Bacterial Pattern Recognition Receptors Research Articles

A bacterial immunity protein directly senses two disparate phage proteins.

Eukaryotic innate immune systems use pattern recognition receptors to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators, known as bacteriophages1-6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have been found to sense only a single trigger during infection, suggesting a one-to-one relationship between bacterial pattern recognition receptors and their ligands7-11. Here we demonstrate that the antiphage defence protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the notable versatility of an immune sensory domain, which may be a common property of antiphage defence systems that enables them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbour both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phages can fully evade CapRelSJ46 defence only when both triggers are mutated. Our work shows how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow the detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defence systems and complex coevolutionary relationships between them and their phage-encoded triggers.

A30 MICROBIOTA AND NOD2 REGULATE SMALL INTESTINAL RESTITUTION THROUGH FETAL-LIKE INTESTINAL STEM CELLS

Abstract Background Inflammatory bowel disease is characterized by chronic inflammation of the gastrointestinal tract, resulting in recurrent injury to the intestinal epithelium. Restitution of the small intestinal epithelium is a coordinated response that involves the dedifferentiation of epithelial cell lineages, proliferation of Lgr5+ intestinal stem cells, and fetal-like stem cell reversion. While gut microbiota are critical mediators of intestinal inflammation, their impact on epithelial restitution remains unclear. Aims We aim to identify the how microbes regulate small intestinal epithelial restitution following damage. Our hypothesis is that gut microbiota accelerate restitution through pattern recognition receptor-driven signals following fetal-like stem cell reversion. Methods Irradiation (IR, 12Gγ) was used to induce a synchronized small intestinal epithelial restitution response in mice. Intestinal restitution kinetics were assessed transcriptionally (scRNA-Seq, qPCR) and histologically. Small intestinal organoids were used to assess epithelial restitution kinetics in vitro. Results ScRNA-Seq of small intestinal epithelial cells following IR from germ-free mice (GF), and specific pathogen-free mice (SPF) mice revealed that microbiota induced greater expression of fetal-like stem cell reversion markers, Ly6a and Clu, and greater expression of the proliferation marker, Pcna. These results were supported histologically as irradiated SPF mice observed an increase in fetal-like stem cells marked by Ly6a and Clu and an increase BrdU+ proliferating cells. ScRNA-seq and in situ hybridization highlighted that fetal-like intestinal stem cells upregulate expression of Nod2, a bacterial pattern recognition receptor. Using intestinal organoids to assess the function of Nod2, we observed that muramyl dipeptide driven Nod2-signlaing potentiates an interferon gene signature following IFNγ and TNFα co-stimulation. Further supporting the role for Nod2 in intestinal restitution, intestinal epithelium specific Nod2KO mice decreased BrdU+ proliferating cells post-IR compared to littermate controls. Conclusions Microbiota promote small intestinal restitution following IR through Nod2-signaling in fetal-like intestinal stem cells. Funding Agencies CIHR

Soluble LPS receptor CD14 is increased in saliva of patients with geographic tongue

Objectives The soluble bacterial pattern recognition receptor, sCD14 augments inflammatory responses in oral cavity. The aim of the study was to investigate whether patients with geographic tongue (GT) with and without fissured tongue (FT) have impaired inflammatory regulation, manifesting as increased levels of sCD14 in the saliva. Material and Methods An enzyme-linked immunosorbent assay was used to measure the amount of sCD14 in whole and parotid saliva of patients diagnosed with GT (GT whole, n = 21; GT parotid, n = 23) and control subjects (GT whole, n = 25; GT parotid, n = 18). The levels of sCD14 were also evaluated according to our previous clinical assessment of GT based on the number of lesions detected on the tongue, as ‘mild’ (a single lesion), ‘moderate’ (2–5 lesions), or ‘severe’ (≥6 lesions). Diagnosis of FT was established when multiple grooves or fissures were observed on the dorsal and lateral surfaces of the tongue. Results GT patients had significantly higher sCD14 levels in whole (p<.05) and parotid saliva (p<.001), compared with controls. GT patients with FT had significantly increased sCD14 levels only in parotid saliva. A gradual increase in sCD14 levels in parotid and unstimulated saliva was seen in GT patients with multiple tongue lesions compared with single lesions. Conclusions GT patients had increased sCD14 in both parotid and unstimulated saliva. sCD14 seems to increase local inflammatory responses, which suggests its involvement in the pathophysiology of GT.

