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PubMed2005

Helicobacter pylori induces interleukin-8 secretion by Toll-like receptor 2- and Toll-like receptor 5-dependent and -independent pathways.

Helicobacter pylori is an important human pathogen that causes gastritis and is strongly associated with gastric ulcers, gastric adenocarcinomas, and mucosa-associated lymphoid tissue lymphomas. In response to H. pylori, interleukin-8 (IL-8) is secreted from host cells to attract components of the innate and adaptive immune systems to the site of infection. Toll-like receptor 2 (TLR2) and TLR5 have been shown to recognize H. pylori and to initiate signaling pathways that result in enhanced activation of NF-kappaB. Here, we evaluated the contribution of mitogen-activated protein kinase signaling pathways to TLR2-dependent and TLR5-dependent secretion of IL-8. Secretion of IL-8 from H. pylori-infected HEK293 cells was augmented by the expression of TLR2 or TLR5. While H. pylori infection resulted in the activation of ERK, JNK, and p38, the enhanced IL-8 secretion from TLR2- and TLR5-expressing cells coincided with increased p38 activation and phosphorylation of the transcription factor ATF2. When p38 activity was inhibited in TLR2- or TLR5-expressing cells, H. pylori-dependent IL-8 secretion returned to the level observed in infected parental HEK293 cells that did not express TLR2 or TLR5; inhibition of p38 had no effect on IL-8 secretion from infected parental HEK cells. In contrast, inhibition of JNK and/or ERK resulted in substantially less IL-8 secretion from infected cells, independent of TLR2 or TLR5 expression. Based on these data, we propose that H. pylori induces IL-8 secretion through a dual mechanism that includes a TLR2/5-independent component involving the activities of JNK and ERK and a TLR2/5-dependent component that requires p38 activity.

Activating Transcription Factor 2↗
PubMed2006

Expression of immune responsive genes in cell lines from two different Anopheline species.

Malaria infection results in increased expression of immune responsive genes, including those encoding antimicrobial peptides such as Gambicin (Gam1) and Cecropin A (Cec1). Understanding how these genes are regulated will provide insights how the mosquito immune system is activated by Plasmodium. We previously have shown that Cec1 was primarily regulated by the Imd-Relish (REL2) pathway in the Anopheles gambiae Sua1B cell line. We show here that expression of Defensin A (Def1) and Gam1 was reduced after RNA interference against components of the Imd-REL2 pathway in An. gambiae cell lines. Interestingly, promoter reporters of these antimicrobial peptides were expressed at very low level in the cell line MSQ43 from Anopheles stephensi. Surprisingly, over-expression of either NF-kappaB transcription factor REL1 or REL2 alone is sufficient to induce the expression of Cec1, Gam1 and Def1. These results suggest that expression of these antimicrobial peptides (AMP) in vivo may be regulated by both the Toll and Imd pathways. We also show here for the first time that Tep4, a gene encoding a thioester containing protein, is regulated by REL2. Taken together, these results suggest that there are significant overlaps of genes regulated by the Toll-Rel1 and Imd-Rel2 pathways. Further, the different expression patterns in two different Anopheline cell lines provide a platform to identify other key positive and negative regulators of the antimicrobial peptide genes.

Animals↗
PubMed2001

Identification of new signaling components in the Drosophila genome sequence.

The availability of the complete sequence of the Drosophila genome and the assignment of putative reading frames, provides an opportunity to search for new members in families of proteins generating signaling cascades. The six major pathways that dictate patterning were examined: receptor tyrosine kinases, transforming growth factor beta (TGF beta), Wnt, Toll, Hedgehog and Notch. Several new components were identified for the first four pathways, including ligands, receptors, cytoplasmic components and transcription factors. Most notable is the identification of a vascular endothelial growth factor (VEGF) receptor tyrosine kinase, two insulin/insulin growth factor I (IGF I) receptors without cytoplasmic protein kinase domains, and a family of proteins similar to Rhomboid (a protein involved in cleavage of TGF alpha-like ligands). A new TGF beta family ligand, two new Wnts and a Frizzled receptor were also identified. Finally, for the Toll pathway, two new potential Spatzle-like ligands and two new receptors were identified.

