Expression of Toll-like receptor 2 in duodenal biopsies from dogs with inflammatory bowel disease is associated with severity of disease

There is growing evidence that aberrant innate immune responses towards the bacterial flora of the gut play a role in the pathogenesis of canine inflammatory bowel disease (IBD). Toll-like receptors (TLR) play an important role as primary sensors of invading pathogens and have gained significant attention in human IBD as differential expression and polymorphisms of certain TLR have been shown to occur in ulcerative colitis (UC) and Crohn's disease (CD). The aim of the current study was to evaluate the expression of two TLR important for recognition of commensals in the gut. TLR2 and TLR4 mRNA expression in duodenal biopsies from dogs with IBD was measured and correlated with clinical and histological disease severity. Endoscopic duodenal biopsies from 20 clinical cases and 7 healthy control dogs were used to extract mRNA. TLR2 and TLR4 mRNA expression was assessed using quantitative real-time PCR. TLR2 mRNA expression was significantly increased in the IBD dogs compared to controls, whereas TLR4 mRNA expression was similar in IBD and control cases. In addition, TLR2 mRNA expression was mildly correlated with clinical severity of disease, however, there was no correlation between TLR2 expression and histological severity of disease.

Evidence for dissociation of TLR mRNA expression and TLR agonist-mediated functions in bovine macrophages

Toll-like receptors are of key importance in the recognition of and response to infectious agents by cells of the innate immune system. TLR mRNA expression and TLR-mediated functions were determined in bovine macrophages (MPhi) infected with bovine viral diarrhea virus (BVDV) or stimulated with interferon-gamma (IFN-gamma) in order to see whether they are correlated under these conditions. As parameters quantitative real time RT-PCR (QRT-PCR) for TLR2, TLR3 and TLR4, NO and TNF production were measured. Triggering of bovine MPhi with bona fide TLR2 and TLR4 agonists (lipopolysaccharide, lipoteichoic acid, peptidoglycan, lipopetide) led to NO and TNF production but neither TLR3 nor TLR9 agonists (double-stranded RNA, CpG DNA) showed this effect. The mRNA expression of TLR2, TLR3 and TLR4 was neither influenced by MPhi costimulation with IFN-gamma nor by MPhi preinfection with BVDV nor by the ligands themselves. However, NO production induced by TLR2 or TLR4 agonists was strongly modulated either by IFN-gamma costimulation or BVDV preinfection. Thus costimulation of MPhi with IFN-gamma resulted in an increase of both NO synthesis and TNF expression by cells stimulated simultaneously by TLR2 or TLR4 agonists. Preinfection of bovine MPhi by BVDV resulted in upregulation of TLR2- and TLR4-mediated NO synthesis. Collectively, these data show that TLR-mediated functions may be modulated by viral infection or activation via IFN-gamma of MPhi whereas the mRNA concentrations of relevant TLR members were not significantly influenced. Thus, the amount of TLR2, TLR3 and TLR4 mRNA transcripts is stable at least under the conditions tested. More importantly, modulation of TLR-mediated responses was dissociated from mRNA expression of TLR members.

Human monocytoid cells as a model to study Toll-like receptor-mediated activation

THP-1 2A9, a subclone of the monocytoid cell line THP-1 and known to be exquisitely sensitive to LPS, was tested for TNF production following triggering by excess doses of TLR ligands. TLR2, TLR4 and TLR5 agonists, but neither TLR3 nor TLR9 agonists, induced TNF production. When used at lower concentrations, priming by calcitriol strongly influenced the sensitivity of cells to LPS and different TLR2 triggers (lipoteichoic acid (LTA), trispalmitoyl-cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam3Cys) and peptidoglycan (PGN)). Priming by calcitriol failed to modulate TLR2 and TLR4 mRNA and cell surface expression of these receptors. TNF signals elicited by TLR2 agonists were blocked by the TLR-specific antibody 2392. CD14-specific antibodies showed variable effects. CD14-specific antibodies inhibited TNF induction by LTA. High concentrations partially inhibited TNF induction by Pam3Cys. The same antibodies failed to inhibit TNF induction by PGN. Thus, THP-1 2A9 cells respond by TNF production to some, but not all TLR agonists, and the wide variety of putative TLR2 agonists interact to variable degrees also with other cell-surface-expressed binding sites such as CD14. THP-1 2A9 cells might provide a model by which to investigate in more detail the interaction of pathogen-associated molecular patterns and monocytoid cell-surface-expressed pattern recognition receptors.

