OBJECTIVE: To analyze expression of TLR9 in human nasal epithelial cells by semi-quantitative reverse transcription-polymerase chain reaction, immunohistochemistry and flow cytometry. METHOD: Human primary nasal epithelial cells (HNECs) were cultured, and then analyze the expression of TLR9 in primary cultured HNECs measured by semi-quantitative RT-PCR and immunohistochemistry and flow cytometry. RESULT: Primary cultured HNECs were observed by 400 x optical microscopes. Round or irregular cells stick to the bottom of cell culture plates. RT-PCR showed that mRNA expressions of TLR9 were found in primary nasal epithelial cells by 1% agarose gel electrophoresis. Interestingly, by analyzing with gray level of GAPDH, expression of TLR9 mRNA in HNECs was higher than positive control PBMCs. CONCLUSION TLR9 is expressed by HNECs. Expression of TLR9 mRNA in nasal epithelial cells is higher than in PBMCs.
Cells, Cultured ; Epithelial Cells ; metabolism ; Humans ; Nasal Mucosa ; cytology ; metabolism ; RNA, Messenger ; genetics ; Toll-Like Receptor 9 ; genetics ; metabolism

Recent findings show that Toll-like receptors (TLRs) expressed in immune cells play a crucial role in the innate immune response and the subsequent induction of adaptive immune responses against microbial infection on tissue injury. Furthermore, expression of TLRs in cancer cells is associated with tumor proliferation and invasion. To explore the role of TLRs expression in cervical carcinogenesis in Uighur women, we detected the expressions of TLR3, TLR4, TLR7, and TLR9 in 25 normal cervical tissues, 64 cervical intraepithelial neoplasia (CIN) tissues, and 63 cervical squamous cell carcinoma (CSCC) tissues using immunohistochemical staining, as well as human papillomavirus type 16 (HPV16) infection using PCR. All samples used in this study were from Xinjiang Uighur women. We found the expression levels of TLR4, TLR7, and TLR9 were significantly higher in CIN and CSCC than in normal controls (P < 0.05). Up-regulation of TLR4 and TLR7 were correlated with tumor differentiation but not FIGO stage or lymph node metastasis (P > 0.05). Up-regulation of TLR9 was correlated with lymph node metastasis (P < 0.05) but not tumor differentiation or FIGO stage (P > 0.05). We also analyzed the correlation between the expressions of TLRs and HPV16 infection and found that the expressions of TLR4 and TLR9 significantly correlated with HPV16 infection in CIN (r = 7.434, P = 0.006; r = 7.123, P = 0.008) and CSCC (r = 6.423, P = 0.001; r = 8.478, P = 0.004), whereas the expression of TLR3 was not significantly different in any of the three groups and had no significant correlation with HPV16 infection. Our results suggest that high expression of TLR4, TLR7, and TLR9 may play important roles in the development and progression of CIN and CSCC in Uighur women, and the expressions of TLR4 and TLR9 can be up-regulated by HPV16 infection.
Carcinoma, Squamous Cell ; metabolism ; pathology ; virology ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; virology ; China ; ethnology ; Female ; Gene Expression Regulation, Neoplastic ; Human papillomavirus 16 ; isolation & purification ; Humans ; Lymphatic Metastasis ; Neoplasm Staging ; Papillomavirus Infections ; genetics ; pathology ; Toll-Like Receptor 3 ; metabolism ; Toll-Like Receptor 4 ; metabolism ; Toll-Like Receptor 7 ; metabolism ; Toll-Like Receptor 9 ; metabolism ; Toll-Like Receptors ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; metabolism ; pathology ; virology

Toll-like receptors (TLRs) family may play important roles in inflammatory bowel disease. This study examined the expression of TLR2, TLR4 and TLR9 in the colonic tissues of patients with ulcerative colitis (UC) and explored their roles in the pathogenesis of UC. Colonic biopsies were taken from the colon of 30 patients with mild or moderate UC (at active phase) and 10 healthy controls during colonoscopy. TLR2, TLR4 and TLR9 protein expression levels were immunohistochemically detected. The mRNA expression levels of TLR2, TLR4 and TLR9 were assessed by reverse transcription polymerase chain reaction (RT-PCR). The disease activity index (DAI), colonoscopic and histologic grades and fecal microbial flora were determined. Histological examination showed that the intestinal mucous membrane of UC patients underwent acute inflammation changes. Immunohistochemistry exhibited that the expression levels of TLR2, TLR4 and TLR9 in colon epithelia and inflammatory cells were higher in UC patients than in control group (P<0.01). The mRNA expression levels of TLR2, TLR4 and TLR9 were increased in UC patients but were not detected in the normal controls. Expression levels of TLR2, TLR4 and TLR9 were positively correlated, and bore close correlation with DAI, colonoscopic and histologic grades and fecal microbial flora. An important mechanism of UC might be that abnormal activation of mucosal immunity by intestinal dysbacteriosis caused dysregulation of TLRS that mediates innate immunity.
Colitis, Ulcerative ; genetics ; metabolism ; pathology ; Colon ; metabolism ; microbiology ; Colonoscopy ; Feces ; microbiology ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; Intestinal Mucosa ; metabolism ; microbiology ; Male ; Reverse Transcriptase Polymerase Chain Reaction ; Severity of Illness Index ; Toll-Like Receptor 2 ; biosynthesis ; genetics ; Toll-Like Receptor 4 ; biosynthesis ; genetics ; Toll-Like Receptor 9 ; biosynthesis ; genetics

