Toll-like Receptor 9 Expression Levels Research Articles

CpG ODN 1668 as TLR9 agonist mediates humpback grouper (Cromileptes altivelis) antibacterial immune responses.

Cromileptes altivelis (humpback grouper) is the main farmed species in the southern coastal area of China owing to its important economic value. Toll-like receptor 9 (TLR9) belongs to the toll-like receptor (TLR) family and functions as a pattern recognition receptor, recognising unmethylated oligodeoxynucleotides containing the CpG motif (CpG ODNs) in bacterial and viral genomes, thereby activating host immune response. In this study, the C. altivelis TLR9 (CaTLR9) ligand CpG ODN 1668 was screened and found to significantly enhance the antibacterial immunity of humpback grouper in vivo and head kidney lymphocytes (HKLs) in vitro. In addition, CpG ODN 1668 also promoted the cell proliferation and immune gene expression of HKLs and strengthened the phagocytosis activity of head kidney macrophages. However, when the CaTLR9 expression was knocked down in the humpback group, the expression levels of TLR9, myeloid differentiation factor 88 (Myd88), tumour necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-1β (IL-1β), IL-6, and IL-8 were significantly reduced, and the antibacterial immune effects induced by CpG ODN 1668 were mostly abolished. Therefore, CpG ODN 1668 induced antibacterial immune responses in a CaTLR9-dependent pathway. These results enhance the knowledge of the antibacterial immunity of fish TLR signalling pathways and have important implications for exploring natural antibacterial molecules in fish.

CpG ODN 2102 promotes antibacterial immune responses and enhances vaccine-induced protection in golden pompano (Trachinotusovatus).

CpG oligodeoxynucleotides (ODNs) are oligodeoxynucleotides containing CpG motifs and can be recognized by toll-like receptor 9 (TLR9), activating the host's immune responses. In this study, ten different CpG ODNs were designed and synthesized to study the antibacterial immune responses of CpG ODNs in golden pompano (Trachinotus ovatus). Results showed that CpG ODN 2102 significantly improved the immunity of golden pompano against bacteria. Besides, CpG ODN 2102 promoted the proliferation of head kidney lymphocytes and activated the head kidney macrophages. When TLR9-specific small interfering RNA (siRNA) was used to interfere with TLR9 expression, the immune responses were decreased. Moreover, the expression levels of myeloid differentiation primary response 88 (Myd88), p65, tumor necrosis factor receptor-associated factor 6 (TRAF6), and tumor necrosis factor-alpha (TNF-α) in the TLR9-knockdown golden pompano kidney (GPK) cells were significantly reduced. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) promoter activity of the TLR9-knockdown GPK cells was also significantly reduced. In vivo, the antibacterial immune effects induced by CpG ODN 2102 in golden pompano were mostly abolished when TLR9 expression was knocked down. These results suggested that TLR9 was involved in the immune responses induced by CpG ODN 2102. CpG ODN 2102 also enhanced the protective effect of the Vibrio harveyi vaccine pCTssJ, where the survival rate of golden pompano was significantly improved by 20%. In addition, CpG ODN 2102 enhanced the messenger RNA (mRNA) expression levels of TLR9, Myxovirus resistance (Mx), interferon γ (IFN-γ), TNF-α, interleukin (IL)-1β, IL-8, major histocompatibility complex class (MHC) Iα, MHC IIα, Immunoglobulin D (IgD), and IgM. Therefore, TLR9 was involved in the antibacterial immune responses induced by CpG ODN 2102 and CpG ODN 2102 possessed adjuvant immune effects. These results enlarged our knowledge of the antibacterial immunity of fish TLRs signaling pathway and had important implications for exploring natural antibacterial molecules in fish and developing new vaccine adjuvants.

TLR9 induces colitis-associated colorectal carcinogenesis by regulating NF-κB expression levels.

