TLR7 mRNA Levels Research Articles

Toll-Like Receptor 7-Expressed Macrophages Are Involved in the Pathogenesis of Esophageal Achalasia and Esophagogastric Junction Outflow Obstruction.

Esophageal achalasia is a typical esophageal motility disorder (EMD). Although viral infections have been hypothesized to play a role in the pathogenesis of esophageal achalasia, its etiology remains unclear. This study used esophageal muscle layer specimens collected during per-oral endoscopic myotomy (POEM) procedures to investigate the association between esophageal achalasia and esophagogastric junction outflow obstruction (EGJOO) and pattern recognition receptors. Patients with esophageal achalasia and EGJOO who underwent POEM were allocated to the EMD group. Biopsies of the inner circular muscle were conducted during the POEM procedure. The control group comprised individuals diagnosed with esophageal squamous cell carcinoma who underwent surgical resection. Expression of pattern recognition receptors, including Toll-like receptor (TLR) 7, was examined by polymerase chain reaction. Immunohistochemical staining was performed to determine TLR7 expression sites in the esophageal muscle layer, and the relationship between TLR7 mRNA expression and clinical score was investigated. Our analysis revealed a notable upregulation of TLR7 mRNA levels within the muscle layer of esophageal achalasia and EGJOO, in contrast to those of control specimens. In contrast, the correlation between TLR7 and clinical score was not significant. Immunohistochemical staining revealed increased numbers of TLR7-expressing macrophages between the muscle layers. TLR7-expressing macrophages are involved in the innate immune response underlying esophageal achalasia and EGJOO. This result will lead to the elucidation of new pathogenetic mechanisms and the development of novel therapeutic targets.

Prolonged alcohol consumption influences microRNA expression in the nucleus accumbens of the rat brain.

The microRNA (miR) species analyzed in this study are involved in molecular mechanisms of TLR4 and TLR7 signaling, mediating the development of neuroinflammation and neurodegeneration. We have investigated the expression levels of miR-let7b, miR-96, miR-182, miR-155, and the mRNA content of HMGB1, TLR3, TLR4 in the nucleus accumbens (NAc) of the brain of rats exposed to long-term alcoholization. The long-term alcoholization caused a decrease in miR-let7b, miR-96, miR-182, and TLR7 mRNA levels; this was accompanied by an increase in miR-155, TLR4, and Hmgb1 mRNA levels in the NAc of rat brain. TLR7 is functionally linked to miR-let7b. The data of a simultaneous decrease in miR-let7b and TLR7 mRNA are of interest for further studies; they may indicate on the lack of functionally significant links between Hmgb1 and the miR-let7b-TLR7 system in NAc. The existing evidence of a functional relationship between TLR4 with miR-155 and miR-182 and our observations on their expression changes during chronic alcoholization are very interesting and require further investigation. The suggestion about the development of neuroinflammatory process in NAc under prolonged alcohol exposure are relevant for studying the level of TLR gene expression in NAc, as well as the expression of miR species, which may have a functional relationship with the TLR system.

Alpha-pyrrolidinopentiothiophenone (α-PVT) activates the TLR–NF-κB–MAPK signaling pathway and proinflammatory cytokine production and induces behavioral sensitization in mice

Synthetic cathinones are chemical derivatives of cathinone, a structural analog to amphetamine. It has been shown that synthetic cathinones have abuse potentials similar to psychomotor stimulants such as amphetamine and induce neuroinflammation. Among the novel synthetic cathinones, α-pyrrolidinopentiothiophenone (α-PVT) has been known to produce rewarding and reinforcing effects in rodent models. However, it has not yet been determined whether α-PVT induces neuroinflammation in vivo. In the present study, mice were exposed to repeated saline or α-PVT (20 mg/kg, intraperitoneally) for 7 days to test changes in locomotor activity and neuroinflammation-related factors in the striatum of mice. Repeated administration of α-PVT significantly induced locomotor sensitization. In addition, repeated α-PVT administration significantly increased the number of microglial cells, accompanied by marked increases in TLR1, TLR4, TLR6, and TLR7 mRNA levels in the striatum of mice. Furthermore, acute or repeated α-PVT administration increased the levels of phosphorylated NF-κB, ERK, p38, and JNK MAPK activation and repeated α-PVT, but not acute, increased the levels of TNF-α and IL-6 mRNA in the striatum of mice. Finally, systemic administration of TAK-242 (5 mg/kg, i.p.) or MPLA (50 μg/kg, i.p.), each an inhibitor or activator of TLR4, did not change α-PVT-induced behavioral sensitization in mice. These results suggest that the activation of TLR4 by repeated α-PVT administration may lead to neuroinflammation via TLR-mediated NF-κB and MAPK signaling pathways and the production of TNF-α and IL-6 in the striatum of mice, at least without the regulation of behavioral sensitization.