OBJECTIVETo investigate the effects of tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on the expression of pattern recognition receptors (PRRs) on the surface of mouse alveolar macrophages.

METHODSAlveolar macrophages from mouse were cultured in DMEM supplemented with 10% (V/V) endotoxin-free calf serum. After the alveolar macrophages were stimulated with TNF alpha and IFN gamma (concentration, 20 ng/ml) for 3 h, 6 h and 12 h, the expression of PRRs, including cluster of differentiation 14 (CD14), scavenger receptor (SR), toll-like receptor 4 (TLR4), TLR2 and TLR9 mRNA and proteins were examined by RT-PCR and immunohistochemistry.

RESULTSThe expressions of CD14, TLR2 and TLR9 receptors, which were related with cellular activation, were up-regulated by the stimulation of TNF alpha and IFN gamma (P < 0.05), while SR, which was related with cellular defense action, was down-regulated (P < 0.05). Although the expression of TLR4 was up-regulated, there was no statistical significance (P > 0.05).

CONCLUSIONSThe cytokines such as TNF alpha and IFN gamma could also produce feedback regulation on the expression of PRRs at the levels of genes and proteins. Such regulation on the PRRs expression would be significant for further amplification of inflammation cascade and eventually leading to uncontrolled inflammation.

Animals ; Cells, Cultured ; Interferon-gamma ; pharmacology ; Lipopolysaccharide Receptors ; biosynthesis ; genetics ; Macrophages, Alveolar ; metabolism ; Mice ; RNA, Messenger ; genetics ; Receptors, Pattern Recognition ; biosynthesis ; Toll-Like Receptor 2 ; biosynthesis ; genetics ; Toll-Like Receptor 4 ; biosynthesis ; genetics ; Toll-Like Receptor 9 ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; pharmacology

Dendritic cells (DCs) comprise two functionally distinct subsets: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). pDCs are specialized in rapid and massive secretion of type I interferon (IFN-I) in response to nucleic acids through Toll like receptor (TLR)-7 or TLR-9. In this report, we characterized a CD56(+) DC population that express typical pDC markers including CD123 and BDCA2 but produce much less IFN-I comparing with pDCs. In addition, CD56(+) DCs cluster together with mDCs but not pDCs by genome-wide transcriptional profiling. Accordingly, CD56(+) DCs functionally resemble mDCs by producing IL-12 upon TLR4 stimulation and priming naïve T cells without prior activation. These data suggest that the CD56(+) DCs represent a novel mDC subset mixed with some pDC features. A CD4(+)CD56(+) hematological malignancy was classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN) due to its expression of characteristic molecules of pDCs. However, we demonstrated that BPDCN is closer to CD56(+) DCs than pDCs by global gene-expression profiling. Thus, we propose that the CD4(+)CD56(+) neoplasm may be a tumor counterpart of CD56(+) mDCs but not pDCs.
Biomarkers ; metabolism ; CD56 Antigen ; genetics ; immunology ; Cell Lineage ; genetics ; immunology ; Dendritic Cells ; immunology ; metabolism ; pathology ; Gene Expression ; Hematologic Neoplasms ; genetics ; immunology ; pathology ; Humans ; Immunophenotyping ; Interferon Type I ; biosynthesis ; metabolism ; Interleukin-12 ; biosynthesis ; metabolism ; Interleukin-3 Receptor alpha Subunit ; genetics ; immunology ; Lectins, C-Type ; genetics ; immunology ; Membrane Glycoproteins ; genetics ; immunology ; Myeloid Cells ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; genetics ; immunology ; Terminology as Topic ; Toll-Like Receptor 4 ; genetics ; immunology ; Toll-Like Receptor 7 ; genetics ; immunology ; Toll-Like Receptor 9 ; genetics ; immunology