Chronic colorectal inflammation has been associated with colorectal cancer (CRC); however, its exact molecular mechanisms remain unclear. The present study aimed to investigate the effect of Toll-like receptor 9 (TLR9) on the development of colitis-associated CRC (CAC) through its regulation of the NF-κB signaling pathway. By using a CAC mouse model and immunohistochemistry, the present study discovered that the protein expression levels of TLR9 were gradually upregulated during the development of CRC. In addition, the expression levels of TLR9 were revealed to be positively correlated with NF-κB and Ki67 expression levels. In vitro, inhibiting TLR9 expression levels using chloroquine decreased the cell viability, proliferation and migration of the CRC cell line HT29, and further experiments indicated that this may occur through downregulating the expression levels of NF-κB, proliferating cell nuclear antigen and Bcl-xl. In conclusion, the findings of the present study suggested that TLR9 may serve an important role in the development of CAC by regulating NF-κB signaling.

Toll-like receptor-9 (TLR-9) deficiency alleviates optic nerve injury (ONI) by inhibiting inflammatory response in vivo and in vitro

Traumatic optic neuropathy is a common clinical problem. Damage to the optic nerve leads to shear stress and triggers secondary swelling within the optic canal. The study aims to explore the role of the inflammatory response following optic nerve injury (ONI) in toll-like receptor-9 knockout mice (TLR-9-/-) compared to wild-type mice (WT). At first, TLR-9-/- and WT mice were subjected to ONI. We then found that ONI significantly up-regulated TLR-9 expression levels in retinal tissues of WT mice. The retinal degeneration after ONI was alleviated in TLR-9-/- mice, as evidenced by the increased number of retinal ganglion cells (RGCs) and thickness of inner retinal layer (IRL). TUNEL staining and immunofluorescence staining of BRN3A indicated that TLR-9 knockout effectively improved the survival of RGCs. ONI-enhanced expression of Iba-1 and TMEM119 was markedly reduced in TLR-9-/- mice, indicating the suppression of microglial activation. Moreover, production of pro-inflammatory regulators, including inducible nitric oxide synthase (iNOS), macrophage chemo-attractant protein (MCP)-1, cyclooxygenase-2 (COX-2), interleukin (IL)-1β, IL-18 and tumor necrosis factor-α (TNF-α), was significantly decreased in TLR-9-/- mice following ONI. TLR-9 knockout-attenuated inflammation was mainly through repressing myeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase 4 (IRAK4). Furthermore, ONI greatly up-regulated the protein expression levels of phosphorylated (p)-IKKα, p-IκBα and p-nuclear factor (NF)-κB, whereas being repressed in TLR-9-/- mice. The effects of TLR-9 on ONI were verified in lipopolysaccharide (LPS)-stimulated retinal microglial cells transfected with small interfering RNA TLR-9 (siTLR-9). As expected, promoting TLR-9 with its agonist markedly restored inflammation in TLR-9 knockdown cells stimulated by LPS. Therefore, all findings above suggested that suppressing TLR-9 showed neuroprotective effects against ONI through reducing inflammatory response, and TILR-9 might be a promising therapeutic target to develop effective strategies for the treatment of optic neuropathies.

Investigating the TLR9 mRNA Expression Level in Different Histological Types of Colorectal Polyps.

Toll-like receptor 9 (TLR9) is a cellular DNA receptor of the innate immune system which plays a pivotal role in inflammatory response. Recently, changing expression levels of TLR9 has been observed in a wide range of cancer cells; however, there is little information about colorectal polyps. Herein, we assessed the mRNA expression of TLR9 in different colorectal polyp types compared to normal group in order to investigate its expression level during CRC initiation. Fifty-four biopsy samples from colorectal polyp patients and from 20 healthy subjects were collected. The mucosal mRNA expression level of TLR9 gene was identified by real time PCR. Fold change of gene expression was evaluated by 2-ΔΔct method. There was a significant relationship between the lower expression of TLR9 gene in the polyp cases compared to normal individuals (P value = 0.0005), Also, decreased TLR9 mRNA expression was obtained in adenomas in contrast to hyperplastic and normal groups (P value = 0.0008). Based on the current results, we hypothesized that aberrant surface expression of TLR9 on tumor cells may promote the growth and invasion of colorectal polyps. Further, TLR9 modulation may have an important impact on the development of novel therapeutic strategies.