COVID-19 susceptibility, severity, clinical outcome and Toll-like receptor (7) mRNA expression driven by TLR7 gene polymorphism (rs3853839) in middle-aged individuals without previous comorbidities

BackgroundToll-like receptors are implicated in the pathophysiology of the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory disease (MERS), according to several studies. The whole-genome sequencing of SARS-CoV-2 revealed that the TLR7 gene could be implicated in the virus's pathogenesis since the virus includes ssRNA patterns that could bind to TLR7. AimThe purpose of this study was to look into the function of the TLR7 (rs3853839) C/G polymorphism and the expression of TLR7 mRNA transcript in the development, severity and progression of COVID-19. Subjects and methodsA case-control study included 285 participants who were divided into two groups: 150 middle-aged people with COVID 19 who had no previous co-morbidities and 135 healthy volunteers who served as controls. TaqMan test was used to genotype the TLR7 (rs3853839) C/G polymorphism, and real-time PCR was used to determine the relative expression of its mRNA transcript. The level of IL-6 in serum was determined using the ELISA method as an indicator of cytokine storm and COVID-19 severity. ResultsThe GG genotype was shown to be much more common in COVID-19 patients (38.7%) than controls (4.4%), with an OR of 19.86 (95% CI: 7.85; 50.22) and was linked to disease severity and poor clinical outcomes (hospitalization, respiratory failure, cardiac complications, ICU admission and mechanical ventilation).As a result, the G allele was considerably higher in cases (57.0%), while the C allele was significantly higher in controls (p = 0.001). The GG genotype was found to be substantially more common in patients who were severely/critically unwell. TLR7 mRNA expression levels were significantly higher in COVID-19 patients (2.44 ± 0.89) than in controls (1.06 ± 0.46) (p = 0.001). TLR7 mRNA levels were highest in COVID 19 patients with the GG genotype (rs3853839). Patients with the GG genotype had considerably lower WBC counts, but significantly higher serum ferritin, CRP, IL-6 and D dimer levels (P = 0.045, 0.001, 0.023, 0.033, 0.001, respectively). ConclusionThe GG form of the TLR7 SNP (rs3853839) could be a genetic risk factor for COVID-19 infection, severe illness and poor clinical outcome. TLR7 mRNA expression was also elevated in COVID-19 patients who were severely/critically unwell and had a bad outcome, suggesting that they could be used as COVID-19 prognostic biomarkers.

Nephropathogenic Infectious Bronchitis Virus Mediates Kidney Injury in Chickens via the TLR7/NF-κB Signaling Axis.

Nephropathogenic infectious bronchitis virus (NIBV) is one of the most important viral pathogens in the world poultry industry. Here, we used RT–qPCR, WB and immunofluorescence to explore the interaction between NIBV and the host innate immune system of the kidney. Multiple virions were found in the kidney tissues of the disease group under electron microscopy, and pathological changes such as structural damage of renal tubules and bleeding were observed by HE staining. In addition, we found that the mRNA levels of TLR7, TRAF6, and IKKβ were upregulated after NIBV infection. IRF7 mRNA levels decreased significantly at 5 dpi and increased significantly at 11 to 18 dpi. The NF-κB P65 mRNA level increased significantly at 5 to 18 dpi and decreased at 28 dpi. However, NIBV infection-induced NF-κB P65 protein levels were downregulated at multiple time points. Moreover, we demonstrated that the cytokine (IFN-γ, IL-8, and IL-6) mRNA and protein expression levels were increased significantly at multiple time points after NIBV infection. Furthermore, immunofluorescence analysis showed that NF-κB P65 and IFN-γ were mainly located in the nuclear or perinuclear region. The positive signal intensity of NF-κB P65 was significantly lower than that of the normal group at 1 to 5 dpi, and there was no significant change in the subsequent time period. The positive signal intensity of IFN-γ decreased significantly at 5 dpi, and increased significantly at 11 to 28 dpi. In conclusion, we found that NIBV promoted cytokine release through the TLR7/NF-κB signaling axis, thus causing kidney injury.

