Sampling the cultivation parameter space for the bacterial production of TLR1 intracellular domain reveals the multiple optima.

T7 expression system is an extremely popular approach for the recombinant protein production in Escherichia coli for structural and functional studies and therapeutic applications. There are many useful tools and successful techniques that allow expressing the desired protein in this system. However, high yield of soluble protein often requires a systematic optimization of a wide range of cell cultivation parameters. Here we analyze the effect of three key cultivation parameters - chemical inductor, temperature and time of post-induction culturing on the expression level of TLR1 intracellular TIR domain in a soluble form. In addition, the influence of Triton X-100 detergent on the protein solubility during the cell lysis was investigated. We show that a high expression level of the correctly folded soluble protein can be obtained under different combinations of cultivation parameters.

Regulation of epithelial cell expressed C3 in the intestine - Relevance for the pathophysiology of inflammatory bowel disease?

The complement system not only plays a critical role in efficient detection and clearance of bacteria, but also in intestinal immune homeostasis as mice deficient for key complement components display enhanced intestinal inflammation upon experimental colitis. Because underlying molecular mechanisms for this observation are unclear, we investigated the crosstalk between intestinal epithelial cells (IEC), bacteria and the complement system in the course of chronic colitis. Surprisingly, mouse intestinal epithelial cell lines constitutively express high mRNA levels of complement component 3 (C3), Toll-like receptor 2 (Tlr2) and Tlr4. Stimulation of these cells with lipopolysaccharide (LPS), but not with flagellin, LD-muramyldipeptide or peptidoglycan, triggered increased C3 expression, secretion and activation. Stimulation of the C3aR on these cell lines with C3a resulted in an increase of LPS-triggered pro-inflammatory response. Tissue biopsies from C57BL/6J mice revealed higher expression of C3, Tlr1, Tlr2 and Tlr4 in colonic primary IECs (pIECs) compared to ileal pIECs, while in germ-free mice no differences in C3 protein expression was observed. In DSS-induced chronic colitis mouse models, C3 mRNA expression was upregulated in colonic biopsies and ileal pIECs with elevated C3 protein in the lamina propria, IECs and the mucus. Notably, increased C3b opsonization of mucosa-attached bacteria and decreased fecal full-length C3 protein was observed in DSS-treated compared to untreated mice. Of significant interest, non-inflamed and inflamed colonic biopsy samples from CD but not UC patients displayed exacerbated C3 expression compared to controls. These findings suggest that a novel TLR4-C3 axis could control the intestinal immune response during chronic colitis.

GM-CSF Down-Regulates TLR Expression via the Transcription Factor PU.1 in Human Monocytes.

Toll-like receptors (TLR) are crucial sensors of microbial agents such as bacterial or viral compounds. These receptors constitute key players in the induction of inflammation, e.g. in septic or chronic inflammatory diseases. Colony-stimulating factors (CSFs) such as granulocyte-macrophage-CSF (GM-CSF) or granulocyte-CSF (G-CSF) have been extensively investigated in their capacity to promote myelopoiesis in febrile neutropenia or to overcome immunosuppression in patients suffering from sepsis-associated neutropenia or from monocytic immunoincompetence. We report here that GM-CSF, downregulates TLR1, TLR2 and TLR4 in a time- and dose-dependent fashion in human monocytes. Diminished pathogen recognition receptor expression was accompanied by reduced downstream p38 and extracellular-signal-regulated kinase (ERK) signaling upon lipoteichoic acid (LTA) and lipopolysaccharide (LPS) binding-and accordingly led to impaired proinflammatory cytokine production. Knockdown experiments of the transcription factors PU.1 and VentX showed that GM-CSF driven effects on TLR regulation is entirely PU.1 but not VentX dependent. We further analysed monocyte TLR and CD14 expression upon exposure to the IMID® immunomodulatory drug Pomalidomide (CC-4047), a Thalidomide analogue known to downregulate PU.1. Indeed, Pomalidomide in part reversed the GM-CSF-mediated effects. Our data indicate a critical role of PU.1 in the regulation of TLR1, 2, 4 and of CD14, thus targeting PU.1 ultimately results in TLR modulation. The PU.1 mediated immunomodulatory properties of GM-CSF should be taken into consideration upon usage of GM-CSF in inflammatory or infection-related conditions.

