Investigation of the [−/A]8 and C1236T genetic variations within the human toll-like receptor 3 gene for association with multiple sclerosis

Multiple sclerosis (MS) is a serious cause of neurological disability among young adults. The clinical course remains difficult to predict, and the pathogenesis of the disease is still modestly understood. Autoimmunity is thought to be a key aspect of the disease, with autoreactive T cells thought to mediate central nervous system (CNS) inflammation to some extent. Toll-like receptors are known to mediate cellular recognition of pathogens by way of patterns of molecular presentation. Toll-like receptor 3 is coded by the gene TLR3 and is recognized as an important factor in virus recognition and is known to be involved in the expression of neuroprotective mediators. We set out to investigate two variations within the TLR3 gene, an 8 bp insertion-deletion \[-/A](8) and a single base-pair variation C1236T, in subjects with MS and matched healthy controls to determine whether significant differences exist in these markers in an Australian population. We used capillary gel electrophoresis and TaqMan genotyping assay techniques to resolve genotypes for each marker, respectively. Our work found no significant difference between frequencies for TLR3 \[-/A](8) by genotype (chi(2)=1.03, p=0.60) or allele (chi(2)=1.09, p=0.30). Similarly, we found no evidence for the association of TLR3 C1236T by genotype (chi(2)=0.35, p=0.84) or allele frequency (chi(2)=0.31, p=0.58). This work reveals no evidence to suggest that these markers are associated with MS in the tested population. Although the role of TLR3 and the wider toll-like receptor family remain significant in neurological and CNS inflammatory disorders, our current work does not support a role for the two tested variants in this gene with regard to MS susceptibility.

Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses : implications for laminitis

Equine laminitis, a disease of the lamellar structure of the horse’s hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Tolllike receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-�), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-� and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-�, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.

Toll-like receptor 4 and CD14 polymorphisms in ankylosing spondylitis: Evidence of a weak association in finns

Objective To investigate the association of CD14 and Toll-like receptor (TLR4) with ankylosing spondylitis (AS). Methods A promoter variant in CD14 and 2 coding polymorphisms in TLR4 were investigated in UK and Finnish families with AS and in a UK case-control study. A metaanalysis of published TLR4 and CD14 studies was performed. Results In the Finnish study the CD74-260bp T variant showed an association (p = 0.006), and the common 2-marker TLR4 haplotype showed a weak association (global p = 0.03), with AS. No associations were seen in the UK based studies or in the metaanalyses. Conclusion CD14 and TLR4 showed an association with AS in the Finns only.

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-alpha) secretion by macrophages was essential for robust SHH activation, as TNF-alpha(-/-) macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-alpha or blockade of TNF-alpha receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.

The WNT Signaling Pathway Contributes to Dectin-1-Dependent Inhibition of Toll-Like Receptor-Induced Inflammatory Signature

Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of beta-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-alpha) secretion by macrophages was essential for robust SHH activation, as TNF-alpha(-/-) macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-alpha or blockade of TNF-alpha receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.

Virulent Salmonella enterica infections can be exacerbated by concomitant infection of the host with a live attenuated S. enterica vaccine via Toll-like receptor 4-dependent interleukin-10 production with the involvement of both TRIF and MyD88.

During systemic disease in mice, Salmonella enterica grows intracellularly within discrete foci of infection in the spleen and liver. In concomitant infections, foci containing different S. enterica strains are spatially separated. We have investigated whether functional interactions between bacterial populations within the same host can occur despite the known spatial separation of the foci and independence of growth of salmonellae residing in different foci. In this study we have demonstrated that bacterial numbers of virulent S. enterica serovar Typhimurium C5 strain in mouse tissues can be increased by the presence of the attenuated aroA S. Typhimurium SL3261 vaccine strain in the same tissue. Disease exacerbation does not require simultaneous coinjection of the attenuated bacteria. SL3261 can be administered up to 48 hr after or 24 hr before the administration of C5 and still determine higher tissue numbers of the virulent bacteria. This indicates that intravenous administration of a S. enterica vaccine strain could potentially exacerbate an established infection with wild-type bacteria. These data also suggest that the severity of an infection with a virulent S. enterica strain can be increased by the prior administration of a live attenuated vaccine strain if infection occurs within 48 hr of vaccination. Exacerbation of the growth of C5 requires Toll-like receptor 4-dependent interleukin-10 production with the involvement of both Toll/interleukin-1 receptor-domain-containing adaptor inducing interferon-beta and myeloid differentiation factor 88.

Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella)

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. To investigate grass carp immune system responding to GCRV (grass carp reovirus) infection, the full-length cDNA sequence and genomic organization of grass carp TLR3 (CiTLR3) was identified and characterized. The full-length genome sequence of CiTLR3 is composed of 5668 nucleotides, including five exons and four introns. The full-length of CiTLR3 cDNA is 3681 bp in length and encodes a polypeptide of 904 amino acids with an estimated molecular mass of 102,765 Da and a predicted isoelectric point of 8.35. Analysis of the deduced amino acid sequence indicated that CiTLR3 has four main structural domains, including a signal peptide sequence, 14 LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/interleukin-1 receptor) domain. It is most similar to the crucian carp (Carassius auratus) TLR3 amino acid sequence with an identity of 99%. Quantitative RT-PCR analysis showed that CiTLR3 transcripts were significantly up-regulated starting at day 1 and continued through day 7 following GCRV infection (P < 0.05). These data implied that CiTLR3 is involved in antiviral defense, provide molecular and functional information for grass carp TLR3, and implicate their role in mediating immune protection against grass carp viral diseases. (C) 2009 Elsevier Ltd. All rights reserved.

Toll-like receptor 4 signaling pathway can be triggered by grass carp reovirus and Aeromonas hydrophila infection in rare minnow Gobiocypris rarus

Toll-like receptor 4 (TLR4) is critical for LPS recognition and cellular responses. It also recognizes some viral envelope proteins. Detection mostly results in the inflammation rather than specific antiviral responses. However, it's unclear in fish. In this report, a TLR4 gene (named as GrTLR4b) was cloned and characterized from rare minnow Gobiocypris rarus. The full length of GrTLR4b cDNA consists of 2766 nucleotides and encodes a polypeptide of 818 amino acids with an estimated molecular mass of 94,518 Da and a predicted isoelectric point of 8.41. The predicted amino acid sequence comprises a signal peptide, six leucine-rich repeat (LRR) motifs, one leucine-rich repeat C-terminal (LRRCT) motif, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic region of 167 amino acids containing one Toll - interleukin 1 - receptor (TIR) motif. It's closely similar to the zebrafish (Danio rerio) TLR4b amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed GrTLR4b mRNA was constitutive expression in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus or Aeromonas hydrophila, GrTLR4b expressions were up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). These data implied that TLR4 signaling pathway could be activated by both viral and bacterial infection in rare minnow. (C) 2009 Elsevier Ltd. All rights reserved.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappa B, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappa B activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1). antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. (C) 2007 Elsevier Ltd. All rights reserved.

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated Suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare. (C) 2008 Elsevier B.V. All rights reserved.

Toll-like receptor 3 regulates Mx expression in rare minnow Gobiocypris rarus after viral infection

Toll-like receptor 3 (TLR3) plays a key role in activating immune responses during viral infection. To study the genes involved in the regulatory function of TLR3 in the rare minnow Gobiocypris rarus after viral infection, a full-length cDNA of TLR3 (GrTLR3) with a splice variant (GrTLR3s) was identified by homologous cloning and RACE techniques. The antiviral effector molecule Mx gene was cloned and partially sequenced. The mRNA expression levels of GrTLR3, GrTLR3s, and Mx were studied in different tissues before and after virus infection by real-time quantitative RT-PCR. The transcripts of all three genes in liver were significantly increased following GCRV infection (P<0.05). The mRNA levels in liver were upregulated at 24 h post-injection for GrTLR3 and GrTLR3s, and at 12 h for Mx. The upregulated expression levels were several folds for GrTLR3s, tens of folds for GrTLR3, and hundreds of folds for Mx. By semi-quantitative RT-PCR, GrTLR3 and Mx expressed at all the developmental stages, whereas GrTLR3s could only be detected at later developmental stages. Using RNAi and transgenic techniques, GrTLR3 mediated Mx expression but GrTLR3s did not. The time-dependent upregulation of receptor and effector, and the Mx over-expression dependent on TLR3, indicated that GrTLR3 regulated Mx expression in viral infection through a configuration change in rare minnow, and its splice variant did not contribute to the process.