Animals↗
PubMed2006

Structure-activity relationship studies on the immune stimulatory effects of base-modified CpG toll-like receptor 9 agonists.

Synthetic oligodeoxynucleotides containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are able to stimulate potent immune responses through a signaling pathway involving Toll-like receptor 9 (TLR9). We have investigated the structure-activity relationship (SAR) of base-modified CpG oligonucleotides with TLR9 by measuring TLR9 activation by 20-mer oligonucleotides having just a single human recognition motif (5'-GTCGTT-3') in functional cell-based TLR9 assays. Substitution of guanine by hypoxanthine and 6-thioguanine resulted in activity similar to the unmodified parent molecule, whereas purine, 2-aminopurine, 2,6-diaminopurine, and 8-oxo-7,8-dihydroguanine substitution resulted in approximately 40-60 % reduction in activity, and 7-deazaguanine substitution led to the strongest (80 %) reduction in TLR9 stimulation. Furthermore, none of the investigated modifications at C5 and N4 of cytosine were well tolerated with respect to human TLR9 stimulation. Our results are compatible with a SAR model in which guanine is recognized by the Hoogsteen site, and C5 is most critical for recognition of cytosine. In addition, we found significant species-specific differences between human and murine TLR9 recognition, which demonstrates the importance of choosing appropriate assay systems for SAR studies.

Adjuvants, Immunologic↗
PubMed2003

Toll receptors modulate allergic responses: interaction with dendritic cells, T cells and mast cells.

PURPOSE OF REVIEW: The discovery of Toll-like receptors has generated much interest in understanding the impact of innate immunity on adaptive immune responses, including allergic diseases. RECENT FINDINGS: Recent studies suggest that Toll-like receptor pathways may mediate interactions between dendritic cells, T lymphocytes and mast cells, thus modulating allergic immune responses. Toll-like receptor signaling triggers dendritic cell maturation, which primes naive T lymphocytes towards specific T helper cell types 1 and 2 immune responses. Although a T helper cell type 1/2 balance may be important in modulating allergic responses, T regulatory cells that suppress certain immune responses may be critical in immune regulation. SUMMARY: With the identification of different subsets of dendritic cells and the discovery of Toll-like receptors on T regulatory cells and mast cells, the manipulation of Toll-like receptor signaling may lead to novel therapeutic options in allergic diseases.

Dendritic Cells↗
PubMed2005

A toll-like receptor 2-responsive lipid effector pathway protects mammals against skin infections with gram-positive bacteria.

flake (flk), an N-ethyl-N-nitrosourea-induced recessive germ line mutation of C57BL/6 mice, impairs the clearance of skin infections by Streptococcus pyogenes and Staphylococcus aureus, gram-positive pathogens that elicit innate immune responses by activating Toll-like receptor 2 (TLR2). Positional cloning and sequencing revealed that flk is a novel allele of the stearoyl coenzyme A desaturase 1 gene (Scd1). flake homozygotes show reduced sebum production and are unable to synthesize the monounsaturated fatty acids (MUFA) palmitoleate (C(16:1)) and oleate (C(18:1)), both of which are bactericidal against gram-positive (but not gram-negative) organisms in vitro. However, intradermal MUFA administration to S. aureus-infected mice partially rescues the flake phenotype, which indicates that an additional component of the sebum may be required to improve bacterial clearance. In normal mice, transcription of Scd1-a gene with numerous NF-kappaB elements in its promoter--is strongly and specifically induced by TLR2 signaling. Similarly, the SCD1 gene is induced by TLR2 signaling in a human sebocyte cell line. These observations reveal the existence of a regulated, lipid-based antimicrobial effector pathway in mammals and suggest new approaches to the treatment or prevention of infections with gram-positive bacteria.

Animals↗
PubMed2003

Calprotectin release from human neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide via the CD-14-Toll-like receptor-nuclear factor kappaB pathway.