RNA receptors, TLR3 and TLR7, are potentially associated with SLE clinical features.

The influence of intracellular Toll-like-receptors (TLR), recognized as nucleic acid sensors, in the immunopathogenesis of systemic lupus erythematosus (SLE) is increasingly explored. Yet, the results of both functional and genetic studies remain conflictual. We evaluated the association between TLR3 and TLR7 genes selected variants and SLE and investigated the possible relationship with clinical and serological parameters. Then, we studied the genetic expression of these receptors, and if the TLR7 gene evades X chromosome inactivation (XCI). Our study covers 106 cases and 200 controls, genotyped using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. TLR3 and TLR7 expression level was assessed by qPCR carried, respectively, on renal tissues and PBMC, and methylation status was evaluated by methylation-specific PCR. Results were statistically analysed using Shesis software, χ2 , and Mann-Whitney test. Significant associations with SLE susceptibility were found for the TLR3 rs3775291, rs5743305 and rs3775294 polymorphisms. Further subgroup analysis, TLR3 rs3775291 and rs3775294 polymorphisms were significantly associated with lupus nephritis (LN) and even correlate with the presence of auto-antibodies binding RNA molecules. SLE and LN were more common in men with rs3853839-G variant within TLR7 gene versus those carrying the C allele. Moreover, the role of the G allele in the TLR7 expression up-regulation was confirmed. However, gene expression analysis showed no significant differences in TLR3 and TLR7 mRNA levels between LN patient biopsies and healthy tissues (p>.05). When comparing patients and controls, no statistical difference was observed in XCI pattern. Otherwise, notable associations were raised between TLR3 and TLR7 gene variants and clinical and serological lupus features pointing towards the role of genetic background in the physiopathogenesis of the disease.

TLR7 in B cells promotes renal inflammation and Gd-IgA1 synthesis in IgA nephropathy.

TLR7 has been linked to the pathogenesis of glomerulonephritis, but its precise roles are not clear. In this study, we evaluated the roles of TLR7 in IgA nephropathy (IgAN). TLR7 proteins were abundant in CD19+ B cells infiltrated in the kidneys of patients with IgAN. The intensities of both intrarenal TLR7 and CD19 proteins were closely associated with kidney function (estimated glomerular filtration rate [eGFR] and serum creatinine concentration) and renal histopathology (tubular atrophy, leukocyte infiltration, tubulointerstitial fibrosis, and global glomerulosclerosis) in patients with IgAN. Meanwhile, TLR7 mRNA levels were significantly increased in peripheral blood B cells of patients with IgAN. TLR7+CD19+ B cells expressed inflammatory cytokines (IL-6 and IL-12) in kidneys and produced high levels of IgA1 and galactose deficient-IgA1 (Gd-IgA1) in peripheral blood of patients with IgAN. Mechanistically, TLR7 activated B cells to produce high levels of Gd-IgA1 via the TLR7-GALNT2 axis in IgAN. Protein levels of GALNT2 were increased by overexpression of TLR7, while they were reduced by TLR7 knockdown in B cells. GALNT2 overexpression augmented Gd-IgA1 production in B cells derived from patients with IgAN. Taken together, high TLR7 expression in B cells has dual roles in the development and progression of IgAN, by facilitating renal inflammation and Gd-IgA1 antibody synthesis.

One-Week Nebulization of Mycobacterium vaccae Can Protect Against Pulmonary Respiratory Syncytial Virus Infection in Balb/c Mice.

Background: Respiratory syncytial virus (RSV) infection is the most common cause of acute lower respiratory tract infection in children, leading to their death. Currently, no effective prevention and treatment methods for RSV infection are available. RSV and many other unknown viruses pose a serious threat to human health. Our previous study demonstrated that Mycobacterium vaccae nebulization can protect against allergic asthma. As RSV infection and asthma are closely related, we hypothesized that M. vaccae could protect against pulmonary RSV infection. Therefore, we evaluated the effect of M. vaccae on RSV infection in Balb/c mice. Methods: The mice were randomized into three groups: normal, RSV, and M. vaccae. One week before the RSV infection model was established, the mice in the M. vaccae group were nebulized with M. vaccae. On the fourth day after RSV infection, airway responsiveness, airway inflammation, pulmonary RSV infection, mRNA levels of pulmonary toll-like receptor (TLR) 7 and TLR8, and pulmonary NF09, acetylcholine, and epidermal growth factor regulator (EGFR) expression levels in all mice were measured. Results: The airway inflammation in the M. vaccae group was alleviated compared with that in the RSV group. In the M. vaccae group, the pulmonary mRNA level of RSV and the pulmonary expression levels of NF09, acetylcholine, and EGFR were decreased considerably, whereas the mRNA levels of TLR7 and TLR8 were increased significantly. Conclusions: One-week nebulization of M. vaccae can protect against RSV infection in Balb/c mice. The mechanism involves the regulation of neurotransmitters and expression of TLR7, TLR8, and EGFR.