TLR1 expression in mouse brain was increased in a KA-induced seizure model.

OBJECTIVE: Toll-like receptors (TLRs) that mediate inflammatory responses play an important role in epilepsy; however, whether TLR1 is also involved in epileptogenesis remains unclear. Thus, in this study, we investigated the extent and pattern of TLR1 expression in epileptic tissues. METHODS: One-hundred and thirty-two mice were intra-cerebroventricularly injected with PBS or kainic acid (KA) and were examined at 1, 3, 8 and 24 h. The expression pattern and distribution of TLR1 were examined by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot analysis and immunohistochemistry staining. RESULTS: The mRNA and protein levels of TLR1 were significantly upregulated in the hippocampus and temporal cortex of epileptic mice compared with those of controls. TLR1 expression was increased as early as 1 h following KA treatment and peaked at 8 and 24 h. Immunohistochemistry staining demonstrated that TLR1 was distributed in the CA1-3, dentate gyrus and hilus regions of the hippocampus and different cortical regions. Immunofluorescent staining further revealed that TLR1 was primarily expressed in the neurons, microglia, and astrocytes of epileptogenic tissue. SIGNIFICANCE: These results demonstrate that cortical and hippocampal sub-regional expression of TLR1 is altered during epileptogenesis in a time- and location-specific manner, suggesting a close association with the process of epilepsy.

γ-Irradiated cancer cells promote tumor growth by activation of Toll-like receptor 1-mediated inducible nitric oxide synthase in macrophages.

RT is commonly used to treat malignant tumors. However, tumor regrowth is a major limitation to RT as an antitumor treatment. In the present study, we investigated the tumor-promoting effects of high-dose (or ablative) RT treatments on tumor-bearing mice. We focused on the role of macrophages that interact with IR-CCs in the TME, which cause tumor regrowth. We observed that CT26(H-2(d)) tumor growth was enhanced by i.v. injection of IR-CT26 cells compared with NR control CT26 cells. The levels of iNOS gene expression and NO production from RAW264.7 macrophages (H-2(d)) in response to the interaction with IR-CT26 cells were higher than with NR-CT26 cells. When CT26 tumor-bearing mice were treated i.v. with L-NMMA, a NOS inhibitor, the reduction in in vivo tumor growth was higher in the IR-CT26-injected group compared with the NR-CT26-injected control group. In vivo CT26 tumor growth was decreased after transplanting PEM extracted from L-NMMA-treated, tumor-bearing mice. Although iNOS activity was reduced by inhibiting TLR1 expression with TLR1-siRNA, it was enhanced by TLR1 overexpression. Transcriptional activation and protein expression levels of iNOS were also decreased in the presence of TLR1-siRNA but increased as a result of TLR1 overexpression. These results demonstrate that postradiotherapeutic tumor regrowth may be caused by interaction of IR-CCs with macrophages that induce TLR1-mediated iNOS expression and NO production. Our data suggest that iNOS in macrophages could be a useful target to regulate postradiotherapeutic responses in hosts and subsequently limit tumor regrowth.

The host response: Toll-like receptor expression in periprosthetic tissues as a biomarker for deep joint infection.