Adaptations of intestinal macrophages to an antigen-rich environment

Intestinal macrophages, preferentially located in the subepithelial lamina propria, represent in humans the largest pool of tissue macrophages. To comply with their main task, i.e. the efficient removal of microbes and particulate matter that might have gained access to the mucosa from the intestinal lumen while maintaining local tissue homeostasis, several phenotypic and functional adaptations evolved. Most notably, microbe-associated molecular pattern (MAMP) receptors, including the lipopolysaccharide receptors CD14 and TLR4, but also the Fc receptors for IgA and IgG are absent on most intestinal Mø. Here we review recent findings on the phenotypic and functional adaptations of intestinal Mø and their implications for the pathogenesis of inflammatory bowel diseases.

TLR expression on neutrophils at the pulmonary site of infection: TLR1/TLR2-mediated up-regulation of TLR5 expression in cystic fibrosis lung disease

Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam(3)CSK(4). Ab-blocking experiments revealed that the effect of Pam(3)CSK(4) was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

Chemokine receptor 4 (CXCR4) is part of the lipopolysaccharide "sensing apparatus"

Recognition of bacterial lipopolysaccharide (LPS) by the innate immune system involves at least three receptor molecules: CD14, TLR4 and MD-2. Additional receptor components such as heat shock proteins, chemokine receptor 4 (CXCR4), or CD55 have been suggested to be part of this activation cluster; possibly acting as additional LPS transfer molecules. Our group has previously identified CXCR4 as a component of the "LPS-sensing apparatus". In this study we aimed to elucidate the role that CXCR4 plays in innate immune responses to LPS. Here we demonstrate that CXCR4 transfection results in responsiveness to LPS. Fluorescence correlation spectroscopy experiments further showed that LPS directly interacts with CXCR4. Our data suggest that CXCR4 is not only involved in LPS binding but is also responsible for triggering signalling, especially mitogen-activated protein kinases in response to LPS. Finally, co-clustering of CXCR4 with other LPS receptors seems to be crucial for LPS signalling, thus suggesting that CXCR4 is a functional part of the multimeric LPS "sensing apparatus".

Using distinct molecular signatures of human monocytes and dendritic cells to predict adjuvant activity and pyrogenicity of TLR agonists

We present a systematic study that defines molecular profiles of adjuvanticity and pyrogenicity induced by agonists of human Toll-like receptor molecules in vitro. Using P(3)CSK(4), Lipid A and Poly I:C as model adjuvants we show that all three molecules enhance the expansion of IFNgamma(+)/CD4(+) T cells from their naïve precursors following priming with allogeneic DC in vitro. In contrast, co-culture of naive CD4(+) T cells with allogeneic monocytes and TLR2/TLR4 agonists only resulted in enhanced T cell proliferation. Distinct APC molecular signatures in response to each TLR agonist underline the dual effect observed on T cell responses. Using protein and gene expression assays, we show that TNF-alpha and CXCL10 represent DC-restricted molecular signatures of TLR2/TLR4 and TLR3 activation, respectively, in sharp contrast to IL-6 produced by monocytes upon stimulation with P(3)CSK(4) and Lipid A. Furthermore, although all TLR agonists are able to up-regulate proIL-1beta specific gene in both cell types, only monocyte activation with Lipid A results in detectable IL-1beta release. These molecular profiles, provide a simple screen to select new immune enhancers of human Th1 responses suitable for clinical application.

Do common genetic variants in endotoxin signaling pathway contribute to predisposition to alcoholic liver cirrhosis?

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), produced by endotoxin-activated Kupffer cells, play a key role in the pathogenesis of alcoholic liver cirrhosis (ALC). Alleles TNFA -238A, IL1B -31T and variant IL1RN*2 of repeat polymorphism in the gene encoding the IL-1 receptor antagonist increase production of TNF-alpha and IL-1beta, respectively. Alleles CD14 -159T, TLR4 c.896G and TLR4 c.1196T modify activation of Kupffer cells by endotoxin. We confirmed the published associations between these common variants and genetic predisposition to ALC by means of a large case-control association study conducted on two Central European populations. METHODS: The study population comprised a Czech sample of 198 ALC patients and 370 controls (MONICA project), and a German sample of 173 ALC patients and 331 controls (KORA-Augsburg), and 109 heavy drinkers without liver disease. RESULTS: Single locus analysis revealed no significant difference between patients and controls in all tested loci. Diplotype [IL1RN 2/ 2; IL1B -31T+] was associated with increased risk of ALC in the pilot study, but not in the validation samples. CONCLUSIONS: Although cytokine mediated immune reactions play a role in the pathogenesis of ALC, hereditary susceptibility caused by variants in the corresponding genes is low in Central European populations.