Effect of human breast milk on innate immune response: Up-regulation of bacterial pattern recognition receptors and innate cytokines in THP-1 monocytic cells

Human breast milk (HBM) contains many bioactive components that protect infants from various microorganisms. Pattern recognition receptors on phagocytic cells recognize microbial pathogens and promote the innate immune system. This study aimed to evaluate the effect of HBM on the expression of pattern recognition receptors and innate cytokines in the monocytic cell line THP-1 and the phagocytic activity of RAW264.7 macrophages. Expression levels of specific mRNAs in THP-1 cells were quantitated using reverse transcription-polymerase chain reaction. Phagocytic activity was measured by fluorescence microscopy to detect the uptake of fluorescent dye-labeled carboxylate-modified polystyrene latex beads in RAW264.7 macrophages. HBM stimulated the phagocytic activity of RAW264.7 macrophages. HBM increased mRNA expression of pattern recognition receptors, including the cluster of differentiation 14 and toll-like receptor 2 and 4, and various innate cytokines, including tumor necrosis factor α, interleukin-1β, C-X-C motif chemokine 8, and C-C motif chemokine ligand 2, in THP-1 monocytic cells. Furthermore, milk oligosaccharides in HBM, such as lacto- N-fucopentaose I, enhanced the expression of pattern recognition receptors and various innate cytokines. HBM is able to modulate the innate immune response by upregulating the expression of pattern recognition receptors and various innate cytokines in monocytes/macrophages.

An immune-responsive PGRP-S1 regulates the expression of antibacterial peptide genes in diamondback moth, Plutella xylostella (L.)

Peptidoglycan recognition proteins (PGRPs) are family of pattern recognition receptors (PRRs) and triggers the innate immune system (IIS) against the microbial infection. Although PGRPs have been intensively studied in model insects, they remain uncharacterized in most of the non-model insects. Here, we cloned and characterized a full-length cDNA of PGRP, from P. xylostella (PxPGRP-S1), which encodes a protein of 239 amino acids with PGRP domain, Ami2 domain and transmembrane region. The phylogenetic analysis revealed that the PxPGRP-S1 was closely related to the unigene of Plutella xylostella. Quantitative real-time PCR and immunohistochemistry revealed that PxPGRP-S1 is mainly expressed in the fat body of the healthy larva. The expression of PxPGRP-S1 was significantly upregulated in the midgut at 24 h postinfection by Bacillus thuringiensis. Silencing of the PxPGRP-S1 expression by RNAi, significantly decrease the expression of the antimicrobial peptides (AMPs) in the 4th instar larvae of P. xylostella. Similarly injection of an anti-PxPGRP-S1 serum caused the low expression of the AMPs in P. xylostella. Additionally, PxPGRP-S1 depleted P. xylostella by oral administration of bacterial expressed dsRNA decreased the resistance against B. thuringiensis challenge, leads to high mortality. Together, our result indicates that PxPGRP-S1, served as a bacterial pattern recognition receptor (PRR) and triggers the expression of AMPs in P. xylostella.

