Fecal luminal factors from patients with irritable bowel syndrome induce distinct gene expression of colonoids.

BACKGROUND: Alteration of the host-microbiota cross talk at the intestinal barrier may participate in the pathophysiology of irritable bowel syndrome (IBS). Therefore, we aimed to determine effects of fecal luminal factors from IBS patients on the colonic epithelium using colonoids. METHODS: Colon-derived organoid monolayers, colonoids, generated from a healthy subject, underwent stimulation with fecal supernatants from healthy subjects and IBS patients with predominant diarrhea, phosphate-buffered saline (PBS), or lipopolysaccharide (LPS). Cytokines in cell cultures and fecal LPS were measured by ELISA and mRNA gene expression of monolayers was analyzed using Qiagen RT2 Profiler PCR Arrays. The fecal microbiota profile was determined by the GA-map™ dysbiosis test and the fecal metabolite profile was analyzed by untargeted liquid chromatography/mass spectrometry. KEY RESULTS: Colonoid monolayers stimulated with fecal supernatants from healthy subjects (n = 7), PBS (n = 4) or LPS (n = 3) presented distinct gene expression profiles, with some overlap (R2 Y = 0.70, Q2  = 0.43). Addition of fecal supernatants from healthy subjects and IBS patients (n = 9) gave rise to different gene expression profiles of the colonoid monolayers (R2 Y = 0.79, Q2  = 0.64). Genes (n = 22) related to immune response (CD1D, TLR5) and barrier integrity (CLDN15, DSC2) contributed to the separation. Levels of proinflammatory cytokines in colonoid monolayer cultures were comparable when stimulated with fecal supernatants from either donor types. Fecal microbiota and metabolite profiles, but not LPS content, differed between the study groups. CONCLUSIONS: Fecal luminal factors from IBS patients induce a distinct colonic epithelial gene expression, potentially reflecting the disease pathophysiology. The culture of colonoids from healthy subjects with fecal supernatants from IBS patients may facilitate the exploration of IBS related intestinal micro-environmental and barrier interactions.

Genetic Variation in Toll-Like Receptor 5 and Colonization with Flagellated Bacterial Vaginosis-Associated Bacteria.

Bacterial vaginosis (BV) is a vaginal dysbiotic condition linked to negative gynecological and reproductive sequelae. Flagellated bacteria have been identified in women with BV, including Mobiluncus spp. and BV-associated bacterium-1 (BVAB1), an uncultivated, putatively flagellated species. The host response to flagellin mediated through Toll-like receptor 5 (TLR5) has not been explored in BV. Using independent discovery and validation cohorts, we examined the hypothesis that TLR5 deficiency-defined by a dominant negative stop codon polymorphism, rs5744168-is associated with an increased risk for BV and increased colonization with flagellated bacteria associated with BV (BVAB1, Mobiluncus curtisii, and Mobiluncus mulieris). TLR5 deficiency was not associated with BV status, and TLR5-deficient women had decreased colonization with BVAB1 in both cohorts. We stimulated HEK-hTLR5-overexpressing NF-κB reporter cells with whole, heat-killed M. mulieris or M. curtisii and with partially purified flagellin from these species; as BVAB1 is uncultivated, we used cervicovaginal lavage (CVL) fluid supernatant from women colonized with BVAB1 for stimulation. While heat-killed M. mulieris and CVL fluid from women colonized with BVAB1 stimulate a TLR5-mediated response, heat-killed M. curtisii did not. In contrast, partially purified flagellin from both Mobiluncus species stimulated a TLR5-mediated response in vitro We observed no correlation between vaginal interleukin 8 (IL-8) and flagellated BVAB concentrations among TLR5-sufficient women. Interspecies variation in accessibility of flagellin recognition domains may be responsible for these observations, as reflected in the potentially novel flagellin products encoded by Mobiluncus species versus those encoded by BVAB1.

Flagellin attenuates experimental sepsis in a macrophage-dependent manner.