Intestinal macrophages: differentiation and involvement in intestinal immunopathologies

Intestinal macrophages, preferentially located in the subepithelial lamina propria, represent the largest pool of tissue macrophages in humans. As an adaptation to the local antigen- and bacteria-rich environment, intestinal macrophages exhibit several distinct phenotypic and functional characteristics. Notably, microbe-associated molecular pattern receptors, including the lipopolysaccharide (LPS) receptors CD14 and TLR4, and also the Fc receptors for IgA and IgG are absent on most intestinal macrophages under homeostatic conditions. Moreover, while macrophages in the intestinal mucosa are refractory to the induction of proinflammatory cytokine secretion, they still display potent phagocytic activity. These adaptations allow intestinal macrophages to comply with their main task, i.e., the efficient removal of microbes while maintaining local tissue homeostasis. In this paper, we review recent findings on the functional differentiation of monocyte subsets into distinct macrophage populations and on the phenotypic and functional adaptations that have evolved in intestinal macrophages in response to their antigen-rich environment. Furthermore, the involvement of intestinal macrophages in the pathogenesis of celiac disease and inflammatory bowel diseases is discussed.

Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps

Neutrophil extracellular traps (NETs) represent extracellular structures able to bind and kill microorganisms. It is believed that they are generated by neutrophils undergoing cell death, allowing these dying or dead cells to kill microbes. We show that, following priming with granulocyte/macrophage colony-stimulating factor (GM-CSF) and subsequent short-term toll-like receptor 4 (TLR4) or complement factor 5a (C5a) receptor stimulation, viable neutrophils are able to generate NETs. Strikingly, NETs formed by living cells contain mitochondrial, but no nuclear, DNA. Pharmacological or genetic approaches to block reactive oxygen species (ROS) production suggested that NET formation is ROS dependent. Moreover, neutrophil populations stimulated with GM-CSF and C5a showed increased survival compared with resting neutrophils, which did not generate NETs. In conclusion, mitochondrial DNA release by neutrophils and NET formation do not require neutrophil death and do also not limit the lifespan of these cells.

Regulation of visfatin by microbial and biomechanical signals in PDL cells

OBJECTIVES This in vitro study was established to examine whether visfatin thought to be a link between periodontitis and obesity is produced by periodontal ligament (PDL) cells and, if so, whether its synthesis is modulated by microbial and/or biomechanical signals. MATERIALS AND METHODS PDL cells seeded on BioFlex® plates were exposed to the oral pathogen Fusobacterium nucleatum ATCC 25586 and/or subjected to biomechanical strain for up to 3 days. Gene expression of visfatin and toll-like receptors (TLR) 2 and 4 was analyzed by RT-PCR, visfatin protein synthesis by ELISA and immunocytochemistry, and NFκB nuclear translocation by immunofluorescence. RESULTS F. nucleatum upregulated the visfatin expression in a dose- and time-dependent fashion. Preincubation with neutralizing antibodies against TLR2 and TLR4 caused a significant inhibition of the F. nucleatum-upregulated visfatin expression at 1 day. F. nucleatum stimulated the NFκB nuclear translocation. Biomechanical loading reduced the stimulatory effects of F. nucleatum on visfatin expression at 1 and 3 days and also abrogated the F. nucleatum-induced NFκB nuclear translocation at 60 min. Biomechanical loading inhibited significantly the expression of TLR2 and TLR4 at 3 days. The regulatory effects of F. nucleatum and/or biomechanical loading on visfatin expression were also observed at protein level. CONCLUSIONS PDL cells produce visfatin, and this production is enhanced by F. nucleatum. Biomechanical loading seems to be protective against the effects of F. nucleatum on visfatin expression. CLINICAL RELEVANCE Visfatin produced by periodontal tissues could play a major role in the pathogenesis of periodontitis and the interactions with obesity and other systemic diseases.