OBJECTIVES: Calprotectin is a cytosolic protein with antibacterial action in leukocytes and its level increases in some inflammatory diseases, including periodontal diseases, rheumatoid arthritis and ulcerative colitis. Recently, we found that the lipopolysaccharide of Porphyromonas gingivalis (P-LPS) induced calprotectin release from human neutrophils. P-LPS, a major virulence factor of periodontal pathogens, is known to induce the production and release of inflammatory cytokines through CD14, Toll-like receptor (TLR) and nuclear factor kappaB (NF-kappaB). In the present study, we investigated whether calprotectin release by P-LPS is induced via the CD14-TLR-NF-kappaB pathway and the cellular mechanism of calprotectin release in human neutrophils. MATERIAL AND METHODS: Human neutrophils were isolated from the peripheral blood of healthy donors and pre-incubated in medium containing antibodies against CD14, TLR2 and TLR4, or several inhibitors of NF-kappaB, microtubules and microfilaments, and then incubated with P-LPS. The calprotectin amount in the culture medium was determined using ELISA, and the nuclear extracts from cells were used for the examination of NF-kappaB binding activity using electrophoretic mobility shift assays. RESULTS: P-LPS increased calprotectin release from neutrophils and its induction was inhibited by anti-CD14 and anti-TLR2 antibodies, but not by two anti-TLR4 antibodies. NF-kappaB inhibitors suppressed P-LPS-induced NF-kappaB binding activity and calprotectin release. The inhibitors of microtubule and microfilament polymerization significantly decreased P-LPS-induced calprotectin release. CONCLUSION: These results suggest that calprotectin release is induced by P-LPS via the CD14-TLR2-NF-kappaB signal pathway in human neutrophils and may be dependent on microtubule and microfilament systems.

Actin Cytoskeleton↗
PubMed2004

Herpes simplex virus type-1 induces IFN-alpha production via Toll-like receptor 9-dependent and -independent pathways.

Type I IFN production in response to the DNA virus herpes simplex virus type-1 (HSV-1) is essential in controlling viral replication. We investigated whether plasmacytoid dendritic cells (pDC) were the major tissue source of IFN-alpha, and whether the production of IFN-alpha in response to HSV-1 depended on Toll-like receptor 9 (TLR9). Total spleen cells or bone marrow (BM) cells, or fractions thereof, including highly purified pDC, from WT, TLR9, and MyD88 knockout mice were stimulated with known ligands for TLR9 or active HSV-1. pDC freshly isolated from both spleen and BM were the major source of IFN-alpha in response to oligodeoxynucleotides containing CpG motifs, but in response to HSV-1 the majority of IFN-alpha was produced by other cell types. Moreover, IFN-alpha production by non-pDC was independent of TLR9. The tissue source determined whether pDC responded to HSV-1 in a strictly TLR9-dependent fashion. Freshly isolated BM pDC or pDC derived from culture of BM precursors with FMS-like tyrosine kinase-3 ligand, produced IFN-alpha in the absence of functional TLR9, whereas spleen pDC did not. Heat treatment of HSV-1 abolished maturation and IFN-alpha production from all TLR9-deficient DC but not WT DC. Thus pDC and non-pDC produce IFN-alpha in response to HSV-1 via both TLR9-independent and -dependent pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The toll-like receptor-nuclear factor kappaB pathway in rheumatoid arthritis.

The study of the role cytokines play in the pathogenesis of rheumatoid arthritis (RA) has provided a whole new range of targets for drug development. Many of them (e.g. TNF, IL-1, IL-6, IL-15 and IL-18) are already being targeted in the clinic with success using neutralizing monoclonal antibodies or soluble cytokine receptors. Targeting TNF, in particular, has shown great efficacy in controlling both the inflammation and structural damage of the joints, setting a new gold standard for the treatment of RA. However, what triggers the production of inflammatory cytokines such as TNF in RA remains to be determined. In this article, we review evidence suggesting that the transcription factor Nuclear Factor kappaB (NF-kappaB) is essential for the expression of both inflammatory cytokines and tissue destructive enzymes in RA. Also, we discuss whether Toll-like receptors (TLRs), major receptors involved in pathogen recognition and potent activators of the NF-kappaB pathway, are involved in triggering the inflammatory and joint destructive process in RA and whether they constitute sensible targets for monoclonal antibodies/soluble receptors and small molecule inhibitors. We conclude that although the TLR- NF-kappaB pathway offers ample opportunities for therapeutic intervention, future drugs to be approved will need to match or exceed the efficacy and safety of anti-TNF agents, with safety the most difficult aspect to predict.