BACKGROUND: Toll-like receptors (TLRs) 1 and 6 are consistent molecular indicators of the host inflammatory response against bacterial infection. Our aims were to determine whether TLR elevation could be detected in infected periprosthetic tissues and to assess the utility of these biomarkers as tests for detecting a periprosthetic joint infection. METHODS: Fifty-nine patients undergoing revision total joint arthroplasty (twenty-seven hips and thirty-two knees) were prospectively evaluated for periprosthetic joint infection according to currently recommended diagnostic criteria. Nine patients were excluded because of insufficient work-up, leaving fifty available for study. Of these, twenty-one were categorized as infected and twenty-nine as noninfected. Periprosthetic tissues were collected intraoperatively, and total RNA was extracted by standard techniques. Expression of TLR messenger RNAs was assessed by first-strand complementary DNA synthesis from 1 µg of total RNA followed by real-time PCR (polymerase chain reaction). Results were normalized relative to the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Expression of TLRs 1, 6, and 10 in the infected and noninfected groups was compared with use of the Student t test. The receiver operating characteristic curve, area under the curve (AUC), sensitivity, specificity, positive likelihood ratio (LR+), and negative likelihood ratio (LR-) were calculated to determine the accuracy of each TLR for predicting periprosthetic joint infection at its optimal diagnostic threshold. RESULTS: Mean TLR1 mRNA expression was significantly elevated in infected compared with noninfected samples (0.600 compared with 0.005, p = 0.0003); the same was true of TLR6 (0.208 compared with 0.0165, p = 0.0059) but not of TLR10 (0.00019 compared with 0.00014, p = 0.6238). The AUC was 0.995 for TLR1, 0.883 for TLR6, and 0.546 for TLR10. The optimal threshold for diagnosing periprosthetic joint infection was 0.0924 for TLR1 (sensitivity = 95.2%, specificity = 100%, LR+ = 13.80, LR- = 0.91) and 0.0215 for TLR6 (sensitivity = 85.7%, specificity = 82.8%, LR+ = 4.98, LR- = 0.83). CONCLUSIONS: In our pilot study, TLR1 expression in periprosthetic tissues most accurately predicted periprosthetic joint infection. This measure of the host response may be particularly helpful in detecting culture-negative infections and avoiding false positives resulting from contamination. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

The oxidative stress product carboxyethylpyrrole potentiates TLR2/TLR1 inflammatory signaling in macrophages.

Oxidative stress is key in the pathogenesis of several diseases including age-related macular degeneration (AMD), atherosclerosis, diabetes, and Alzheimer's disease. It has previously been established that a lipid peroxidation product, carboxyethylpyrrole (CEP), accumulates in the retinas of AMD patients. Retinal infiltrating macrophages also accumulate in the retinas of both AMD patients and in a murine model of AMD. We therefore investigated the ability of CEP-adducts to activate innate immune signaling in murine bone-marrow derived macrophages (BMDMs). We found that CEP specifically synergizes with low-dose TLR2-agonists (but not agonists for other TLRs) to induce production of inflammatory cytokines. Moreover, CEP selectively augments TLR2/TLR1-signaling instead of TLR2/TLR6-signaling. These studies uncover a novel synergistic inflammatory relationship between an endogenously produced oxidation molecule and a pathogen-derived product, which may have implications in the AMD disease process and other oxidative stress-driven pathologies.

Toll-like receptors in human chondrocytes and osteoarthritic cartilage.

BACKGROUND AND PURPOSE: Degenerating cartilage releases potential danger signals that react with Toll-like receptor (TLR) type danger receptors. We investigated the presence and regulation of TLR1, TLR2, and TLR9 in human chondrocytes. METHODS: We studied TLR1, TLR2, TLR4, and TLR9 mRNA (qRT-PCR) and receptor proteins (by immunostaining) in primary mature healthy chondrocytes, developing chondrocytes, and degenerated chondrocytes in osteoarthritis (OA) tissue sections of different OARSI grades. Effects of a danger signal and of a pro-inflammatory cytokine on TLRs were also studied. RESULTS: In primary 2D-chondrocytes, TLR1 and TLR2 were strongly expressed. Stimulation of 2D and 3D chondrocytes with a TLR1/2-specific danger signal increased expression of TLR1 mRNA 1.3- to 1.8-fold, TLR2 mRNA 2.6- to 2.8-fold, and TNF-α mRNA 4.5- to 9-fold. On the other hand, TNF-α increased TLR1 mRNA] expression 16-fold, TLR2 mRNA expression 143- to 201-fold, and TNF-α mRNA expression 131- to 265-fold. TLR4 and TLR9 mRNA expression was not upregulated. There was a correlation between worsening of OA and increased TLR immunostaining in the superficial and middle cartilage zones, while chondrocytes assumed a CD166(×) progenitor phenotype. Correspondingly, TLR expression was high soon after differentiation of mesenchymal stem cells to chondrocytes. With maturation, it declined (TLR2, TLR9). INTERPRETATION: Mature chondrocytes express TLR1 and TLR2 and may react to cartilage matrix/chondrocyte-derived danger signals or degradation products. This leads to synthesis of pro-inflammatory cytokines, which stimulate further TLR and cytokine expression, establishing a vicious circle. This suggests that OA can act as an autoinflammatory disease and links the old mechanical wear-and-tear concept with modern biochemical views of OA. These findings suggest that the chondrocyte itself is the earliest and most important inflammatory cell in OA.