Photodynamic Therapy for Genital Warts Causes Activation of Local Immunity.

5-aminolevulinic acid photodynamic therapy (PDT) for genital warts is effective, safe, and can prevent recurrence. It is believed that PDT can induce immune responses, but the mechanism is not completely understood. The objectives of this article are to confirm the effect of PDT for genital warts on local immunity and to investigate the recruitment and significance of immune cells in tissues. Local immune changes in T lymphocytes (CD3+, CD4+, CD8+), plasmacytoid dendritic cells (pDCs) (CD123+), and myeloid dendritic cells (CD1a+) after PDT in patients were evaluated by immunohistochemistry staining. Changes in mRNA levels of IFN-γ, IFN-α, IFN-ß, interferon-stimulated gene 15 kDa (ISG-15), Mx2, Toll-like receptor 9 (TLR9), and interferon regulatory factor 7 (IRF7) were analyzed by real-time quantitative polymerase chain reaction. At 4 hours after PDT, CD4+ increased, accompanied by increased levels of mRNA expression of IFN-γ, but CD4+ and mRNA expression levels of IFN-γ were decreased at 24 hours after PDT. CD123+ pDCs showed an increasing trend. CD1a+ LCs in the epidermis gradually decreased, and DCs in the epidermis gradually increased. CD3+ infiltrated and migrated to the superficial dermis, but CD8+ did not change significantly after PDT. The mRNA expression levels of IFN-α, IFN-ß, ISG-15, Mx2, TLR9, and IRF7 showed an increasing trend after PDT. As compared with the patients without significantly increased IFN-α and IFN-ß after PDT sessions, patients with significant increases needed fewer sessions of PDT for remission. PDT for genital warts can activate T lymphocyte-mediated, DC-related, and pDC-related immunity. The clinical efficacy of PDT for genital warts may be related to the increased levels of IFN-α and IFN-ß after treatment.

Impact of ketamine intervention for acute lung injury on RAGE and TLR9.

The purpose of this study was to explore the benefits of ketamine intervention for acute lung injury (ALI) and its effects on the receptor for advanced glycation end-product (RAGE) and toll-like receptor 9 (TLR9). Lipopolysaccharide (LPS, 3 mg/kg) was used to induce ALI rat model. Forty healthy Sprague-Dawley rats (6-8 weeks) were assigned into control, model, low ketamine (5 mg/kg), and high ketamine (50 mg/kg) groups. After 24 h, these rats were sacrificed and lungs were collected. The pathological score, lung W/D ratio, the percentage of leukocytes and epithelial in bronchoalveolar lavage fluids (BALF), the expression levels of RAGE, TLR9, and other inflammation markers in serum and lungs were significantly higher in the Model group, indicating a good ALI model. Ketamine intervention restored all these parameters, with more benefits in the High dose group. The high dose ketamine decreased the degree of ALI by inhibiting the expression of RAGE, TLR9, TNF-α, NF-κB, IL-6 and MPO in tissues.

Intrauterine growth restriction increases circulating mitochondrial DNA and Toll-like receptor 9 expression in adult offspring: could aerobic training counteract these adaptations?