Investigation of toll-like receptor tolerance in the regulation of inflammation

Inflammation is a complex and highly organised immune response to microbes and tissue injury. Recognition of noxious stimuli by pathogen recognition receptor families including Toll-like receptors results in the expression of hundreds of genes that encode cytokines, chemokines, antimicrobials and regulators of inflammation. Regulation of TLR activation responses is controlled by TLR tolerance which induces a global change in the cellular transcriptional expression profile resulting in gene specific suppression and induction of transcription. In this thesis the plasticity of TLR receptor tolerance is investigated using an in vivo, transcriptomics and functional approach to determine the plasticity of TLR tolerance in the regulation of inflammation. Firstly, using mice deficient in the negative regulator of TLR gene transcription, Bcl-3 (Bcl-3-/-) in a model of intestinal inflammation, we investigated the role of Bcl-3 in the regulation of intestinal inflammatory responses. Our data revealed a novel role for Bcl-3 in the regulation of epithelial cell proliferation and regeneration during intestinal inflammation. Furthermore this data revealed that increased Bcl-3 expression contributes to the development of inflammatory bowel disease (IBD). Secondly, we demonstrate that lipopolysaccharide tolerance is transient and recovery from LPS tolerance results in polarisation of macrophages to a previously un-described hybrid state (RM). In addition, we identified that RM cells have a unique transcriptional profile with suppression and induction of genes specific to this polarisation state. Furthermore, using a functional approach to characterise the outcomes of TLR tolerance plasticity, we demonstrate that cytokine transcription is uncoupled from cytokine secretion in macrophages following recovery from LPS tolerance. Here we demonstrate a novel mechanism of regulation of TLR tolerance through suppression of cytokine secretion in macrophages. We show that TNF-α is alternatively trafficked towards a degradative intracellular compartment. These studies demonstrate that TLR tolerance is a complex immunological response with the plasticity of this state playing an important role in the regulation of inflammation.

An investigation into the role of Bcl-3 in toll-like receptor signalling

Through the recognition of potentially harmful stimuli, Toll-like receptors (TLRs) initiate the innate immune response and induce the expression of hundreds of immune and pro-inflammatory genes. TLRs are critical in mounting a defence against invading pathogens however, strict control of TLR signalling is vital to prevent host damage from excessive or prolonged immune activation. In this thesis the role of the IκB protein Bcl (B-cell lymphoma)-3 in the regulation of TLR signalling is investigated. Bcl3-/- mice and cells are hyper responsive to TLR stimulation and are defective in LPS tolerance. Bcl-3 interacts with and blocks the ubiquitination of homodimers of the NF-κB subunit, p50. Through stabilisation of inhibitory p50 homodimers, Bcl-3 negatively regulates NF-κB dependent inflammatory gene transcription following TLR activation. Firstly, we investigated the nature of the interaction between Bcl-3 and p50 and using peptide array technology. Key amino acids required for the formation of the p50:Bcl-3 immunosuppressor complex were identified. Furthermore, we demonstrate for the first time that interaction between Bcl-3 and p50 is necessary and sufficient for the anti-inflammatory properties of Bcl-3. Using the data generated from peptide array analysis we then generated cell permeable peptides designed to mimic Bcl-3 function and stabilise p50 homodimers. These Bcl-3 derived peptides are potent inhibitors of NF-κB dependent transcription activity in vitro and provide a solid basis for the development of novel gene-specific approaches in the treatment of inflammatory diseases. Secondly, we demonstrate that Bcl-3 mediated regulation of TLR signalling is not limited to NF-κB and identify the MAK3K Tumour Progression Locus (Tpl)-2 as a new binding partner of Bcl-3. Our data establishes role for Bcl-3 as a negative regulator of the MAPK-ERK pathway.