Comparative transcriptomics between species attributes reactogenicity pathways induced by the capsular group B meningococcal vaccine, 4CMenB, to the membrane-bound endotoxin of its outer membrane vesicle component

The capsular group B meningococcal (MenB) four component vaccine (4CMenB) has been licensed for the prevention of invasive disease caused by MenB. The vaccine causes fever in infants, particularly when given in combination (concomitant) with other routinely-administered vaccines (routine), such as the standard diphtheria, tetanus, pertussis (DTP)-containing vaccine. To assess the suitability of a mouse immunisation model to study this phenomenon, we monitored temperature in mice after a second dose of routine vaccines, with or without 4CMenB, and compared the results with those in humans. Using this mouse model, we explored the reactogenicity of 4CMenB components by measuring changes in temperature, cytokines, and gene expression induced by 4CMenB, one of its components, wild-type or attenuated endotoxin outer membrane vesicles (OMVs), or lipopolysaccharide (LPS). A significant rise (p < 0.01) in temperature was observed in mice immunised with 4CMenB, wild-type OMVs, and LPS. RNA-sequencing of mouse whole blood revealed a gene signature shared by the 4CMenB, OMV, and LPS groups consisting of bacterial pattern recognition receptors and neutrophil activation marker genes. Sequencing of neutrophils isolated after concomitant 4CMenB identified cells expressing the OMV-associated genes Plek and Lcp1. Immunisation with 4CMenB or OMVs led to increased IL-6 in serum and significant upregulation (p < 0.0001) of prostaglandin-synthesising enzymes on brain tissue. These data demonstrate the suitability of a mouse model for assessing vaccine reactogenicity and strongly indicate that the fever following vaccination with 4CMenB in human infants is induced by endotoxin contained in the OMV component of the vaccine.

Aberrant Expression of Bacterial Pattern Recognition Receptor NOD2 of Basophils and Microbicidal Peptides in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease, associated with basophil infiltration into skin lesions and Staphylococcus aureus (S. aureus)-induced inflammation. Pattern recognition receptors (PRRs), including microbicidal peptide human neutrophil α-defensins (HNP) and dermcidin, can exert immunomodulating activity in innate immunity and skin inflammation. We investigated the plasma concentration of HNP and dermcidin, the expression of bacterial toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors of basophils and plasma concentration and ex vivo induction of AD-related inflammatory cytokines and chemokines using ELISA and flow cytometry, in AD patients and control subjects. Plasma concentrations of HNP, dermcidin and AD-related Th2 chemokines CCL17, CCL22 and CCL27 were significantly elevated in AD patients compared with controls (all p < 0.05). Plasma concentrations of CCL27 and CCL22 were found to correlate positively with SCORing atopic dermatitis (SCORAD), objective SCORAD, % area affected, lichenification and disease intensity, and CCL27 also correlated positively with pruritus in AD patients (all p < 0.05). Protein expressions of NOD2 but not TLR2 of basophils were significantly down-regulated in AD patients compared with controls (p = 0.001). Correspondingly, there were lower ex vivo % inductions of allergic inflammatory tumor necrosis factor-α, IL-6 and CXCL8 from peripheral blood mononuclear cells upon NOD2 ligand S. aureus derived muramyl dipeptide stimulation in AD patients comparing with controls. The aberrant activation of bacterial PRRs of basophils and anti-bacterial innate immune response should be related with the allergic inflammation of AD.

Yeast-containing feed additive alters gene expression profiles associated with innate immunity in whole blood of a rodent model.

Feeding a yeast-containing additive (YCA; OmniGen-AF) improves immune responses in ruminant livestock and reduces subsequent production losses. The objective was to identify molecular pathways by which dietary YCA may modify immune responses using a rodent model. Thirty-seven healthy, unchallenged CD rats received a diet containing 0 (control; n = 5, only 28 d), 0.5% (n = 15) or 1% (n = 17) YCA for 7 (n = 4/group), 14 (n = 3 or 4/group), 21 (n = 3 or 4/group) or 28 (n = 5/group) d. At the end of the feeding periods, whole blood was collected and the isolated RNA was analyzed for the expression of 84 genes involved in innate and cell-mediated adaptive immune responses. Three bacterial pattern recognition receptors TLR1 (0.5%: + 2.01; 1%: + 2.38), TLR6 (0.5%: + 2.11; 1%: + 2.34) and NOD2 (0.5%: + 2.32; 1%: + 2.23), two APC surface receptors CD1D1 (0.5%: + 1.75; 1%: + 2.33) and CD80 (0.5%: +2.45; 1%: +3.00), and the cell signaling molecule MAPK8 (0.5%: +1.87; 1%: +2.35) were significantly up-regulated by YCA at both inclusion rates. In conclusion, feeding YCA may potentially increase recognition and responses to bacterial pathogens and T-cell activation and differentiation and thereby maintain health and prevent production losses.