BACKGROUND: Sepsis is the leading cause of death among critically ill patients, and no specific therapeutic agent is currently approved for the treatment of sepsis. METHODS: We assessed the effects of flagellin administration on survival, bacterial burden, and tissue injury after sepsis. In addition, we examined the effects on phagocytosis and bacterial killing in monocytes/macrophages. RESULTS: Therapeutic administration of flagellin increased bacterial clearance, decreased organ inflammation and injury, and reduced immune cell apoptosis after experimental sepsis, in a Toll-like receptor 5 (TLR5)-dependent manner. Macrophages, but not neutrophils, mediated the beneficial effects of flagellin on experimental sepsis, and flagellin induced macrophage polarization into M1 in septic mice. Flagellin treatment could directly enhance phagocytosis and bacterial killing of macrophages, but not neutrophils. Subsequent studies demonstrated that flagellin could promote phagosome formation and increase reactive oxygen species (ROS) levels in macrophages. Finally, we found that the expression of TLR5 was significantly elevated on the surface of circulating monocytes, but not neutrophils, from patients with sepsis. Higher expression levels of TLR5 on monocytes were associated with increased mortality, documented bacteremia, and higher Sequential Organ Failure Assessment scores of the septic patients. Moreover, flagellin treatment rescued the impaired phagocytosis and bacterial killing ability of monocytes/macrophages from patients who died of sepsis. CONCLUSIONS: These novel findings not only established the potential value of application of flagellin as an immunoadjuvant in treating sepsis, but also provided new insights into targeted therapeutic strategy on the basis of monocyte TLR5 expression in septic patients.

The expression and localization of Toll-like receptors 2, 4, 5 and 9 in the epididymis and vas deferens of a adult tom cats.

Toll-like receptors (TLRs) are important molecules, which provide protection against infections of the reproductive tract. This study demonstrates for the first time the expression and localization patterns of TLRs in the caput, corpus and cauda segments of the epididymal duct (ED) and the vas deferens (VD) of adult domestic cats using immunohistochemistry and western blotting. While immunoblot analyses revealed relatively similar protein levels for TLRs 2, 4, 5, and 9 in three segments of the ED, the protein levels of TLR2 and TLR4 in the VD were found to be significantly higher than those measured in the ED segments (P < 0.05). On the other hand, immunostaining showed that TLRs exhibited regional- and cell-specific localization patterns. TLR2 and TLR5 were immunolocalized to the nucleus and cytoplasm of the principal cells in all ducts. TLR4 was restricted to the stereocilia, and TLR9 was located in the cytoplasm of the principal cells. Narrow cells displayed positive immunoreactions for TLR4 and TLR5. The basal cells of the different ED segments were positive for all four TLRs. TLR2, TLR5 and TLR9 were detected in the cytoplasmic droplets of the spermatozoa. TLR4 and TLR9 were detected along the entire length of the sperm tail, whilst TLR2 and TLR5 were absent in the midpiece. TLR2 and TLR5 were also detected in the equatorial segment of the sperm head. These results suggest that TLR2, TLR4, TLR5 and TLR9 are important not only for the protection of the ED, VD and spermatozoa but also for the maturation and storage of spermatozoa in the ED and VD, respectively.

Toll-like receptor 5 and 7 expression in adenoid cystic carcinoma of major salivary glands.

Adenoid cystic carcinoma (ACC) of the salivary glands has a poor long-term prognosis and high metastatic rate. Toll-like receptors (TLRs) have been related to tumour progression but have also tumour growth-inhibiting responses. To the best of our knowledge, they have not been studied previously in ACC. We studied the immunoexpression of TLR 5 and 7 in ACC of the major salivary glands. From a cohort of 54 patients with ACC of the major salivary glands treated at the Department of Otorhinolaryngology-Head and Neck Surgery, Helsinki University Hospital, Helsinki, Finland in 1974-2009, there were 34 primary tumours and six metastases available for immunohistochemical analysis. Immunohistochemical expression of TLR 5 and 7 were correlated to clinicopathological findings and patient survival. Both TLR 5 and 7 were expressed in ACCs and their metastases, mostly on the cell membranes. The expression was heterogeneous in individual tumours. TLR 5 was expressed less in male samples, and TLR 7 had lower expression in ACCs with solid growth pattern. No correlation with survival was found. In the normal salivary gland, the TLR 5 and 7 expression was mainly negative. Both TLR 5 and 7 are expressed in salivary adenoid cystic carcinoma on the cell membranes as well as in cytoplasm.