Reconstituted high-density lipoprotein modulates activation of human leukocytes

An anti-inflammatory effect of reconstituted High Density Lipoprotein (rHDL) has been demonstrated in atherosclerosis and in sepsis models. An increase of adhesion molecules as well as tissue factor expression on endothelial cells in response to inflammatory or danger signals are attenuated by the treatment with rHDL. Here we show the inhibitory effect of rHDL on the activation of human leukocytes in a whole blood assay as well as on monocyte-derived human dendritic cells (DC). Multiplex analysis of human whole blood showed that phytohaemagglutinin (PHA)-induced secretion of the cytokines IL-1β, IL-1RA, IL-2R, IL-6, IL-7, IL-12(p40), IL-15 and IFN-α was inhibited. Furthermore, an inhibitory effect on the production of the chemokines CCL-2, CCL-4, CCL-5, CXCL-9 and CXCL-10 was observed. Activation of granulocytes and CD14+ monocytes by PHA is inhibited dose-dependently by rHDL shown as decreased up-regulation of ICAM-1 surface expression. In addition, we found a strong inhibitory effect of rHDL on toll-like receptor 2 (TLR2)- and TLR4-mediated maturation of DC. Treatment of DC with rHDL prevented the up-regulation of cell surface molecules CD80, CD83 and CD86 and it inhibited the TLR-driven activation of inflammatory transcription factor NF-κB. These findings suggest that rHDL prevents activation of crucial cellular players of cellular immunity and could therefore be a useful reagent to impede inflammation as well as the link between innate and adaptive immunity.

EFFICACY AND MECHANISM OF â-DEFENSIN2 FUSED ANTIGEN PROTEIN VACCINES

Vaccines which use the strategy of fusing adjuvant murine â-defensin2 (mBD2) to an antigen in order to elicit stronger anti-antigen immune responses are referred to as murine â-defensin2 (mBD2) vaccines. Previous studies have validated the potential of mBD2 vaccines, thus in this study we focus on increasing vaccine efficacy as well as mechanism elucidation. Initially, we demonstrate superior IFN-ã release levels by antigen specific effector T cells when antigen is crosspresented by dendritic cells (DC) which absorbed mBD2 vaccine (mBD2 fused antigen protein) over antigen alone. We move unto an in vivo model and note significant increases in the expansion of antigen specific class I T cells but not class II T cells when receiving mBD2 vaccine over antigen alone. Further, knowing mBD2’s link with CC chemokine receptor 6 (CCR6) and Toll-like receptor 4 (TLR4) we note that this enhanced class I T cell expansion is CCR6 independent but TLR4 dependent. With anti-tumor responses desired, we demonstrate in tumor protection experiments with mice, compelling tumor protection when combining adoptive T cell therapy and mBD2 vaccine immunization. We further note that mBD2 vaccines are not limited by the antigen and characterize a viable strategy for enhancing tumor antigen immunogenicity.

Hepatic and renal cytochrome p450 gene regulation during citrobacter rodentium infection in wild-type and toll-like receptor 4 mutant mice.

Citrobacter rodentium is the rodent equivalent of human enteropathogenic Escherichia coli infection. This study investigated regulation of hepatic and renal cytochrome P450 (P450) mRNAs, hepatic P450 proteins, cytokines, and acute phase proteins during C. rodentium infection. Female C3H/HeOuJ (HeOu) and C3H/HeJ (HeJ) mice [which lack functional toll-like receptor 4 (TLR4)] were infected with C. rodentium by oral gavage and sacrificed 6 days later. Hepatic CYP4A10 and 4A14 mRNAs were decreased in HeOu mice (<4% of control). CYP3A11, 2C29, 4F14, and 4F15 mRNAs were reduced to 16 to 55% of control levels, whereas CYP2A5, 4F16, and 4F18 mRNAs were induced (180, 190, and 600% of control, respectively). The pattern of P450 regulation in HeJ mice was similar to that in HeOu mice for most P450s, with the exception of the TLR4 dependence of CYP4F15. Hepatic CYP2C, 3A, and 4A proteins in both groups were decreased, whereas CYP2E protein was not. Renal CYP4A10 and 4A14 mRNAs were significantly down-regulated in HeOu mice, whereas other P450s were unaffected. Most renal P450 mRNAs in infected HeJ mice were increased, notably CYP4A10, 4A14, 4F18, 2A5, and 3A13. Hepatic levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNFalpha) mRNAs were significantly increased in infected HeOu mice, whereas only TNFalpha mRNA was significantly increased in HeJ mice. Hepatic alpha1-acid glycoprotein was induced in both groups, whereas alpha-fibrinogen and angiotensinogen were unchanged. These data indicate that hepatic inflammation induced by C. rodentium infection is mainly TLR4-independent and suggest that hepatic P450 down-regulation in this model may be cytokine-mediated.