Analysis of nucleotide-binding oligomerization domain proteins in a murine model of pneumococcal meningitis.

BackgroundThe innate immune system recognizes pathogens via its pattern recognition receptors. The objective of this study was to investigate the role of the nucleotide-binding oligomerization domain (NOD) proteins, a family of the novel bacterial pattern recognition receptors, in host responses to the gram-positive bacteria Streptococcus pneumoniae.MethodsSprague–Dawley rats were infected via intracisternal injections of viable S. pneumoniae, and rats in the control group were injected with sterile saline. After infection, real-time PCR was performed to determine the presence of mRNAs encoding NOD1 and NOD2. Quantitative analyses of the NOD1, NOD2 and NF-kB proteins were also performed western blotting following challenge infections with viable S. pneumoniae. The TNF-α and IL-6 levels in brain homogenates were evaluated using enzyme-linked immunosorbent assays (ELISAs).ResultsThe results revealed up-regulations of the mRNA and protein levels of NOD2 within the CNS of rats with S. pneumoniae meningitis. Moreover, the activation of NF-κB in the brain tissues following infection with live S. pneumoniae was also significantly increased, which indicates that NOD2 mediated NF-κB activation in experimental pneumococcal meningitis. Similarly, TNF-α and IL-6 levels were increased in the brain following in vivo S. pneumoniae administration.ConclusionsThese results suggest that NOD2 is involved in the host response to the gram-positive bacteria S. pneumoniae in the CNS and that NOD2 might play an important role in the initiation and/or progression of CNS inflammation associated with pneumococcal meningitis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0648-3) contains supplementary material, which is available to authorized users.

Structure-Function Analysis of Grass Clip Serine Protease Involved in Drosophila Toll Pathway Activation

Grass is a clip domain serine protease (SP) involved in a proteolytic cascade triggering the Toll pathway activation of Drosophila during an immune response. Epistasic studies position it downstream of the apical protease ModSP and upstream of the terminal protease Spaetzle-processing enzyme. Here, we report the crystal structure of Grass zymogen. We found that Grass displays a rather deep active site cleft comparable with that of proteases of coagulation and complement cascades. A key distinctive feature is the presence of an additional loop (75-loop) in the proximity of the activation site localized on a protruding loop. All biochemical attempts to hydrolyze the activation site of Grass failed, strongly suggesting restricted access to this region. The 75-loop is thus proposed to constitute an original mechanism to prevent spontaneous activation. A comparison of Grass with clip serine proteases of known function involved in analogous proteolytic cascades allowed us to define two groups, according to the presence of the 75-loop and the conformation of the clip domain. One group (devoid of the 75-loop) contains penultimate proteases whereas the other contains terminal proteases. Using this classification, Grass appears to be a terminal protease. This result is evaluated according to the genetic data documenting Grass function.

Constitutive Expression of a Bacterial Pattern Recognition Receptor, CD14, in Human Salivary Glands and Secretion as a Soluble Form in Saliva

Constitutive Expression of a Bacterial Pattern Recognition Receptor, CD14, in Human Salivary Glands and Secretion as a Soluble Form in Saliva

S07 IL23R, ATG16L1, IRGM, and OCTNs mRNA expression in inflamed and non-inflamed mucosa of IBD patients