Animals↗
PubMed2006

Induction of dendritic cell maturation by pertussis toxin and its B subunit differentially initiate Toll-like receptor 4-dependent signal transduction pathways.

OBJECTIVE: Pertussis toxin (PT) has the capacity to activate dendritic cells (DCs) for the augmentation of cell-mediated immune responses. To investigate the mechanism(s) by which PT activates DCs, we investigated the effects of PT and its B-oligomer (PTB) on the maturation of human and mouse DCs and determined whether PT could act as a pathogen-associated molecular pattern to activate one of the Toll-like receptors (TLRs). METHODS: The effects of PT and PTB on the maturation of human and mouse DCs were analyzed in terms of surface marker expression, cytokine production, antigen-presenting capacity, and intracellular signaling. The participation of TLR4 in PT-induced signaling was determined by comparing the effect of PT on DCs derived from TLR4-deficient and wild-type mice, as well as by measuring PT-induced NF-kappaB activation in HEK293 cells transiently transfected to express various TLRs. RESULTS: Although both promoted phenotypic and functional maturation DCs, however, unlike PT that induced DC production of interleukin (IL)-6, tumor necrosis factor-alpha, IL-12, and interferon-inducible protein, PTB was capable of stimulating the production of interferon-inducible protein. Bone marrow-derived DCs from C3H/HeJ mice with defective TLR-4 alleles were unresponsive to PT and PTB, whereas DCs from C3H/HeN mice responded. In addition, PT induced NF-kappaB activation and IL-8 production in HEK293 cells transfected with a combination of TLR4 and MD2 but not in nontransfected or TLR2-transfected HEK293 cells. Comparison of the patterns of cytokine induction and intracellular signaling events in DCs treated by PT and PTB revealed that although PT, like lipopolysaccharide, triggered both MyD88-dependent and -independent pathways, PTB preferentially triggered MyD88-independent pathways. Interestingly, mouse splenocyte proliferation in response to PT and PTB was only partially dependent on TLR4. CONCLUSION: The data identify PT as another pathogen-associated molecular pattern that induces DC maturation in a TLR4-dependent manner. Unlike PT, which triggers both MyD88-dependent and -independent pathways, PTB only triggers the MyD88-independent pathway in DCs.

Animals↗
PubMed2005

Differential induction of the toll-like receptor 4-MyD88-dependent and -independent signaling pathways by endotoxins.