The Bacterial Component Flagellin Induces Anti-Sepsis Protection Through TLR-5, IL-1RN and VCAN During Polymicrobial Sepsis in Mice.

BACKGROUND: The present study was designed to observe the effects of the bacterial component flagellin on anti-sepsis protection through TLR-5, VCAN and IL-1RN. METHODS: A clinically relevant model of sepsis was induced by cecal ligation and puncture (CLP). An in vitro culture of endothelial cells was analyzed. RESULTS: Flagellin induced anti-sepsis protection through inhibition of inflammation and induction of endothelial proliferation by down-regulating the expression of TLR 3, TLR 4, and IL-1RN and promoting the expression of VCAN in mice 24 h post-CLP. In vitro, flagellin promoted the proliferation of endothelial cells. These effects could be inhibited by transfection of endothelial cells with VCAN siRNA or IL-1RN over-expression constructs. VCAN expression decreased after transfection of the cells with an IL-1RN over-expression construct and increased after transfection of the cells with an IL-1RN siRNA construct. IL-1RN expression remained unchanged after transfection of the cells with VCAN over-expression or siRNA constructs. CONCLUSIONS: These data suggest that flagellin pretreatment promoted anti-sepsis protection through the TLR-5, IL-1RN and VCAN pathway. This pathway is necessary to mediate endothelial repair and thereby promote survival following sepsis challenge.

Investigation of the utility of complementary electrochemical detection techniques to examine the in vitro affinity of bacterial flagellins for a toll-like receptor 5 biosensor.

An initial investigation of the fabrication of a novel biosensor utilizing toll-like receptor 5 (TLR5) has been conducted. The detection assay using this sensor platform has been carried out using two complementary electrochemical techniques. The electrochemical properties of the modified bare gold surface following TLR5 immobilization were characterized. The electrochemical response to changes in the sensor film resistance and electron charge-transfer permittivity triggered by independent exposures to flagellins from Salmonella typhimurium (S. typhimurium) and Bacillus subtilis (B. subtilis) were examined and observed. The quantified film resistance data gathered using electrochemical impedance spectroscopy (EIS) over a macroscopic scale are in significant agreement with the corresponding electron charge-transfer permittivity measured locally by scanning electrochemical microscopy (SECM). Unlike other sensors that exploit pathogen recognition elements, TLR5 biosensors have the potential to carry out broad-spectrum detection of flagellated bacterial pathogens in near real time. This broad-spectrum detection platform is a significant step toward the development of fast, inexpensive clinical tools for early warning diagnoses and immediate on-site treatment.

A specific Toll-like receptor profile on T lymphocytes and values of monocytes correlate with bacterial, fungal, and cytomegalovirus infections in the early period of allogeneic stem cell transplantation.