In both main forms of Inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD), intestinal commensal microbiota are thought to trigger the disease in genetically susceptible individuals. Approximately one third of patients with CD have a mutation in the NOD2 gene that encodes an intracellular bacterial pattern-recognition receptor located within Paneth-cells. The maturation of Paneth cells is regulated via the canonical Wnt cascade. The Nkd1 (naked cuticle homologue 1), gene, a Wnt antagonist, is located near NOD2 at 16q12, and it has been found weakly associated with CD in WTCCC dataset (rs8047222 P<1x10-5). Aims: To investigate rs4785433, rs730055, and rs8047222 SNPs of NKD1 and to search for likely correlation with clinical phenotypes in both adult and pediatric onset IBD patients. Methods: 1185 CD patients (670 male) with a mean age at diagnosis of 27 yrs (range 1-80) (334 < 19 yrs at diagnosis), 1385 UC patients (803 male) with a mean age at diagnosis of 31 yrs (range 1-83) (246 < 19 yrs at diagnosis), and 645 healthy controls (HC), were genotyped for NKD1 gene by using the TaqMan allelic discriminationmethod. Genotype frequencies were comparedwith respect to age at diagnosis, disease localization and behaviour (Montreal classification), need of resective surgery, response to medical therapy (steroids, immunosuppressive drugs), and NOD2 status. Results: A significant allele and genotype association of the rs8047222 with UC (P=0.01, OR=0.83, CI=0.73-0.96; P=0.02, OR=0.79; CI=0.65-0.96) was shown, especially in adult-onset cohort (P<0.002). After stratifying the cohort on the basis of NOD2 genotypes (NOD2-=no major variants; NOD2+=at last one variant), a significant genotype association in NOD2patients (P=0.004; OR= 0.74; CI=0.61-0.91) was also demonstrated. No significant difference for either allele and genotype frequencies with rs8047222 was found for CD compared with controls, however, a significant genotype association in NOD2patients (P=0.03, OR=0.77, CI=0.62-0.97) was found. At genotype-phenotype evaluation, a reduced freq uency of distal (E1) vs extensive (E3) colitis in UC (47% vs 78%; P=0.006; OR=0.25, CI=0.09-0.70), and colon (56%) vs ileum localization in CD(65%)(P=0.02, OR=0.69, CI=0.49-0.95) was demonstrated. No significant difference for either allele and genotype frequencies for rs4785433, rs730055 SNPs for IBD was found. Conclusions: The rs8047222 SNP on NKD1 gene has a significant protective role in both UC and CD, possibly influenced by NOD2 status, with interaction with disease localization. Nkd1 might be responsible for the residual linkage at 16q12 locus in NOD2-negative CD patients.