The biological response to endotoxin mediated through the Toll-like receptor 4 (TLR4)-MD-2 receptor complex is directly related to lipid A structure or configuration. Endotoxin structure may also influence activation of the MyD88-dependent and -independent signaling pathways of TLR4. To address this possibility, human macrophage-like cell lines (THP-1, U937, and MM6) or murine macrophage RAW 264.7 cells were stimulated with picomolar concentrations of highly purified endotoxins. Harvested supernatants from previously stimulated cells were also used to stimulate RAW 264.7 or 23ScCr (TLR4-deficient) macrophages (i.e., indirect induction). Neisseria meningitidis lipooligosaccharide (LOS) was a potent direct inducer of the MyD88-dependent pathway molecules tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 3alpha (MIP-3alpha), and the MyD88-independent molecules beta interferon (IFN-beta), nitric oxide, and IFN-gamma-inducible protein 10 (IP-10). Escherichia coli 55:B5 and Vibrio cholerae lipopolysaccharides (LPSs) at the same pmole/ml lipid A concentrations induced comparable levels of TNF-alpha, IL-1beta, and MIP-3alpha, but significantly less IFN-beta, nitric oxide, and IP-10. In contrast, LPS from Salmonella enterica serovars Minnesota and Typhimurium induced amounts of IFN-beta, nitric oxide, and IP-10 similar to meningococcal LOS but much less TNF-alpha and MIP-3alpha in time course and dose-response experiments. No MyD88-dependent or -independent response to endotoxin was seen in TLR4-deficient cell lines (C3H/HeJ and 23ScCr) and response was restored in TLR4-MD-2-transfected human embryonic kidney 293 cells. Blocking the MyD88-dependent pathway by DNMyD88 resulted in significant reduction of TNF-alpha release but did not influence nitric oxide release. IFN-beta polyclonal antibody and IFN-alpha/beta receptor 1 antibody significantly reduced nitric oxide release. N. meningitidis endotoxin was a potent agonist of both the MyD88-dependent and -independent signaling pathways of the TLR4 receptor complex of human macrophages. E. coli 55:B5 and Vibrio cholerae LPS, at the same picomolar lipid A concentrations, selectively induced the MyD88-dependent pathway, while Salmonella LPS activated the MyD88-independent pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2007

Innate immune response to adenoviral vectors is mediated by both Toll-like receptor-dependent and -independent pathways.

Recombinant adenoviral vectors have been widely used for gene therapy applications and as vaccine vehicles for treating infectious diseases such as human immunodeficiency virus disease. The innate immune response to adenoviruses represents the most significant hurdle in clinical application of adenoviral vectors for gene therapy, but it is an attractive feature for vaccine development. How adenovirus activates innate immunity remains largely unknown. Here we showed that adenovirus elicited innate immune response through the induction of high levels of type I interferons (IFNs) by both plasmacytoid dendritic cells (pDCs) and non-pDCs such as conventional DCs and macrophages. The innate immune recognition of adenovirus by pDCs was mediated by Toll-like receptor 9 (TLR9) and was dependent on MyD88, whereas that by non-pDCs was TLR independent through cytosolic sensing of adenoviral DNA. Furthermore, type I IFNs were pivotal in innate and adaptive immune responses to adenovirus in vivo, and type I IFN blockade diminished immune responses, resulting in more stable transgene expression and reduction of inflammation. These findings indicate that adenovirus activates innate immunity by its DNA through TLR-dependent and -independent pathways in a cell type-specific fashion, and they highlight a critical role for type I IFNs in innate and adaptive immune responses to adenoviral vectors. Our results that suggest strategies to interfere with type I IFN pathway may improve the outcome of adenovirus-mediated gene therapy, whereas approaches to activate the type I IFN pathway may enhance vaccine potency.

Adenoviridae↗
PubMed2002

Sequential activation of signaling pathways during innate immune responses in Drosophila.

Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. We found that these transcriptional patterns can be dissected into distinct groups. We demonstrate that, in addition to signaling through the Toll and Imd pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, our results may imply a close link between the control of tissue repair and antimicrobial processes.

Animals↗
PubMed2003

Toll-like receptor-mediated activation of mast cells: implications for allergic disease?

Toll-like receptors have a critical role in innate immunity and host defence. However their role in allergic disease has not been studied in great detail. The presence of these receptors on mast cells opens up new possibilities concerning the role of Toll-like receptors in the pathogenesis of asthma and atopic dermatitis. The current review examines the biology of Toll-like receptors expressed on mast cells. In particular, mast cell expression of Toll-like receptors and the diverse responses observed following Toll-like receptor-mediated activation are considered. Several pathogens such as Staphylococcus aureus and respiratory syncytial virus are known to contribute to the development or maintenance of allergic disease and also express potent activators of the Toll-like receptor pathways. The importance of such interactions and the full role of pathogens in chronic allergic disease remain to be elucidated. The unusual ability of Toll-like receptor 2 activators to selectively induce leukotriene production by mast cells opens up new possibilities concerning mechanisms of disease exacerbation during infection.

Animals↗
PubMed1997

Antimicrobial peptide defense in Drosophila.