BACKGROUND: Bacterial, fungal, and viral infections often affect non-relapse mortality after allogeneic stem cell transplantation (alloSCT). Recovery from infections depends on a balanced integration between innate and adaptive immune responses. In this complex interplay, a key role is played by Toll-like receptors (TLRs), which are sensors of pathogen-associated molecular patterns. To our knowledge, no previous study deals with both expression and function of all human TLRs together, in relation to infections in the setting of alloSCT. METHODS: We prospectively evaluated 9 TLRs by flow cytometry on T lymphocytes and monocytes of 35 patients in relation to infectious events from day +30 to day +120. Tumor necrois factor-alpha, interleukin-4, interferon-gamma, and monocyte chemoattractant protein-1 induction upon TLR activation was assessed by enzyme-linked immunosorbent assay on cell supernatants. RESULTS: In multivariate Cox regression analysis, levels of TLR-9 expression on T lymphocytes (P = 0.01) and values of natural killer cells (P = 0.01) correlated negatively with bacterial infections, whereas cytomegalovirus (CMV) infection resulted as a positive predictor. We observed a trend for negative correlation between TLR-7 levels on T lymphocytes and fungal infections (P = 0.07). Values of monocytes were negatively associated with CMV infection (P = 0.03), whereas levels of TLR-5 on T lymphocytes were positive predictors (P = 0.01). Age (P = 0.03) and bacterial infections (P = 0.006) negatively influenced overall survival. Monocyte values were positive predictors of survival (P = 0.003). CONCLUSIONS: Bacterial, fungal, and CMV infections were associated with a different expression of some TLRs on T lymphocytes. The protective role of TLR-7 and TLR-9 seemed dominant over other TLRs involved in recognizing fungi and bacteria. We also observed an atypical involvement of TLR-5 in CMV infection. The dominant and atypical role of some TLRs could depend on their pleiotropic functions and the changing inflammatory environment of transplanted patients. A specific TLR profile and an adequate count of monocytes could improve survival, promoting an effective control of infections, and balanced immune responses. If our findings will be confirmed by further studies, these immunological variables could be useful as parameters to predict susceptibility to infections.

Molecular cloning and functional analysis of duck Toll-like receptor 5.

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the single-exon TLR5 gene of the Maya breed of Common Shelduck (Tadorna tadorna). The TLR5 open reading frame is 2580 bp in length and encodes an 859-amino acid protein. The putative amino acid sequence of duck TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat domains, a leucine-rich repeat C-terminal domain, a transmembrane domain, and an intracellular Toll-interleukin-1 receptor domain. The duck TLR5 gene was highly expressed in the lung, bone marrow, spleen, and liver; moderately expressed in kidney, small intestine, large intestine, and brain. A plasmid expressing duck TLR5 was constructed and transfected into HEK293T cells, and expression was confirmed by indirect immunofluorescence assay. HEK293T cells transfected with duck TLR5- and NF-κB-luciferase-containing plasmids significantly responded to flagellin from Salmonella typhimurium, indicating that it is a functional TLR5 homolog.

Immunohistochemical detection of Helicobacter pylori without association of TLR5 expression in oral squamous cell carcinoma.

BACKGROUND: Approximately 15% of human deaths from cancer are associated with chronic viral or bacterial infections. Helicobacter pylori (HP), a flagellated, Gram-negative, spiral, microaerophilic bacteria is considered to be the most common chronic bacterial infection in humans. Toll-like receptor 5 (TLR5) is involved in recognition of bacterial flagella and is thought to promote tumour growth through inflammation-dependent mechanisms in epithelial cells. METHODS: Expression of HP and TLR5 was analysed in OSCC specimen (n = 191) by immunohistochemistry. TLR5 expression specificity was conducted by Western blotting in cancer cell lines (BICR3, BICR56). TLR5-stained sections were scanned and digitally analysed using ImageJ and the immunomembrane plug-in. HP expression and TLR5 expression were associated with clinicopathological characteristics and impact on survival. RESULTS: Helicobacter pylori detection was significantly associated with recurrence of the tumour, whereas TLR5 expression was not. Multivariate analysis demonstrated HP expression as an independent prognostic factor (P = 0.0260). TLR5 specificity was confirmed by Western blot analysis. CONCLUSIONS: For the first time, this study provides evidence that immunohistochemically detected HP expression in OSCC is associated with reduced disease-free survival in a large patient cohort. Although TLR5 was not associated with any clinicopathological characteristics or impact on survival, investigation of the TLR family seems to be reasonable due to the possible existence of other pathogenic bacterial or viral compounds in oral cavity cancer.

Increased Toll-like receptor 5 expression indicates esophageal columnar dysplasia.