Copy number of the X-linked genes TLR7 and CD40L influences innate and adaptive immune responses.

The number of the X chromosome-linked genes has been previously suggested to influence immune responses and the development of autoimmune diseases. In the present study, we aimed at evaluating the level of expression of CD40L (an X-linked gene involved in adaptive immunity) and TLR7 (an X-linked gene involved in innate immunity) in a variety of different karyotypes. Those included males, females and patients with X chromosome aneuploidy. Healthy females (46, XX; n=10) and healthy males (46, XY; n=10) were compared to females with Turner syndrome (TS) (45, X; n=11) and males with Klinefelter syndrome (KS) (47, XXY; n=5). Stimulation of peripheral blood mononuclear cells (PBMCs) with PMA and ionomycin resulted in higher percentage of CD3+CD40L+ T cells (P<0.001) and higher level expression of CD40L in T cell (P<0.001) in female and KS patients compared with male and TS patients. TLR7-mediated IFN-alpha production by HLADR+CD3- CD19- cells was significantly upregulated in healthy women compared with healthy males, TS and KS patients (P<0.001). TLR7 agonist-stimulated PBMCs from healthy females and KS patients expressed significantly higher levels of TLR7 mRNA than those from male and TS patients (P<0.05). The increased expression of the X-linked genes TLR7 and CD40L in healthy females and KS patients suggests that the presence of two X chromosomes plays a major role in enhancing both innate and adaptive immune responses. These results may contribute to the explanation of sex-based differences in immune biology and the sex bias in predisposition to autoimmune diseases.

Abstract 4425: Comprehensive study of TGF-β pathway-driven functional molecular characterization of human hepatocellular cancer

Abstract Background: TGF-β plays a complex role in cancer- from tumor suppression to immune modulation to tumor promotion that are unclearly defined to date. Recent clinical studies show that targeting TGF-β improves survival up to 21 months, yet prognostic significances are undefined. Moreover, the relationships between patterns of mutations and transcriptomic phenotypes for the TGF-β pathway are unclear. Methods: 1. We analyzed the transcriptome of 488 hepatocellular cancers and screened for mutations in the TGF-b pathway in 202 HCCs from The Cancer Genome Atlas (TCGA). 2. Next we correlated mouse models of HCC with a functional analysis of drivers. Results: 1. Transcriptomic analyses revealed aberrant TGF-β superfamily profiles in 72% of hepatocellular cancers, with mutations in 38% of patients. 2. Significantly, HCCs characterized by the “inactivated” TGF-β signature were associated with a significantly poorer survival particularly in early stage HCCs, compared to HCCs with the “activated” TGF-β signature (p = 0.0027). 3. We observed the greatest number of functional mutations in the SPTBN1 gene (6%), which encodes a tumor suppressor TGF-β/Smad3 adaptor protein. 4. We found a strong association between DNA damage response genes and the TGF-β pathway at both transcriptomic and genomic levels. 5. We also observed a strong correlation between VD related genes and TGF-β pathway genes in the TCGA genomic analysis. 6. subsequent VD deprivation synergistically with TGF-β inactivation promotes liver tumor development. 7. Through a transcriptomic and functional analysis we observed that TLR7 mRNA levels are increased 4-fold in liver tissues from Smad3+/- mice, and TLR7 is a direct target of Smad3. Conclusions: The TGF-β pathway plays a pivotal role in liver tumorigenesis and the molecular signatures we characterize here have prognostic significance. In specific populations, VD deficiency and TGF-β disruption synergistically promote liver tumor growth, possibly through regulating TLR7 expression. The additional association with the DNA repair pathway supports new approaches to biomarker driven targeting of TGF-β, improving survival of liver cancer. Citation Format: Jian Chen, Jiun-Sheng Chen, Jianping Zhang, YoungJin Gi, Lior Katz, Ji-Hyun Shin, YunSeong Jeong, Mitch Belkin, Wilma Jogunoori, Bibhuti Mishra, Jon White, Shoujun Gu, Milind Javle, Xiaoping Su, John Stroehlein, Marta Davila, Xuemei Wang, Jeffrey Morris, Patrizia Farci, Rehan Akbani, Lopa Mishra. Comprehensive study of TGF-β pathway-driven functional molecular characterization of human hepatocellular cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4425.