OBJECTIVE: To determine the role of Toll-like receptor 9 (TLR9) in synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG-ODN)-induced species-specific immune responses. METHODS: CpG-ODN was co-cultured with peripheral blood mononuclear cells (PBMC) of humans, macaques and mice. The IFN-alpha in the supernatant was measured by ELISA. The reverse transcription PCR was used to analyze the expression levels of TLR9 mRNA in PBMC. RESULTS: CpG-ODN induced high amounts of IFN-alpha in human PBMC, but had no effect on macaques and mice. The expression of TLR9 mRNA was observed in all human PBMC, and the levels of TLR9 mRNA were significantly up-regulated with the stimulation of CpG-ODN. We did not observe any expression of TLR9 mRNA in PBMC in macaques. CONCLUSION TLR9 underlies the molecular foundation of CpG-ODN-induced species specificity.
Animals ; Humans ; Interferon-alpha ; biosynthesis ; Leukocytes, Mononuclear ; cytology ; immunology ; metabolism ; Macaca mulatta ; Mice ; Oligodeoxyribonucleotides ; pharmacology ; Species Specificity ; Toll-Like Receptor 9 ; biosynthesis ; genetics

The aim of this study was to investigate the influence of opiate abuse on the expression of Toll-like receptor 9 (TLR9) in the peripheral blood mononuclear cells (PBMCs) of HIV-1-infected patients and to elucidate possible mechanisms involved in the enhancement of HIV-1 replication by opiate abuse. A total of 200 participants were enrolled in the study by random selection from methadone treatment centers and voluntary HIV counseling and testing centers in the cities of Nanning, Liuzhou, and Qinzhou. These participants included 50 HIV-positive opiate abusers (Opiates HIV(+) group), 50 HIV-negative opiate abusers (Opiates HIV(-) group), 50 HIV-positive subjects who were not opiate abusers (Non-opiates HIV (+) group), and 50 HIV-negative subjects who were not opiate abusers (Control group). PBMCs were isolated from the peripheral blood samples from the subjects and the expression levels of TLR9 mRNA and protein were determined by q-PCR and western blot respectively. There was no significant difference among the four groups in age, gender, nationality, domicile, marital status, educational background or duration of drug abuse (P > 0.05). The median viral loads of the Opiates HIV(+) were significantly higher than those of the Non-Opiates HIV(+) groups (4.450 x 10(3) and 3.977 x 10(3) copies/mL respectively, P < 0.05). The relative expression levels of TLR9 mRNA in the Opiates HIV(+), Non-Opiates HIV(+), Opiates HIV(-) and Control groups were (2.13 +/- 1.59) x 10(-3), (3.66 +/- 2.22) x 10(-3), (1.96 +/- 1.42) x 10(-3) and (7.66 +/- 4.87) x 10(-3), respectively. The expression of TLR9 mRNA was significantly lower in both HIV-1-infected and -uninfected groups of opiate abusers compared with groups of non-abusers (P < 0.05). There was no significant difference in TLR9 mRNA expression levels between the Opiates HIV(+) group and the Opiates HIV(-) group (P > 0.05). However, in the non-opiate groups, the expression levels of TLR9 mRNA in the HIV(+) group were significantly lower than that of the control group (P< 0.05). Western blot results confirmed that the expression of TLR9 protein was lower in the Opiates HIV(+), Non-Opiates HIV(+), and Opiates HIV(-) groups compared to the control group. These results suggest that opiate abuse can decrease the expression of TLR9 in PBMCs, which may result in the enhancement of HIV-1 infection and replication due to a decline in immune response mediated by the TLR9 pathway.
Adolescent ; Adult ; Female ; HIV Infections ; genetics ; metabolism ; virology ; HIV-1 ; physiology ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Middle Aged ; Opioid-Related Disorders ; genetics ; metabolism ; Toll-Like Receptor 9 ; genetics ; metabolism ; Young Adult