Toll-like receptor 5 (TLR5) is an immune receptor, which recognizes bacterial flagellin. Increased expression has been reported in various premalignant and malignant lesions indicating a role in carcinogenesis. We assessed the expression of TLR5 in normal esophageal squamous epithelium, Barrett's esophagus with and without dysplasia, and in esophageal adenocarcinoma. Specimens with normal esophagus (n=93), gastric (n=75) or intestinal metaplasia (n=53) without dysplasia, and low-grade (n=56) or high-grade dysplasia (n=33) and esophageal adenocarcinoma (n=94) were studied. TLR5 immunohistochemical stainings were analyzed for the proportion of positive cells and the intensity of expression. In normal squamous epithelium, only the basal third showed TLR5 expression. In esophageal gastric or intestinal metaplasia, expression was present in majority of the cells but significantly weaker (p<0.001) than in dysplastic epithelium. In dysplasia, expression extended to the apical cytoplasm, contrasting basolateral expression in non-dysplastic columnar epithelium. Receiver operating characteristic curve analysis showed that moderate to high expression intensity of TLR5 indicates low-grade dysplasia with 86 % sensitivity and 83 % specificity. Carcinomas showed increased expression in comparison with non-dysplastic columnar epithelium, but there was no association with prognosis. Our results indicate that the esophageal columnar dysplasia is associated with clear increase of TLR5 expression and dissolution of regular polarized expression. TLR5 staining provides a possible biomarker for the recognition of low-grade dysplasia. In addition, the findings suggest a role for abnormal expression of TLR5 in the pathogenesis of esophageal adenocarcinoma and suggest importance of altered microbiome in the pathogenesis of complications of Barrett's esophagus.

Increased nucleic Acid receptor expression in chronic periodontitis.

BACKGROUND: Nucleic acid sensing has emerged as one of the important components of the immune system triggering inflammation. The aim of this study is to determine the expression of bacterial DNA sensors, including Toll-like receptor 9 (TLR-9), DNA-dependent activator of interferon-regulatory factors (DAI), and absent in melanoma 2 (AIM2) in chronic periodontitis (CP versus healthy) (H) tissues. METHODS: Thirty-five CP and 27 H gingival biopsies were included. Real-time quantitative polymerase chain reaction was performed to determine mRNA levels of AIM2, DAI, and TLRs (TLR-1 through TLR-9). The difference in gene expression for each sensor between CP and H tissues was calculated using analysis of covariance. The Spearman test was used to determine correlations among innate receptors. The expression of TLR-9, AIM2, and DAI in gingival tissues was further confirmed using immunohistochemistry. RESULTS: The present results reveal statistically significant upregulation of TLR-9 (P <0.006), DAI (P <0.001), and TLR-8 (P <0.01) in CP tissues compared to H sites. Although mRNA expression was not changed significantly between groups for other receptors, the present results reveal significant correlations between receptors (P <0.05), suggesting that cooperation between multiple components of the host immune system may influence the overall response. Immunohistochemistry further confirmed expression of TLR-9, AIM2, and DAI in gingival tissues. CONCLUSIONS: This study highlights a possible role for nucleic acid receptors in periodontal inflammation. Future investigations will determine whether cytoplasmic receptors and their ligands can be targeted to improve clinical outcomes in periodontitis.

Differential expression of toll-like receptor mRNAs in recurrent aphthous ulceration.

BACKGROUND: Toll-like receptors (TLR) are membrane proteins that recognize conserved molecules derived from bacterial, viral, fungal or host tissues. They are responsible for promoting the production of cytokines and chemokines, increasing the expression of costimulatory molecules and influencing the T Helper response (Th) toward either a Th1 or Th2 profile, thereby modulating the regulatory T cell response and controlling the integrity of the epithelial barrier. The key factors responsible for increased susceptibility to recurrent aphthous ulceration (RAU) are unclear, and because TLRs are involved in both immune regulation and control of the epithelial barrier, a deficiency in TLR activity is likely to cause increased susceptibility. METHODS: We investigated the gene expression of TLRs one through 10 in tissue samples and peripheral blood mononuclear cells (PBMC) of RAU patients in comparison to healthy controls using real-time quantitative reverse transcription PCR. RESULTS: The analysis of mRNA expression levels in oral lesion showed significant (P < 0.01) overexpression of the TLR2 (~6-fold) gene and decreased expression of the TLR3 (~5-fold) and TLR5 (~6-fold) genes in comparison with healthy oral mucosa. The analysis of mRNA expression in PBMC indicated a down-regulation of TLR5 gene expression in the cells from RAU patients (P < 0.05; ~2-fold). CONCLUSION: Our results support the hypothesis that a subset of RAU patients has fewer TLR expression that have been tentatively implicated in anti-inflammatory effects. This derangement of TLR gene expression may cause an overlay exuberant inflammation reaction in situations where normal individuals are resistant.