Gene expression and protein localization of TLR-1, -2, -4 and -6 in amniochorion membranes of pregnancies complicated by histologic chorioamnionitis.

OBJECTIVES: To determine whether histologic chorioamnionitis is associated with changes in gene expression of TLR-1, -2, -4 and -6, and to describe the localization of these receptors in fetal membranes. STUDY DESIGN: A total of 135 amniochorion membranes with or without histologic chorioamnionitis from preterm or term deliveries were included. Fragments of membranes were submitted to total RNA extraction. RNA was reverse transcribed and the quantification of TLRs expression measured by real time PCR. RESULTS: All amniochorion membranes expressed TLR-1 and TLR-4, whereas 99.1% of membranes expressed TLR-2 and 77.4% expressed TLR-6. TLR-1 and TLR-2 expressions were significantly higher in membranes with histologic chorioamnionitis as compared to membranes without chorioamnionitis in preterm pregnancies (p=0.003 and p<0.001, respectively). Among the membranes of term pregnancies there were no differences in the expressions of such receptors regardless of inflammatory status. Regarding TLR-4 and TLR-6 expression, there was no difference among membranes with or without histologic chorioamnionitis, regardless gestational age at delivery. TLR-1, TLR-2, TLR-4 and TLR-6 expressions were observed in amniotic epithelial, chorionic and decidual cells. CONCLUSION: Amniochorion membranes express TLR-1, TLR-2, TLR-4 and TLR-6 and increased expression of TLR-1 and TLR-2 is related to the presence of histologic chorioamnionitis in preterm pregnancies. This study provides further evidence that amniochorion membranes act as a mechanical barrier to microorganisms and as components of the innate immune system.

Impaired innate mucosal immunity in aged mice permits prolonged Streptococcus pneumoniae colonization.

Streptococcus pneumoniae is a frequent asymptomatic colonizer of the nasopharyngeal niche and only occasionally progresses toward infection. The burden of pneumococcal disease is particularly high in the elderly, and the mechanisms behind this increased susceptibility are poorly understood. Here we used a mouse model of pneumococcal carriage to study immunosenescence in the upper respiratory tract (URT). Nasal mucosa-associated lymphoid tissue (NALT) showed increased expression of Toll-like receptor 1, interleukin-1ß, NLRp3 inflammasome, and CCL2 in naive elderly compared to young animals. This suggests an increased proinflammatory expression profile in the NALT of aged mice at baseline. Simultaneously, we observed a more tolerogenic profile in respiratory epithelia of naive elderly compared to young adult mice with upregulation of the NF-κß pathway inhibitor peroxisome proliferator-activated receptor gamma (PPARγ). After nasal instillation of pneumococci, pneumococcal colonization was prolonged in elderly mice compared to in young adults. The delay in clearance was associated with absent or delayed upregulation of a proinflammatory mediator(s) in the NALT, diminished influx of macrophages into the URT niche, and absent downregulation of PPARγ in respiratory epithelium, accompanied by diminished expression of cathelicidin (CRAMP) at the site of colonization. These findings suggest that unresponsiveness to pneumococcal challenge due to altered mucosal immune regulation is the key to increased susceptibility to disease in the elderly.