Drosophila responds to a septic injury by the rapid synthesis of antimicrobial peptides. These molecules are predominantly produced by the fat body, a functional equivalent of mammalian liver, and are secreted into the hemolymph where their concentrations can reach up to 100 microM. Six distinct antibacterial peptides (plus isoforms) and one antifungal peptide have been characterized in Drosophila and their genes cloned. The induction of the gene encoding the antifungal peptide relies on the spätzle/Toll/cactus gene cassette, which is involved in the control of dorsoventral patterning in the embryo, and shows interesting structural and functional similarities with cytokine-induced activation of NF-kappa B in mammalian cells. An additional pathway, dependent on the as yet unidentified imd (for immune-deficiency) gene, is required for the full induction of the antibacterial peptide genes. Mutants deficient for the Toll and imd pathways exhibit a severely reduced survival to fungal and bacterial infections, respectively. Recent data on the molecular mechanisms underlying recognition of non-self are also discussed in this review.

Amino Acid Sequence↗
PubMed2002

Caspase-9/-3 activation and apoptosis are induced in mouse macrophages upon ingestion and digestion of Escherichia coli bacteria.

A number of highly virulent, intracellular bacteria are known to induce cell death by apoptosis in infected host cells. In this work we demonstrate that phagocytosis of bacteria from the Escherichia coli laboratory strain K12 DH5alpha is a potent cell death stimulus for mouse macrophages. RAW264.7 mouse macrophages took up bacteria and digested them within 2-4 h as investigated with green fluorescent protein-expressing bacteria. No evidence of apoptosis was seen at 8 h postexposure, but at 24 h approximately 70% of macrophages displayed an apoptotic phenotype by a series of parameters. Apoptosis was blocked by inhibition of caspases or by forced expression of the apoptosis-inhibiting protein Bcl-2. Processing of caspase-3 and caspase-9 but not caspase-8 was seen suggesting that the mitochondrial branch of the apoptotic pathway was activated. Active effector caspases could be detected in two different assays. Because the adapter molecule myeloid differentiation factor 88 (MyD88) has been implicated in apoptosis, involvement of the Toll-like receptor pathway was investigated. In RAW264.7 cells, heat-treated bacteria were taken up poorly and failed to induce significant apoptosis. However, cell activation was almost identical between live and heat-inactivated bacteria as measured by extracellular signal-regulated kinase activation, generation of free radicals, and TNF secretion. Furthermore, primary bone marrow-derived macrophages from wild-type as well as from MyD88-deficient mice underwent apoptosis upon phagocytosis of bacteria. These results show that uptake and digestion of bacteria leads to MyD88-independent apoptosis in mouse macrophages. This form of cell death might have implications for the generation of the immune response.

Animals↗
PubMed2005

Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells.

The objective of the present study was to characterize the innate immune responses induced by in vitro stimulation of bovine primary mammary epithelial cells (bMEC) using gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression of a panel of 22 cytokines, chemokines, beta-defensins and components of the Toll-Like Receptor signaling pathway. Stimulation of bMEC with LPS for 24h elicited a marked increase in mRNA expression for IL-1beta, IL-8, TNFalpha, CXCL6 and beta-defensin while members of the Toll-Like Receptor pathway, although present, were largely unaffected. Surprisingly, stimulation of these cells with LTA for 24 h did not significantly alter the expression of these genes. A time course of the expression of IL-1beta, IL-8, TNFalpha, CXCL6 and beta-defensin was subsequently performed. The mRNA levels of all genes increased rapidly after stimulation for 2-4 h with both LPS and LTA but only the former treatment resulted in sustained responses. In contrast, the increased gene expression for LTA stimulated cells returned to resting levels after 8-16 h with the exception of beta-defensin, which remained up-regulated. The limited and unsustained cytokine response to LTA may explain why mastitis caused by gram-positive bacteria has greater potential for chronic intra-mammary infection than gram-negative infection. It was concluded that bovine mammary epithelial cells have a strong but differential capacity to mount innate immune responses to bacterial cell wall components.

Animals↗