Recurrent aphthous ulcers--a Toll-like receptor-mediated disease?

BACKGROUND: Recurrent aphthous ulcer (RAU) is characterized by acute and painful inflammatory ulcerations, which heal spontaneously but tend to recur. Many pathogens have been proposed as causative agents, but none has been consistently proven. According to our hypothesis, RAU is an autoinflammatory disorder triggered by pathogen-associated molecular patterns (PAMPs) shared by different pathogenic and commensal microbes. METHODS: PAMP-reactive Toll-like receptors (TLRs) were mapped in oral epithelium in healthy controls compared to RAU. RESULTS: In controls, the superficial epithelium formed a TLR(-), a PAMP non-reactive physical barrier zone, but all TLRs were found deeper in the epithelium, usually restricted to suprabasal and basal cell layers. In RAU, the epithelial TLR polarity was lost: TLRs 1, 2, 5, 7, and 8 were found throughout the epithelium, but also TLRs 4, 6, and 10 extended higher up than normally, whereas TLR-3 was almost lost in RAU. In RAU lesions, connective tissue stroma was heavily infiltrated by TLR(+) inflammatory cells. CONCLUSIONS: Normal TLR architecture prevents inflammatory responses against normal microbes but still contains a deep TLR(+) , PAMP-reactive dormant defense zone. In RAU, the TLR(+), PAMP-reactive zone extends to surface or subsurface exposed to microbial PAMPs. TLR reactivity is further enhanced by recruitment of inflammatory leukocytes forming a new deep line of defense. The organization of the TLR system in healthy mucosa and its changes in RAU are compatible with active pathogenic involvement of TLRs, which together with the typical clinical picture and course suggest that RAU is a TLR-mediated disease.

Stimulants of Toll-like receptors 2 and 4 are elevated in saliva of periodontitis patients compared with healthy subjects.

AIM: Because the absorption of stimulants of Toll-like receptor (TLR)2 and TLR4 from the gastrointestinal tract into the circulation has been proposed to promote the development of atherosclerosis and insulin resistance, we aimed to quantify the abundance of stimulants of TLR2 and TLR4 in human saliva. METHODS: A recently developed bioassay based upon measurement of NF-κB activation in TLR-deficient human embryonic kidney (HEK)-293 cells transfected with human TLR2 or TLR4 and calibrated with synthetic bacterial lipopeptide (Pam(3) CSK(4) ) or Escherichia coli lipopolysaccharide (LPS), was used to establish the normal range of TLR stimulants in saliva of 20 healthy subjects and 20 subjects with periodontal disease. RESULTS: Median soluble stimulants of TLR2 and TLR4 were significantly higher in saliva of periodontitis patients compared with saliva of healthy subjects; 3450 versus 77 ng/ml Pam(3) CSK(4) equivalents (p<0.0001) and 138 versus 7 ng/ml LPS equivalents, respectively (p<0.0001). Salivary TLR stimulant levels remained relatively stable in healthy subjects over several days. Six strains of oral Gram-negative bacteria, including Tannerella forsythensis, Lysobacter enzymogenes, Prevotella intermedia, Prevotella oris and Porphyromonas gingivalis, from a panel of nine examined did not stimulate TLR4-dependent signalling. CONCLUSIONS: Elevated salivary TLR stimulants may represent a novel mechanism by which periodontitis increases the risk of developing cardiovascular disease and insulin resistance.