PURPOSE: The -1237T/C polymorphism of the Toll-like receptor 9 (TLR9) gene has been implicated in the susceptibility of inflammatory bowel diseases (IBDs), but the results remain conflicting. We further investigated this association via meta-analysis. MATERIALS AND METHODS: Multiple electronic databases were extensively searched until February, 2015. The strength of association was evaluated by calculating the pooled odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: A total of 2987 cases and 2388 controls from eight studies were analyzed. Overall, association was found between TLR9 -1237T/C polymorphism and the risk of IBDs when all the studies were pooled (recessive model, OR: 1.59, 95% CI: 1.02-2.47, p=0.04; homozygote comparison, OR: 1.62, 95% CI: 1.04-2.52, p=0.03; allele model, OR: 1.13, 95% CI: 1.00-1.27, p=0.05). Stratification by ethnicity indicated an association between TLR9 -1237T/C polymorphism and IBDs risk in Caucasians (recessive model, OR: 1.59, 95% CI: 1.02-2.47, p=0.04; homozygote comparison, OR: 1.62, 95% CI: 1.04-2.52, p=0.03; allele model, OR: 1.12, 95% CI: 1.00-1.27, p=0.05). When stratified by disease type, significant correlation were only found in the Crohn's disease subgroup (recessive model, OR: 1.69, 95% CI: 1.05-2.73, p=0.03; homozygote model, OR: 1.74, 95% CI: 1.07-2.82, p=0.02; allele model, OR: 1.15, 95% CI: 1.01-1.32, p=0.04). CONCLUSION: The present study suggested that the TLR9 -1237T/C polymorphism might act as a risk factor in the development of IBDs, particularly in Caucasians.
Alleles ; European Continental Ancestry Group/genetics ; Genetic Predisposition to Disease/*genetics ; Homozygote ; Humans ; Inflammatory Bowel Diseases/ethnology/*genetics ; Odds Ratio ; Polymorphism, Genetic/*genetics ; Risk Factors ; Toll-Like Receptor 9/*genetics/metabolism

This study was purposed to investigate the effect of high-dose dexamethasone (DXM) on function and Toll like receptor 9 (TLR-9) expression of plasmacytoid dendritic cells (pDC) in peripheral blood of patients with immune thrombocytopenic purpura (ITP). 15 newly diagnosed patients with ITP received high dose DXM at single daily doses of 40 mg for 4 consecutive days. The peripheral blood plasmacytoid dendritic cells from 13 remission patients and 15 normal controls were separated by immunomagnetic beads and then induced by CpG-OND2216. 24 h later, the levels of IFN-α, IL-6 and TNF-α in the supernatant were detected by enzyme linked immunosorbent assay (ELISA). The expression of TLR9 mRNA of pDC was detected by real-time quantitative PCR. The results indicated that the levels of IFN-α, IL-6 and TNF-α produced by pDC in ITP patients were significantly higher than those in normal controls (P < 0.05). After high dose DXM treatment, the levels of IFN-α, IL-6 and TNF-α decreased without significant difference compared with normal controls (P > 0.05). The expression of TLR9 mRNA in pDC of untreated patients was significantly higher than that in control group (P < 0.05), and significantly reduced after treatment without difference from that in control group (P > 0.05). It is concluded that pDC may play an important role in ITP by their TLR9 and secreted cytokines; dexamethasone may down regulate the expression of TLR9, inhibit pDC function, and thus play a therapeutic role.
Adolescent ; Adult ; Case-Control Studies ; Dendritic Cells ; immunology ; metabolism ; Dexamethasone ; administration & dosage ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; blood ; drug therapy ; immunology ; RNA, Messenger ; genetics ; Toll-Like Receptor 9 ; genetics ; metabolism ; Young Adult

Synthetic oligodeoxynucleotides (ODN) with a CpG-motif are recognized by Toll-like receptor 9 (TLR9) and pleiotropic immune responses are elicited. Stimulation of macrophages with TLR9 agonist prevented apoptosis induced by serum deprivation through increased expression of FLICE-like inhibitory protein (FLIP). CpG ODN-mediated anti-apoptosis depended on the TLR9-Akt-FoxO3a signaling pathway. Inhibition of TLR9 by small interfering (si) RNA or an inhibitor suppressed CpG ODN-mediated anti-apoptosis. Analysis of signaling pathways revealed that the anti-apoptotic effect of CpG ODN required phosphorylation of FoxO3a and its translocation from the nucleus to the cytosol. Overexpression of FoxO3a increased apoptosis induced by serum deprivation and CpG ODN blocked these effects through FLIP expression. In contrast, siRNA knock-down of FoxO3a decreased apoptosis by serum deprivation. In addition, Akt activation was involved in CpG ODN-induced phosphorylation of FoxO3a, expression of FLIP, and anti-apoptosis. Taken together, these results demonstrate the involvement of Akt-FoxO3a in TLR9-mediated anti-apoptosis and indicate that FoxO3a is a distinct regulator for FLIP expression.
Animals ; *Apoptosis ; CASP8 and FADD-Like Apoptosis Regulating Protein/*genetics/metabolism ; Cells, Cultured ; Forkhead Transcription Factors/genetics/*metabolism ; Macrophages/metabolism ; Mice ; Mice, Inbred C57BL ; Oligodeoxyribonucleotides/metabolism ; Oncogene Protein v-akt/metabolism ; RNA, Small Interfering/metabolism ; Signal Transduction ; Toll-Like Receptor 9/genetics/*metabolism