Diminished responsiveness to human ß-defensin-3 and decreased TLR1 expression on monocytes and mDCs from HIV-1-infected patients.

hBD-3 is an antimicrobial peptide that may contribute to adaptive immune responses by activating professional APCs via a TLR1/2-dependent mechanism. Patients with HIV disease experience increased susceptibility to mucosal infections, which may, in part, stem from diminished APC function. Our current studies demonstrate a reduced capacity of hBD-3 to induce the expression of a costimulatory molecule, CD80, on monocytes and mDCs from HIV-infected persons compared with cells from healthy controls. Although the expression of TLR1 and TLR2 on monocytes was not a strong predictor of hBD-3 responsiveness in bivariate analyses, monocytes and mDCs from HIV-infected persons expressed significantly lower levels of TLR1. Monocyte expression of the activation marker CD69, in cells from HIV-infected persons with therapeutically controlled viremia, was correlated directly with TLR2 and TLR4 expression but not with TLR1 expression. Overall, these studies suggest that immune activation may affect TLR2 and TLR4 expression but may not fully account for reduced TLR1 expression in monocytes from HIV-infected persons. Impairments in hBD-3 responsiveness and TLR1 expression are likely to contribute to increased risk of mucosal infection in HIV disease.

Cell surface trafficking of TLR1 is differentially regulated by the chaperones PRAT4A and PRAT4B.

The subcellular localization of Toll-like receptors (TLRs) is critical to their ability to function as innate immune sensors of microbial infection. We previously reported that an I602S polymorphism of human TLR1 is associated with aberrant trafficking of the receptor to the cell surface, loss of responses to TLR1 agonists, and differential susceptibility to diseases caused by pathogenic mycobacteria. Through an extensive analysis of receptor deletion and point mutants we have discovered that position 602 resides within a short 6 amino acid cytoplasmic region that is required for TLR1 surface expression. This short trafficking motif, in conjunction with the adjacent transmembrane domain, is sufficient to direct TLR1 to the cell surface. A serine at position 602 interrupts this trafficking motif and prevents cell surface expression of TLR1. Additionally, we have found that ER-resident TLR chaperones, PRAT4A and PRAT4B, act as positive and negative regulators of TLR1 surface trafficking, respectively. Importantly, either over-expression of PRAT4A or knock-down of PRAT4B rescues cell surface expression of the TLR1 602S variant. We also report that IFN-γ treatment of primary human monocytes derived from homozygous 602S individuals rescues TLR1 cell surface trafficking and cellular responses to soluble agonists. This event appears to be mediated by PRAT4A whose expression is strongly induced in human monocytes by IFN-γ. Collectively, these results provide a mechanism for the differential trafficking of TLR1 I602S variants, and highlight the distinct roles for PRAT4A and PRAT4B in the regulation of TLR1 surface expression.

Peptidoglycan induces necrosis and regulates cytokine production in murine trophoblast stem cells.

PROBLEM: Intrauterine bacterial infection during pregnancy may lead to adverse outcome. The objective of this study was to assess whether peptidoglycan (PGN) derived from Gram-positive bacteria induces trophoblast stem (TS) cell death or alters TS cell cytokine production. METHOD OF STUDY: Toll-like receptor (TLR) transcript expression was assessed by RT-PCR. Protein expression was determined by confocal microscopy or flow cytometry. 7-Aminoactinomycin D (7-AAD) staining was used to assess TS cell death. Morphological features of cell death were evaluated by transmission electron microscopy. The presence of cleaved caspase-3 and high mobility group box 1 (HMGB1) protein was examined by Western blot. Cytokine levels in cell supernatants were determined using a mouse cytokine 23-plex panel. RESULTS: Toll-like receptor 2 and TLR4 protein was expressed from the 1-cell stage through the blastocyst stage of murine embryo development. Murine TS cells expressed TLR2 and TLR6 but not TLR1 or TLR4 RNA. Only TLR2 protein was detected at the plasma membrane of TS cells. PGN induced TS cell death by a caspase-3-independent mechanism. The cell death pathway induced by PGN was morphologically consistent with necrosis. Finally, PGN induced HMGB1 release and increased MIP-1ß secretion while inhibiting the constitutive release of RANTES. CONCLUSION: Peptidoglycan-induced TS cell necrosis and the subsequent release of HMGB1 and MIP-1ß may regulate an infection-induced inflammatory response at the maternal-fetal interface and thus may play a role in the pathogenesis of infection-associated pregnancy complications.

Comparative in vivo infection models yield insights on early host immune response to Campylobacter in chickens.

