like receptor-2 and -4 (TLR2 and TLR4) gene in Turkish patients with RHD. This study included a total 84 patients with RHD, ages ranging between 18 and 65, 25 male and 59 female, fulfilling the revised classification criteria of Jones. One hundred forty healthy unrelated persons were selected as a control group. Genotype analysis: DNA was extracted from whole blood. TLR4 gene (Asp 299Gly and Thr399Ile) and TLR2 gene (Arg753Gln and Arg677Trp) polymorphisms were genotyped by the previously reported method. STATISTICAL ANALYSIS: binary logistic regression models were used. Results were expressed as odds ratios (OR) with corresponding 95% confidence intervals (95% CI). Significant level was predefined at 0.05. There was a significant difference for carrying Ile allele in the 399 position in the patients compared to healthy controls (OR = 5.26, 95% CI, 1.40-19.73, p = 0.014). In the TLR4 gene, Asp 299Gly polymorphism did not reach to a statistically significant value (OR = 3.02). We found no Arg753Gln polymorphism of the TLR2 gene in the patient group. There were three heterozygote samples in the healthy group. We did not detect Arg677Trp polymorphism of the TLR2 gene in both patient and control groups.

Adolescent↗
PubMed2006

Expression of nitric oxide synthase isoforms in the mouse kidney: cellular localization and influence by lipopolysaccharide and Toll-like receptor 4.

We determined the cellular mRNA expression of all intrarenal nitric oxide (NO)-producing NO synthase (NOS) isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS) and inducible NOS (iNOS) in kidneys from wild-type mice (WT) and immune deficient Toll-like receptor 4 (TLR4) mutant mice, during normal physiological conditions and during a short-term (6-16 h) endotoxic condition caused by systemically administered lipopolysaccaride (LPS). Investigations were performed by means of in situ hybridization and polymerase chain reaction amplification techniques. In WT, LPS altered the expression rate of all intrarenal NOS isoforms in a differentiated but NOS-isoform coupled expression pattern, with iNOS induction, and up- and down-regulation of the otherwise constitutively expressed NOS isoforms, e.g. eNOS and nNOS and an iNOS isotype. In TLR4 mutants, LPS caused none or a lowered iNOS induction, but altered the expression rate of the constitutive NOS isoforms. It is concluded that the intrarenal spatial relation of individual NOS-isoforms and their alteration in expression provide the basis for versatile NO-mediated renal actions that may include local interactions between NOS isoforms and their individual NO-target sites, and that the NOS-isoform dependent events are regulated by TLR4 during endotoxic processes. These regulatory mechanisms are likely to participate in different pathophysiological conditions affecting NO-mediated renal functions.

Animals↗
PubMed2006

Simvastatin suppresses endotoxin-induced upregulation of toll-like receptors 4 and 2 in vivo.

In addition to lipid lowering effects, statins appear to have pleiotropic immunomodulatory properties. As they particularly affect monocyte functions, we tested the influence of statin treatment on the monocyte activating toll-like receptors (TLR) 4 and 2 in response to lipopolysaccharides (LPS) in vivo. In this double-blind, placebo-controlled study, 20 healthy, male subjects were randomized to receive either simvastatin (80 mg/day) or placebo for 4 days before intravenous LPS administration (20 IU/kg). Simvastatin did not influence the increase in TLR transcripts after LPS administration measured in mRNA isolated from whole blood by quantitative RT-PCR. In contrast, the parallel upregulation of TLR4 and TLR2 on the surface of monocytes determined by flow cytometry was attenuated by more than half after LPS challenge (P<0.02). Suppressed TLR4 and TLR2 expression was associated with diminished circulating concentrations of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. In conclusion, high-dose simvastatin pretreatment blunted TLR4 and TLR2 expression on monocytes in a human endotoxemia model on a posttranscriptional level. This suppressive effect of statins on key receptors of the innate immunity which was associated with a reduction of effector cytokines reveals a potential mechanism for their beneficial effects in sepsis and cardiovascular disease.

Adult↗
PubMed2004

Identification of a novel isoform of MD-2 that downregulates lipopolysaccharide signaling.

MD-2 is an association molecule of Toll-like receptor 4 and is indispensable for the recognition of lipopolysaccharide. Here we report the identification of mRNA for an alternatively spliced form of MD-2, named MD-2B, which lacks the first 54 bases of exon 3. When overexpressed with MD-2, MD-2B competitively suppressed NF-kappaB activity induced by LPS. Regardless of the truncation, however, MD-2B still bound to TLR4 as efficiently as MD-2. Flow cytometric analyses revealed that MD-2B inhibited TLR4 from being expressed on the cell surface. Our data indicate that MD-2B may compete with MD-2 for binding to TLR4 and decrease the number of TLR4/MD-2 complexes on the cell surface, resulting in the inhibition of LPS signaling.

Amino Acid Sequence↗
PubMed2005

Ethanol alters cellular activation and CD14 partitioning in lipid rafts.