It has been demonstrated that intrauterine growth restriction (IUGR) can program increase cardiometabolic risk. There are also evidences of the correlation between IUGR with low-grade inflammation and, thus can contribute to development of several cardiometabolic comorbidities. Therefore, we investigated the influence of IUGR on circulating mitochondrial DNA (mtDNA)/Toll-like receptor 9 (TLR9) and TNF-α expression in adult offspring. Considering that the aerobic training has anti-inflammatory actions, we also investigated whether aerobic training would improve these inflammatory factors. Pregnant Wistar rats received ad libitum or 50% of ad libitum diet throughout gestation. At 8 weeks of age, male offspring from both groups were randomly assigned to control, trained control, restricted and trained restricted. Aerobic training protocol was performed on a treadmill and after that, we evaluated circulating mtDNA, cardiac protein expression of TLR9, plasma and cardiac TNF-α levels, and left ventricle (LV) mass. We found that IUGR promoted an increase in the circulating mtDNA, TLR9 expression and plasma TNF-α levels. Further, our results revealed that aerobic training can restore mtDNA/TLR9 content and plasma levels of TNF-α among restricted rats. The cardiac TNF-α content and LV mass were not influenced either by IUGR or aerobic training. In conclusion, IUGR can program mtDNA/TLR9 content, which may lead to high levels of TNF-α. However, aerobic training was able to normalize these alterations. These findings evidenced that the association of IUGR and aerobic training seems to exert an important interaction effect regarding pro-inflammatory condition and, aerobic training may be used as a strategy to reduce deleterious adaptations in IUGR offspring.

Therapeutic effect of neonatal mouse Cytomegalovirus hepatitis by CpG Oligodeoxynucleotide 2395

Objective To study the treatment effects of newborn mice murine Cytomegalovirus (MCMV)hepatitis by using CpG Oligodeoxynucleotide 2395(CpG ODN2395). Methods Specific pathogen-free BALB/c newborn mice were divided into 3 groups according to the broods randomly: control group, virus group and treatment group.In control group 9 g/L sodium chloride 20×10-6 L was intraperitoneally injected every other day.In virus group MCMV(TCID50=108.31/L) 20×10-6 L was intraperitoneally injected once and 9 g/L sodium chloride solution 20×10-6 L was intraperitoneally injected every other day.In treatment group murine MCMV(TCID50=108.31/L) 20×10-6 L was intraperitoneally injected once and from the first day CpG ODN2395 20 mg/kg was intraperitoneally injected every other day.Growths and development of mice were observed.Murine body weights were measured.Pathology of livers was observed by means of hematoxylin and eosin stain.The levels of serum ALT and IFN-α were measured by adopting enzyme linked immunosorbent assay.MCMV loads in liver were measured by way of polymerase chain reaction.The expression levels of IFN-α mRNA in liver were detected by using reverse transcription polymerase chain reaction.The expression levels of Toll-like receptor 9(TLR9) and myeloid cell differentiation factor 88(MyD88) in liver were detected by adopting Western blot.The results were analyzed by using SPSS 16.0 statistics software. Results 1.Compared with control group and treatment group, growth and development of virus group mice fell behind and on day 7, 14 body weights were lowest(F=18.919, 25.543, all P<0.05). Growth and development of treatment group mice were between control group and virus group.Body weights of treatment group mice were lower than those of control group, and there was statistical difference on day 7(t=3.187, P<0.05).2.Compared with control group and treatment group, the levels of ALT in virus group was highest.ALT levels of treatment group were higher than those of control group and treatment group(F=11.407, 11.791, 154.656, all P<0.05).3.The pathologic change of liver tissue: the HAI of virus group reached the peak on day 3, then decreased gradually.The HAI of treatment group also reached the peak on day 3, but liver damage was obviously less than those of virus group.The liver damage also relieved gradually and the mean of HAI was obviously lower than that of virus group on day 7 and 14.4.MCMV DNA in liver was negative at control group.The magnitude of viral loading in livers of virus group was higher than that of treatment group.5.The levels of IFN-α in treatment group and virus group reached a peak on day 3 and declined gradually on day 7, 14.The levels of IFN-α on treatment group were higher than that of virus group and control group.The levels of IFN-α virus group were higher than those of control group, but had no statistical difference.6.The mRNA expression of IFN-α in livers of treatment group and virus group began to increase on day 3 and reached a peak on day 7, and declined on day 14.The mRNA expression of IFN-α was higher than that of virus group and control group.The mRNA expression of virus group was higher than that of control group.7.The expressions of TLR9 and MyD88 in livers of treatment group were higher than that of virus group and control group.The expressions of TLR9 and MyD88 of virus group were higher than those of control group. Conclusions CpG ODN2395 can obviously improved liver function and histopathological lesions and reduce MCMV DNA load within liver tissues as well.CpG ODN2395 can improve the expression levels of TLR9 in liver and activate secretion interferon alpha by MyD88-dependent pathway, which were likely to play an important role in its treatment of murine CMV infection. Key words: Cytomegalovirus; CpG Oligodeoxynucleotide 2395; Hepatitis; Mouse