Salmonella typhimurium and Campylobacter jejuni pose significant risks to human health and poultry are a major vector for infection. Comparative in vivo infection models were performed to compare the avian host immune response to both bacterial species. Forty-five commercial broiler chickens were orally challenged with either C. jejuni or S. typhimurium whilst 60 similar control birds were mock challenged in parallel. Birds were sacrificed at 0, 6, 20 and 48 h post-infection and cloacal swabs, blood and tissue samples taken. Peripheral blood leukocytes were isolated for flow cytometric analyses and RNA was extracted for gene expression profiling. Colonisation patterns were markedly different between the two bacterial species, with systemic colonisation of Campylobacter outside the gastrointestinal tract. Salmonella infection induced significant changes in circulating heterophil and monocyte/macrophage populations, whilst Campylobacter infection had no effect on the heterophil numbers but caused a significant early increase in circulating monocytes/macrophages. Toll-like receptor 1 (TLR1) gene expression was decreased, and avian beta-defensin (AvBD) gene expression (AvBD3, AvBD10 and AvBD12) was significantly increased in response to Salmonella infection (P < 0.05). In contrast, Campylobacter infection induced increased TLR21 gene expression but significantly reduced expression of seven antimicrobial peptide (AMP) genes (AvBD3, AvBD4, AvBD8, AvBD13, AvBD14, CTHL2 and CTHL3; P < 0.05). Considered together, microbiological, cellular and gene expression profiles indicate that the innate immune system responds differently to Salmonella and to Campylobacter infection. Furthermore, reduction in the expression of AMPs may play a role in the persistence of high level colonisation of the host by Campylobacter.

The type 1 diabetes locus Idd6 controls TLR1 expression.

The Idd6 locus on mouse chromosome 6, which controls the development of type 1 diabetes in the NOD mouse, affects proliferation rates of T cells and the activity of regulatory CD4+CD25+ T cells. Using a transcriptional profiling approach, we show that splenocytes and thymocytes from diabetes-resistant Idd6 NOD.C3H-congenic mouse strains exhibit a constitutive and specific down-regulation of Toll-like receptor 1 (Tlr1) gene expression compared with diabetes prone NOD mice. This phenotype correlates with a diminished proliferation capacity of both CD4+CD25- effector and CD4+CD25+ regulatory T cells upon in vitro stimulation of the TLR1/TLR2 pathway by the ligand palmitoyl-3-cysteine-serine-lysine 4, and with the constitutive down-regulation of Tnf-alpha and IL-6 in macrophages of Idd6- congenic mice. These data suggest that TLR1 is involved in the regulation of mechanisms that impinge on diabetes development in the NOD mouse.

The polymorphism P315L of human toll-like receptor 1 impairs innate immune sensing of microbial cell wall components.

As a pattern recognition receptor, TLR1 mediates innate immune responses to a variety of microbial cell wall components including bacterial lipoproteins. We have previously shown that the central region of the extracellular domain of human TLR1, comprising leucine-rich repeat (LRR) motifs 9-12, is required for the sensing of bacterial lipopeptides. In this study, we have investigated three nonsynonymous single nucleotide polymorphisms (SNPs) located in this region of TLR1 by generating these variants and examining receptor function. We have found that a variant of TLR1 based upon the SNP P315L, located in the loop of LRR motif 11 (LRR11), is greatly impaired in mediating responses to lipopeptides and a variety of other bacterial agonists for this receptor. Despite normal cell surface expression, the P315L variant also fails to bind to GD2.F4, a commonly used anti-TLR1 mAb. Although a number of amino acid substitutions at position 315 impair receptor function, the leucine substitution has the strongest deleterious effect. GD2.F4 inhibits agonist-induced activation of TLR1, supporting a crucial role for the loop of LRR11 in receptor function. These results also suggest that the P315L SNP may predispose certain individuals to infectious diseases for which the sensing of microbial cell components by TLR1 is critical to innate immune defense.

Molecular cloning and expression of a Toll receptor gene homologue from Zhikong Scallop, Chlamys farreri.

Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors, which show homology with the Drosophila Toll protein and play key roles in detecting various non-self substances and then initiating and activating immune system. In this report, the full length of the first bivalve TLR (named as CfToll-1) is presented. CfToll-1 was originally identified as an EST (expressed sequence tag) fragment from a cDNA library of Zhikong scallop (Chlamys farreri). Its complete sequence was obtained by the construction of Genome Walker library and 5' RACE (rapid amplification of cDNA end) techniques. The full length cDNA of CfToll-1 consisted of 4308 nucleotides with a polyA tail, encoding a putative protein of 1198 amino acids with a 5' UTR (untranslated region) of 211bp and a 3'UTR of 500bp. The predicted amino acid sequence comprised an extracellular domain with a potential signal peptide, nineteen leucine-rich repeats (LRR), two LRR-C-terminal (LRRCT) motifs, and a LRR-N-terminal (LRRNT), followed by a transmembrane segment of 20 amino acids, and a cytoplasmic region of 138 amino acids containing the Toll/IL-1R domain (TIR). The deduced amino acid sequence of CfToll-1 was homologous to Drosophila melanogaster Tolls (DmTolls) with 23-35% similarity in the full length amino acids sequence and 30-54% in the TIR domain. Phylogenetic analysis of CfToll-1 with other known TLRs revealed that CfToll-1 was closely related to DmTolls. An analysis of the tissue-specific expression of the CfToll-1 gene by Real-time PCR showed that the transcripts were constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill. The temporal expressions of CfToll-1 in the mixed primary cultured haemocytes were observed after the haemocytes were treated with 1microgml(-1) and 100ngml(-1) lipopolysaccharide (LPS), respectively. The expression of CfToll-1 was up-regulated and increased about 2-fold at 6h with the treatment of 1microgml(-1) LPS. The expression of CfToll-1 was down-regulated with the treatment of 100ngml(-1) LPS. The results indicated that the expression of CfToll-1 could be regulated by LPS, and this regulation was dose-dependent.

[The expression and functionality of Toll-like receptors in human corneal epithelium and tolerated human corneal epithelial cell line].

OBJECTIVE: To investigate the expression and functionality of Toll-like receptors (TLRs) 1 - 9 in human corneal epithelium and cell line THCE (tolerated human corneal epithelial). METHODS: Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was used to determine the expression of TLR1 - 9 mRNA in the specimens of human corneal epithelial cells from 20 persons, 5 males and 10 females, aged 18 - 25, undergoing photorefractive keratectomy (PRK), and in cultured THCE cells. Human peripheral blood mononuclear cells (PBMCs) were used as positive control. The expression of TLR2 protein and TLR4 protein were detected with Western blotting. KEM-2 fluid containing anti-TLR4 antibody was used to block the THEC cells for 30 minutes, then KEM-2 fluid containing PolyI: C or LPS, ligands for TLR 3 and 4, was used to stimulate the THEC cells, 1, 4, and 8 hours after the concentrations of IL-8 in the supernatant were determined by ELISA. RESULTS: The human corneal epithelial cells strongly expressed TLR1, 2, 3, 5, 6, and 9 mRNA, weakly expressed TLR7 and 8 mRNA, and very weakly expressed TLR4 mRNA. Negative expression of TLR3, 4, 6, and 8 mRNA was seen in 1 case, a female aged 22. Weak expression of TLR5 mRNA was seen in 1 case, a female aged 20. The THCE cells showed the same expression pattern as the healthy human corneal epithelial cells, except for the strong expression of TLR9. The human PBMCs, as positive controls, strongly expressed TLR1 approximately 4 and 8 mRNA, weakly expressed TLR5, 6, and 9 mRNA, and very weakly expressed TLR7 mRNA. The human corneal epithelial cells, cultured THEC cells, and PBMCs all expressed TLR2 and 4 proteins. The concentrations of IL-8 in the supernatant of the culture fluid of THEC cells increased along with the time of stimulation of LPS and Poly: C, ligands for TLR3 and 4, and reached to 297.33 pg/ml and 229.67 pg/ml respectively 8 hours after, 10 times and 7 times those of the control group (both P < 0.05). 30 minutes after the blocking of TLR4 with anti-TLR4 antibody LPS was used to stimulate the THEC, the concentration of IL-8 was 88.54 pg/ml, being 30% that of the un-blocked group (P < 0.05). CONCLUSION: Human corneal epithelium expresses TLR1 approximately 9 at different levels. THCE cell line is an excellent line to study TLRs function.