Alcohol consumption interferes with innate immunity. In vivo EtOH administration suppresses cytokine responses induced through Toll-like receptor 4 (TLR4) and inhibits TLR4 signaling. Actually, EtOH exhibits a generalized suppressive effect on signaling and cytokine responses induced by through most TLRs. However, the underlying mechanism remains unknown. RAW264.7 cells were treated with LPS or co-treated with EtOH or with lipid raft-disrupting drugs. TNF-alpha production, IRAK-1 activation, and CD14 partition were evaluated. EtOH or nystatin, a lipid raft-disrupting drug, suppressed LPS-induced production of TNF-alpha. The suppressive effect of EtOH on LPS-induced TNF-alpha production was additive with that of methyl-beta-cyclodextrin (MCD), another lipid raft-disrupting drug. EtOH interfered with IRAK-1 activation, an early TLR4 intracellular signaling event. Cell fractionation analyses show that acute EtOH altered LPS-related partition of CD14, a critical component of the LPS receptor complex. These results suggest a novel mechanism of EtOH action that involves interference with lipid raft clustering induced by LPS. This membrane action of EtOH might be one of the mechanisms by which EtOH acts as a generalized suppressor for TLR signaling.

Animals↗
PubMed2007

Attenuation of obesity-induced adipose tissue inflammation in C3H/HeJ mice carrying a Toll-like receptor 4 mutation.

Obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. We have provided in vitro evidence that saturated fatty acids, which are released from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for Toll-like receptor 4 (TLR4) to induce the inflammatory changes in macrophages. Here we show the attenuation of adipose tissue inflammation in C3H/HeJ mice carrying a functional mutation in the TLR4 gene relative to control C3H/HeN mice during a 16-week high-fat diet. We also find that adiponectin mRNA expression is significantly reduced by co-culture of hypertrophied 3T3-L1 adipocytes and C3H/HeN peritoneal macrophages, which is reversed, when co-cultured with C3H/HeJ peritoneal macrophages. This study provides in vivo evidence that TLR4 plays a role in obesity-related adipose tissue inflammation and thus helps to identify the therapeutic targets that may reduce obesity-induced inflammation and the metabolic syndrome.

Adipocytes↗
PubMed2004

Lipopolysaccharide signaling induces serum amyloid A (SAA) synthesis in human hepatocytes in vitro.

To investigate the role of lipopolysaccharide (LPS) in hepatocyte activation, we examined the expression of Toll-like receptor 4 (TLR4), the putative receptor for LPS in human hepatocytes. TLR4 mRNA and protein expression was confirmed in human hepatocytes. Stimulation of human hepatocytes with LPS results in rapid degradation of IkappaB-alpha and mitogen activated protein kinase activation. Human hepatocytes stimulated by LPS produced serum amyloid A protein. Our data suggest that human hepatocytes utilize components of TLR4 signal transduction pathways in response to LPS and these direct LPS-mediated effects on hepatocytes may contribute to liver inflammation and injury.

Cells, Cultured↗
PubMed2006

Reduced expression and functional impairment of Toll-like receptor 2 on dendritic cells in chronic hepatitis C virus infection.

BACKGROUND: Dendritic cells (DCs) utilize Toll-like receptors (TLRs) to sense virus and initiate immune responses. We aimed at elucidating the roles of TLRs on DCs in hepatitis C virus (HCV) infection. METHODS: Monocyte-derived DCs were obtained from 32 healthy volunteers (HV) and 30 chronically HCV-infected patients (CH). TLR2, TLR3 and TLR4 expressions on immature DCs were quantified by real-time quantitative RT-PCR. We stimulated DCs with specific TLR ligands and examined DC maturation, cytokine production and ability to stimulate allogeneic CD4(+) T cells. RESULTS: TLR2 expression on immature DCs was lower in the CH group, whereas those of TLR3 or TLR4 were not different between the groups. Each TLR ligand induced DC maturation and stimulated them to release comparable levels of IL-12p70, IL-6, IL-10, TNF-alpha and IFN-beta between the groups. TLR2 and TLR4 ligands enhanced DC ability to stimulate T cell proliferation, with the degree due to the TLR2 ligand being lower in the CH group. CONCLUSIONS: In HCV infection, the TLR2 expression on DCs is reduced and TLR2-stimulated DCs show lesser ability to proliferate T cells than healthy counterparts, suggesting that the TLR2 system is involved in HCV-induced immunopathogenesis.

Journal Article↗
PubMed2004

Toll-like receptors 2 and 4 do not contribute to clearance of Chlamydophila pneumoniae in mice, but are necessary for the release of monokines.

Activation of immune cells by Chlamydophila pneumoniae in vitro has been shown to be toll-like receptor (TLR2)-dependent, but TLR4 is also involved to a minor extent. To investigate the role of TLR2 and TLR4 in vivo, a murine model of C. pneumoniae infection was established. Mice were infected intranasally with a low inoculum of 106 C. pneumoniae elementary bodies (EB) and spreading of bacteria was monitored by real-time PCR. The bronchoalveolar lavage (BAL) showed maximal bacterial load on the day of infection and the lung 2 days later. By day 95, C. pneumoniae were eradicated completely. In serum, anti-C. pneumoniae IgG became detectable on day 18 by microimmunofluorescence test. The course of infection was mild with no apparent symptoms, lack of acute phase response and no induction of tumor necrosis factor-alpha and interleukin-6 in BAL, lung supernatants or blood. Infection of TLR2-/- and C3H/HeJ mice revealed no differences in clearance of bacteria and serological responses compared to wild-type controls, even if a dose of 10(7) EB was used. Intracellular replication of C. pneumoniae in the lungs was proven by the efficacy of antibiotic treatment. These findings indicate that in vivo TLR2 and TLR4 are not important for the development of antibodies and elimination of C. pneumoniae.

Animals↗