Cigarette smoke extract modulates human beta-defensin-2 and interleukin-8 expression in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Human gingival epithelial cells (HGECs) are continually exposed to oral bacteria and to other harmful agents. Their responses to stimuli are critical in maintaining periodontal homeostasis. The aim of this study was to investigate the modulating effect of cigarette smoke extract (CSE) on the innate immune responses of HGECs. MATERIAL AND METHODS: Toll-like receptor (TLR) expression of HGECs was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The effect of CSE or nicotine on the expression of the antimicrobial peptide human beta-defensin-2 (hBD-2) and the pro-inflammatory cytokine interleukin (IL)-8 in stimulated HGEC cultures was evaluated by RT-PCR and enzyme-linked immunosorbent assay. RESULTS: The HGECs expressed mRNA of TLRs 1, 2, 3, 5, 6, 9, 10, and minimally of TLR4, but not of TLRs 7 or 8. Stimulation of HGECs with highly purified TLR2, 3 or 5 ligands led to expression of hBD-2 and of IL-8. Enhancement of hBD-2 and IL-8 was observed in HGECs after combined stimulation with Porphyromonas gingivalis lipopolysaccharide (TLR2 ligand) and tumour necrosis factor-alpha, compared with stimulation using either agent alone. After CSE exposure, hBD-2 expression was markedly reduced in stimulated HGEC cultures, whereas IL-8 expression was markedly increased. These effects were also observed, but were markedly attenuated, upon nicotine treatment. CONCLUSION: Human gingival epithelial cells play a critical role in orchestrating the innate immune responses of periodontal tissue via TLR signalling. Our results represent the first demonstration that CSE can modulate HGEC function by suppressing hBD-2 and enhancing IL-8 production, and this may be, in part, a possible mechanism which promotes periodontal disease.

Immunohistochemical localization of Toll-like receptors 1-10 in periodontitis.

BACKGROUND/AIM: In periodontitis, bacteria and pathogen-associated molecular patterns are sensed by Toll-like receptors (TLRs), which initiate intracellular signaling cascades that may lead to host inflammation. In this study, the expression and distribution of TLRs (TLR-1 to TLR-10) were immunohistochemically detected in gingival epithelium and connective tissue. METHODS: Immunohistochemistry was used for the localization of TLRs in gingival tissue samples from 10 patients with chronic periodontitis and 10 healthy controls; these samples were obtained during routine periodontal flap operations and during extraction operations performed for retained wisdom teeth, respectively. For the evaluation, epithelial layers were stratified to basal, spinous, and superficial layers, and the percentages of TLR-positive cells were determined. RESULTS: Both healthy and periodontitis gingival tissues expressed all TLRs except TLR-10. In patients with periodontitis, epithelial cells showed increased TLR expression towards the basal layer. Healthy controls showed more variable cellular TLR expression and distribution between the layers of epithelium. In the connective tissue, consistently higher TLR expression was found within the periodontitis group compared to the healthy group. CONCLUSIONS: For the first time, the cellular expression and distribution of TLR-1 to TLR-10 have been studied in periodontitis, indicating that TLR-1 to TLR-9 are differentially expressed both in connective tissue and epithelial layers. Except for TLR-7 and TLR-8, all the other TLRs showed statistically significant differences between patients with periodontitis and healthy controls, suggesting their involvement in the pathogenesis of periodontitis.

Differential expression of Toll-like receptors in chronic hyperplastic candidosis.

INTRODUCTION: The first step in the host defense against oral candidosis is the recognition of Candida albicans through a set of germ-encoded pathogen recognition receptors, e.g. Toll-like receptors (TLRs). In man, 10 types of such receptors have been identified so far, of which only TLR2, TLR4, and TLR6 have been linked to mediating candidal ligands, e.g. zymosan. METHODS: Biopsies from patients with chronic hyperplastic candidosis (n = 5), leukoplakia (n = 5), and healthy mucosa (n = 5) were immunohistochemically stained with antibodies to the TLRs (TLR1 to TLR9) to distinguish and compare the staining patterns of the epithelial layer in the three categories of tissues. RESULTS: On analysis, the epithelium of all tissues was divided into three layers: basal, middle, and superficial. Two of the five chronic hyperplastic candidosis sections showed high numbers of hyphae compared to yeasts, which paralleled a decrease in the expression of TLR2 and an increase in the staining intensity of TLR4. Leukoplakia and healthy tissue sections demonstrated stronger immunostaining of TLRs, except TLR9 which showed weaker staining in some sections of the former, and in the basal layers of some sections of the latter. DISCUSSION: This study supports the concept of negative regulation of TLRs that are either ligand-bound (e.g. in chronic hyperplastic candidosis), or not stimulated (in healthy tissue). It also augments the opinion that C. albicans, through its hyphae rather than blastospore, may utilize TLRs, i.e. TLR2, to evade the immune system of the host. Leukoplakia seems to be more immunologically alert, which reduces the chances of worsening the already-diseased tissue.