Chloroquine has tumor-inhibitory and tumor-promoting effects in triple-negative breast cancer

Toll-like receptor-9 (TLR9) is an intracellular DNA receptor that is widely expressed in breast and other cancers. We previously demonstrated that low tumor TLR9 expression upon diagnosis is associated with significantly shortened disease-specific survival times in patients with triple-negative breast cancer (TNBC). There are no targeted therapies for this subgroup of patients whose prognosis is among the worst in breast cancer. Due to the previously detected in vitro anti-invasive effects of chloroquine in these cell lines, the present study aimed to investigate the in vivo effects of chloroquine against two clinical subtypes of TNBC that differ in TLR9 expression. Chloroquine suppressed matrix metalloproteinase (MMP)-2 and MMP-9 mRNA expression and protein activity, whereas MMP-13 mRNA expression and proteolytic activity were increased. Despite enhancing TLR9 mRNA expression, chloroquine suppressed TLR9 protein expression in vitro. Daily treatment of mice with intraperitoneal (i.p.) chloroquine (80 mg/kg/day) for 22 days, did not inhibit the growth of control siRNA or TLR9 siRNA MDA-MB-231 breast cancer cells. In conclusion, despite the favorable in vitro effects on TNBC invasion and viability, particularly in hypoxic conditions, chloroquine does not prevent the growth of the triple-negative MDA-MB-231 cells with high or low TLR9 expression levels in vivo. This may be explained by the activating effects of chloroquine on MMP-13 expression or by the fact that chloroquine, by suppressing TLR9 expression, permits the activation of currently unknown molecular pathways, which allow the aggressive behavior of TNBC cells with low TLR9 expression in hypoxia.

Expression of Toll-like Receptor 9 in Cervical Intraepithelial Neoplasia from Mexican Women

Introduction: Cervical cancer is a major health problem, is the second leading cause of cancer death in women worldwide and the first in developing countries. Toll-like receptors play an important role in the immune system through the recognition of pathogen-associated molecular patterns and damage associated with endogenous molecules, so that the role of Toll-like receptors is being explored in cancer. Objective: To evaluate the expression of Toll-like receptor 9 (TLR9) in cervical intraepithelial neoplasia grade I, II and III tissues using immunohistochemical staining. Material and Methods: Retrospective, observational and descriptive study, the expression of TLR9 incervical intraepithelial neoplasia grade I (n=124), II (n=68) and III (n=29) tissues were evaluated using immunohistochemical staining. All samples used in this study were from Mexican women. Results: We found the expression levels of TLR9 was higher in CIN I (p<0.001) and CIN-II (p<0.001) than in CIN-III. Conclusion: The low expression of TLR9 may play important roles in the development and progression of cervical intraepithelial neoplasia in Mexican women.

Tonsillar TLR9 expression and efficacy of tonsillectomy with steroid pulse therapy in IgA nephropathy patients