OC.01.3 ROLE OF NAKED CUTICLE HOMOLOG 1 GENE ON CHR 16Q12 IN INFLAMMATORY BOWEL DISEASE

In both main forms of Inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD), intestinal commensal microbiota are thought to trigger the disease in genetically susceptible individuals. Approximately one third of patients with CD have a mutation in the NOD2 gene that encodes an intracellular bacterial pattern-recognition receptor located within Paneth-cells. The maturation of Paneth cells is regulated via the canonical Wnt cascade. The Nkd1 (naked cuticle homologue 1), gene, a Wnt antagonist, is located near NOD2 at 16q12, and it has been found weakly associated with CD in WTCCC dataset (rs8047222 P<1x10-5). Aims: To investigate rs4785433, rs730055, and rs8047222 SNPs of NKD1 and to search for likely correlation with clinical phenotypes in both adult and pediatric onset IBD patients. Methods: 1185 CD patients (670 male) with a mean age at diagnosis of 27 yrs (range 1-80) (334 < 19 yrs at diagnosis), 1385 UC patients (803 male) with a mean age at diagnosis of 31 yrs (range 1-83) (246 < 19 yrs at diagnosis), and 645 healthy controls (HC), were genotyped for NKD1 gene by using the TaqMan allelic discriminationmethod. Genotype frequencies were comparedwith respect to age at diagnosis, disease localization and behaviour (Montreal classification), need of resective surgery, response to medical therapy (steroids, immunosuppressive drugs), and NOD2 status. Results: A significant allele and genotype association of the rs8047222 with UC (P=0.01, OR=0.83, CI=0.73-0.96; P=0.02, OR=0.79; CI=0.65-0.96) was shown, especially in adult-onset cohort (P<0.002). After stratifying the cohort on the basis of NOD2 genotypes (NOD2-=no major variants; NOD2+=at last one variant), a significant genotype association in NOD2patients (P=0.004; OR= 0.74; CI=0.61-0.91) was also demonstrated. No significant difference for either allele and genotype frequencies with rs8047222 was found for CD compared with controls, however, a significant genotype association in NOD2patients (P=0.03, OR=0.77, CI=0.62-0.97) was found. At genotype-phenotype evaluation, a reduced freq uency of distal (E1) vs extensive (E3) colitis in UC (47% vs 78%; P=0.006; OR=0.25, CI=0.09-0.70), and colon (56%) vs ileum localization in CD(65%)(P=0.02, OR=0.69, CI=0.49-0.95) was demonstrated. No significant difference for either allele and genotype frequencies for rs4785433, rs730055 SNPs for IBD was found. Conclusions: The rs8047222 SNP on NKD1 gene has a significant protective role in both UC and CD, possibly influenced by NOD2 status, with interaction with disease localization. Nkd1 might be responsible for the residual linkage at 16q12 locus in NOD2-negative CD patients.

P.61 IL23R, ATG16L1, IRGM, AND OCTNS MRNA EXPRESSION IN INFLAMED AND NON-INFLAMED MUCOSA OF IBD PATIENTS

In both main forms of Inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD), intestinal commensal microbiota are thought to trigger the disease in genetically susceptible individuals. Approximately one third of patients with CD have a mutation in the NOD2 gene that encodes an intracellular bacterial pattern-recognition receptor located within Paneth-cells. The maturation of Paneth cells is regulated via the canonical Wnt cascade. The Nkd1 (naked cuticle homologue 1), gene, a Wnt antagonist, is located near NOD2 at 16q12, and it has been found weakly associated with CD in WTCCC dataset (rs8047222 P<1x10-5). Aims: To investigate rs4785433, rs730055, and rs8047222 SNPs of NKD1 and to search for likely correlation with clinical phenotypes in both adult and pediatric onset IBD patients. Methods: 1185 CD patients (670 male) with a mean age at diagnosis of 27 yrs (range 1-80) (334 < 19 yrs at diagnosis), 1385 UC patients (803 male) with a mean age at diagnosis of 31 yrs (range 1-83) (246 < 19 yrs at diagnosis), and 645 healthy controls (HC), were genotyped for NKD1 gene by using the TaqMan allelic discriminationmethod. Genotype frequencies were comparedwith respect to age at diagnosis, disease localization and behaviour (Montreal classification), need of resective surgery, response to medical therapy (steroids, immunosuppressive drugs), and NOD2 status. Results: A significant allele and genotype association of the rs8047222 with UC (P=0.01, OR=0.83, CI=0.73-0.96; P=0.02, OR=0.79; CI=0.65-0.96) was shown, especially in adult-onset cohort (P<0.002). After stratifying the cohort on the basis of NOD2 genotypes (NOD2-=no major variants; NOD2+=at last one variant), a significant genotype association in NOD2patients (P=0.004; OR= 0.74; CI=0.61-0.91) was also demonstrated. No significant difference for either allele and genotype frequencies with rs8047222 was found for CD compared with controls, however, a significant genotype association in NOD2patients (P=0.03, OR=0.77, CI=0.62-0.97) was found. At genotype-phenotype evaluation, a reduced freq uency of distal (E1) vs extensive (E3) colitis in UC (47% vs 78%; P=0.006; OR=0.25, CI=0.09-0.70), and colon (56%) vs ileum localization in CD(65%)(P=0.02, OR=0.69, CI=0.49-0.95) was demonstrated. No significant difference for either allele and genotype frequencies for rs4785433, rs730055 SNPs for IBD was found. Conclusions: The rs8047222 SNP on NKD1 gene has a significant protective role in both UC and CD, possibly influenced by NOD2 status, with interaction with disease localization. Nkd1 might be responsible for the residual linkage at 16q12 locus in NOD2-negative CD patients.