Quantitative messenger RNA expression of Toll-like receptors and interferon-alpha1 in gingivitis and periodontitis.

INTRODUCTION: In addition to bacteria, viruses have been reportedly implicated in periodontitis. However, the available data are confined to Toll-like receptor 2 (TLR2) and TLR4, which recognize bacterial products in periodontitis. In the present study, we investigated the expression levels of TLR5, -7, and -9 messenger RNAs (mRNAs) in addition to those of TLR2 and -4, and compared gingivitis and periodontitis. Interferon-alpha1 (IFN-alpha1), which is important for the antiviral response, was also compared. METHODS: Gene expression was analyzed using quantitative real-time polymerase chain reaction for 59 periodontitis and 27 gingivitis tissue samples together with viral serology in some patients. The presence of plasmacytoid dendritic cells (pDCs), a robust producer of IFN-alpha, was immunohistochemically analyzed in an additional seven periodontitis and two gingivitis specimens. RESULTS: The expression levels of TLR2, -4, -7, and -9 were significantly higher in periodontitis lesions than gingivitis lesions. The expression level of TLR5 was comparable to levels of TLR2 and -4; however, no significant difference was found between gingivitis and periodontitis. Although the expression of IFN-alpha1 mRNA was higher in periodontitis lesions compared with gingivitis lesions, the level was quite low. Only a few pDCs were found in some periodontitis specimens. No difference was found for antibody-positivity between gingivitis and periodontitis. CONCLUSION: This is the first study to show that a variety of TLRs are up-regulated in periodontitis lesions compared with gingivitis lesions, suggesting that diverse microbial and possibly viral antigens are involved in the pathogenic mechanisms for periodontal diseases. However, the ligands recognized by the various TLRs in periodontal lesions remain to be determined.

Flagellin-induced tolerance of the Toll-like receptor 5 signaling pathway in polarized intestinal epithelial cells.

Salmonella typhimurium is a gram-negative enteric pathogen that invades the mucosal epithelium and is associated with diarrheal illness in humans. Flagellin from S. typhimurium and other gram-negative bacteria has been shown to be the predominant proinflammatory mediator through activation of the basolateral Toll-like receptor 5 (TLR5). Recent evidence has shown that prior exposure can render immune cells tolerant to subsequent challenges by TLR ligands. Accordingly, we examined whether prior exposure to purified flagellin would render human intestinal epithelial cells insensitive to future contact. We found that flagellin-induced tolerance is common to polarized epithelial cells and prevents further activation of proinflammatory signaling cascades by both purified flagellin and Salmonella bacteria but does not affect TNF-alpha stimulation of the same pathways. Flagellin tolerance is a rapid process that does not require protein synthesis, and that occurs within 1 to 2 h of flagellin exposure. Prolonged flagellin exposure blocks activation of the NF-kappaB, MAPK, and phosphoinositol 3-kinase signaling pathways and results in the internalization of a fraction of the basolateral TLR5 without affecting the polarity or total expression of TLR5. After removal of flagellin, cells require more than 24 h to fully recover their ability to mount a normal proinflammatory response. We have found that activation of phosphoinositol 3-kinase and Akt by flagellin has a small damping effect in the early stages of flagellin signaling but is not responsible for tolerance. Our study indicates that inhibition of TLR5-associated IL-1 receptor-associated kinase-4 activity occurs during the development of flagellin tolerance and is likely to be the cause of tolerance.