Patients with IgA nephropathy (IgAN) often show aggravation of renal injury with macroscopic hematuria after mucosal infections, especially tonsillitis. We previously demonstrated the important role of mucosal Toll-like receptor 9 (TLR9) activation in the pathogenesis of murine IgAN. Moreover, a single nucleotide polymorphism (SNP) in TLR9 was significantly associated with pathological severity in human IgAN. In this study, we investigated correlations between tonsillar TLR9 messenger RNA expression, TLR9 SNP genotypes and clinical outcomes following tonsillectomy with steroid pulse therapy (SPT) in IgAN patients. Tonsillar TLR9 expression was examined in IgAN (n = 49) and control (n = 15) patients who had undergone tonsillectomy. The correlations between tonsillar TLR9 expression level, TLR9 SNP genotypes and clinical outcomes after tonsillectomy with SPT were examined. High expression of tonsillar TLR9 was observed in ∼23% of IgAN patients. These patients showed stronger and earlier remission of hematuria and proteinuria than those with low TLR9 expression. Patients with the TT genotype of TLR9 SNP (rs352140) had more severe renal damage than those with other genotypes. Patients whose serum IgA level decreased more than average after tonsillectomy (large ΔIgA) showed higher cumulative remission rates of proteinuria than patients with a smaller decrease in these levels (small ΔIgA). CT/CC genotypes were more dominant and tonsillar TLR9 expressions significantly higher in large ΔIgA patients than in small ΔIgA patients. In IgAN patients, expression levels of tonsillar TLR9 and TLR9 SNP were well correlated with the efficacy of tonsillectomy with SPT.

Abstract 615: Novel Toll-like Receptor 9 (TLR9) agonist IMO inhibits tumor growth and cooperates with cetuximab in K-Ras mutant colon and pancreatic cancers

Abstract Background. Somatic mutations in the K-Ras gene, leading to constitutive activation of the Ras/MAPK signalling cascade, represent a strong predictive biomarker for resistance to Epidermal Growth Factor Receptor (EGFR) inhibitors in colorectal and pancreatic cancer patients. We previously demonstrated that a novel Toll-like Receptor 9 (TLR9) agonist currently under clinical development, IMO, has a strong in vivo activity in colorectal cancer models interfering with EGFR-related signalling, synergizing with the anti-EGFR monoclonal antibody cetuximab and boosting its ADCC activity. Methods. In this study, we investigated IMO antitumor effect in colon and pancreatic cancer models with resistance to EGFR inhibitors due to K-Ras mutations. We evaluated the in vitro activity of IMO, alone or in combination with cetuximab, on growth, survival and EGFR-dependent signal transduction of cetuximab-sensitive and cetuximab-resistant colon and pancreatic cancer cells. We also investigated the antitumor effect of the combination IMO plus cetuximab on in vivo growth of orthotopically injected pancreatic cancer models. Results. We verified that K-Ras mutant colon and pancreatic cancer cell lines exhibit lower sensitivity to the anti-EGFR drugs cetuximab and erlotinib compared to K-Ras wild-type cell lines. IMO moderately inhibits in vitro growth of colon and pancreatic cancer cells, regardless of both TLR9 expression levels and K-Ras status. Surprisingly, IMO is able to partially restore cetuximab sensitivity of K-Ras mutant cancer cells. In fact, a strong inhibition of cell survival and a total suppression of MAPK phosphorylation/activation was observed with the combination IMO plus cetuximab in resistant cancer cells. IMO capability to interfere with EGFR-dependent signalling in K-Ras mutant, cetuximab-resistant cells seems to be related to modulation of a functional interaction between TLR9 and EGFR occurring at membrane level. The antitumor effect of the combination IMO plus cetuximab was confirmed also in vivo. In the luciferase- tagged AsPC1 (K-Rasmut) pancreatic cancer orthotopic model, IMO plus cetuximab causes a strong reduction of tumor burden and a significant increase in mice survival as compared to single agent treatments. Conclusion. We demonstrated for the first time that the TLR9 agonist IMO is effective in K-Ras mutant colon and pancreatic cancers, strongly inhibiting tumor growth and cooperating with cetuximab. This effect is due to multiple mechanisms of action, including interference with EGFR signalling by modulation of a TLR9/EGFR interaction and enhancement of cetuximab ADCC. Therefore, we suggest that IMO plus cetuximab may be a potentially effective therapeutic strategy for K-Ras mutant, cetuximab-resistant colorectal and pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 615.