Regulation of the Polymeric Immunoglobulin Receptor in Intestinal Epithelial Cells by Enterobacteriaceae: Implications for Mucosal Homeostasis

The commensal microbiota of the human colon profoundly impacts host gene expression and mucosal homeostasis. Secretory IgA antibodies, which influence the composition of the intestinal microbiota and provide immunity against pathogens, are transported across intestinal epithelial cells (IEC) by the polymeric immunoglobulin receptor (pIgR). To compare the effects of different colonic bacteria on pIgR expression, the human IEC line HT-29 was stimulated with various species representing the 4 major phyla of colonic bacteria. Only bacteria from the family Enterobacteriaceae (phylum Proteobacteria) induced expression of pIgR and other target genes of bacterial pattern recognition receptors. HT-29 cells responded to purified ligands for Toll-like receptor (TLR)4 but not TLR2. Expression of pIgR and transport of IgA were significantly reduced in colons of mice deficient in the TLR adaptor MyD88, consistent with a role for TLR signaling in the regulation of pIgR by colonic bacteria. Induction of pIgR expression in HT-29 cells required NF-κB signaling but not MAPK signaling, in contrast to the requirement for both NF-κB and MAPK signaling for induction of pro-inflammatory genes. These results suggest that commensal Enterobacteriaceae may promote intestinal homeostasis by enhancing pIgR expression in IEC.

Absence of soluble CD14 in saliva of young patients with dental caries

It is generally accepted that salivary components are important for dental health, but to date no clear correlation has been found between one or more of these components and the outcome of dental caries. The identification of salivary factors preventing, favoring or signaling dental caries might help to control the disease. In the present study, western blotting analysis of whole saliva from 20 healthy caries-free children showed the presence of the soluble form of CD14, a bacterial pattern-recognition receptor for many bacterial components that is involved in the innate immune response. The identity of the protein was confirmed through N-terminal sequencing by Edman degradation, and by partial sequencing with mass spectrometry of tryptic peptides. Conversely, CD14 was completely absent in the saliva of 20 age-matched patients affected by two to eight carious lesions, but appeared in their saliva a few weeks after dental restoration. These results suggest that the absence of salivary soluble CD14 could represent an useful index of caries activity, and might be used to detect early carious lesions not visible by oral inspection.

Differential activation of NF-kappaB and gene expression in oral epithelial cells by periodontal pathogens.

To investigate the molecular effects of the periodontopathogens Fusobacterium nucleatum (FN) and Porphyromonas gingivalis (PG) on the oral epithelium, the H400 oral epithelial cell line was cultured in the presence of non-viable bacteria. Following confirmation of the presence of transcripts for the bacterial pattern recognition receptors in H400 cells, Toll-like receptors -2, -4 and -9, and components of the NF-kappaB signalling pathway, immunocytochemical analyses were performed showing that NF-kappaB was activated within 1 h of exposure to both periodontopathogens. A significantly greater number of NF-kappaB nuclear translocations were apparent following H400 cell exposure to FN as compared with PG. Gene expression analyses indicated that transcripts known to be regulated by the NF-kappaB pathway, including cytokines/chemokines TNF-alpha, IL-1beta, IL-8, MCP-1/CCL2 and GM-CSF, were up-regulated following 4 and 24 h of exposure to both periodontopathogens. In addition, H400 periodontopathogen exposure resulted in differential regulation of transcripts for several cytokeratin gene family members. Consistent with the immunocytochemical data, microarray results indicated that FN induced a greater number of gene expression changes than PG following 24 h of exposure, 609 and 409 genes, respectively. Ninety-one genes were commonly differentially expressed by both periodontopathogens and represented biological processes commonly associated with periodontitis. Gene expression analyses by reserve transcriptase-polymerase chain reaction (RT-PCR) of molecules identified from the microarray data sets, including Heme oxygenase-1, lysyl oxidase, SOD2, CCL20 and calprotectin components, confirmed their differential expression profiles induced by the two periodontopathogens. FN and PG have clearly different molecular effects on oral epithelial cells, potentially highlighting the importance of the composition of the plaque biofilm in periodontitis pathogenesis.

Lack of association of the 3′-UTR polymorphism in the NFKBIA gene with Crohn’s disease in an Israeli cohort

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract associated with dysregulation of the immune response. It is caused by a combination of environmental and genetic factors. Patients with CD have a TH1-type inflammatory response characterized by nuclear factor kappa B (NFkappaB) activation. Mutations in the bacterial pattern recognition receptors NOD2/CARD15 and Toll-like receptor 4 (TLR4) genes, which lead to activation of NFkappaB under normal circumstances, have been associated with increased susceptibility for CD. NFkappaB plays a critical role in the immune response and is down-regulated by NFkappaB inhibitor alpha (NFKBIA). NFKBIA was found to be a susceptibility gene for German CD patients lacking NOD2/CARD15 mutations. A cohort of 231 Israeli CD patients previously genotyped for the single nucleotide polymorphisms (SNPs) in the CARD15, TLR4 susceptibility genes for CD, was analyzed for the 3'-untranslated region (UTR) SNP of the NFKBIA gene in comparison to 100 healthy ethnically matched controls. We evaluated the contribution of the 3'-UTR SNP in NFKBIA in patients with or without other SNPs in CARD15 to age of onset, disease location, and disease behavior (Vienna classification). We did not identify a significant difference in allele and genotype frequencies between either groups or an effect on phenotype. No interactions were found between NFKBIA and any NOD2. The contribution of population diversity to susceptibility genes for CD plays an important role in disease-associated variants and is important for better understanding of the pathologic mechanisms of the polymorphism.

Constitutive expression of a bacterial pattern recognition receptor, CD14, in human salivary glands and secretion as a soluble form in saliva.

Saliva contains a number of proteins and glycoproteins that protect oral tissues, but little is known about the role of human saliva in innate immunity. Here we showed that human major salivary gland cells constitutively expressed a bacterial pattern recognition receptor, CD14, by immunohistochemistry. Human salivary gland cells in culture express CD14 mRNA and a 55-kDa CD14 protein in, but not on the cells, and secrete a soluble form with the same molecular mass. Human whole saliva contains a 55-kDa CD14, and the concentration of parotid saliva was 10-fold higher than whole saliva, which is comparable to that of serum CD14. Levels of CD14 in unstimulated whole and parotid saliva were unchanged before and after a meal and between unstimulated and stimulated saliva, indicating that saliva CD14 is constitutively secreted into the oral cavity. In contrast, lipopolysaccharide (LPS)-binding protein was below the detectable level. The saliva CD14 is functionally active in that it mediated the activation of CD14-lacking intestinal epithelial cells by LPS in a Toll-like receptor 4-dependent manner. These results suggested that saliva CD14 is important for the maintenance of oral health and possibly intestinal homeostasis.

Proteolysis of CD14 on human gingival fibroblasts by arginine-specific cysteine proteinases from Porphyromonas gingivalis leading to down-regulation of lipopolysaccharide-induced interleukin-8 production.

Arginine-specific cysteine proteinases (gingipains-R) from periodontopathic Porphyromonas gingivalis cleaved CD14, a bacterial pattern recognition receptor, on human gingival fibroblasts (HGF). Consequently, gingipains-R reduced lipopolysaccharide-induced interleukin-8 production by HGF, indicating that gingipains-R inhibited CD14-dependent HGF activation and are involved in immune evasion by the bacterium in periodontal tissues.