Increased Expression of TLR4 and TLR7 but Not Prolactin mRNA by Peripheral Blood Monocytes in Active Celiac Disease.

Celiac disease (CD) is an organ-specific autoimmune disease, and both adaptive and innate immunity are involved in its development. The aim of the study was to determine whether the markers of intestinal mucosal inflammation in CD can be detected in peripheral blood monocytes (PBMs), and whether the immune properties of PBMs change as the clinical signs and symptoms of CD improve after the introduction of a gluten-free diet (GFD). The focus was on changes in mRNA expression of selected toll-like receptors (TLR2, TLR4, TLR7), stress cytokine prolactin (PRL), and proand anti-inflammatory cytokines (TNF-α, IL-6, IL-12, IL-10) in PBMs. The study involved 20 CD patients diagnosed according to the European Society for Pediatric Gastroenterology, Hepatology and Nutrition criteria and Marsh criteria: 10 recently-diagnosed cases (rCD) and 10 on a GFD for a minimum of one year. The control group comprised 10 ageand sex-matched healthy volunteers. PBMs from peripheral blood specimens were separated using immunomagnetic CD14+ beads. Total RNA was isolated using a standard commercial kit. Cytokine and TLR mRNA levels were quantified by relative qPCR with PGK1 as a reference gene. Significantly higher expression of TLR4 and TLR7 mRNA was observed in PBMs from rCD patients compared to the healthy controls (1.63 times higher; p < 0.05). TLR7 mRNA levels in rCDs were also significantly elevated in comparison to the CD-GFD patients (2.11 times higher; p < 0.01). TNF-α mRNA expression tended to be higher in both groups of patients; by contrast, in IL-6 mRNA, a trend to a fourfold decrease was detected in PBMs from the CD-GFD subjects. IL-10, IL-12 and PRL levels did not differ among the groups. The data suggest that the inflammatory process in rCD intestinal mucosa and submucosa reflecting enterocyte damage can be detected in PBMs in peripheral blood. Further, the cytokine and TLR expression profile in PBMs alters after one year of GFD treatment.

Modulation of dendritic cells and toll-like receptors by marathon running

The focus of this study was to assess exercise-induced alterations of circulating dendritic cell (DC) subpopulations and toll-like receptor (TLR) expression after marathon running. Blood sampling was performed in 15 obese non-elite (ONE), 16 lean non-elite (LNE) and 16 lean elite (LE) marathon runners pre- and post-marathon as well as 24 h after the race. Circulating DC-fractions were measured by flow-cytometry analyzing myeloid DCs (BDCA-1+) and plasmacytoid DCs (BDCA-2+). We further analyzed the (TLR) -2/-4/-7 in peripheral blood mononuclear cells (rt-PCR/Western Blot) and the cytokines CRP, IL-6, IL-10, TNF-α and oxLDL by ELISA. After the marathon, BDCA-1 increased significantly in all groups [LE (pre/post): 0.35/0.47%; LNE: 0.26/0.50% and ONE: 0.30/0.49%; all p < 0.05]. In contrast, we found a significant decrease for BDCA-2 directly after the marathon (LE: 0.09/0.01%; LNE: 0.12/0.03% and ONE: 0.10/0.02%; all p < 0.05). Levels of TLR-7 mRNA decreased in all groups post-marathon (LE 44%, LNE 67% and ONE 52%; all p < 0.01), with a consecutive protein reduction (LE 31%, LNE 52%, ONE 42%; all p < 0.05) 24 h later. IL-6 and IL-10 levels increased immediately after the run, whereas increases of TNF-α and CRP-levels were seen after 24 h. oxLDL levels remained unchanged post-marathon. In our study population, we did not find any relevant differences regarding training level or body weight. Prolonged endurance exercise induces both pro- and anti-inflammatory cytokines. Anti-inflammatory cytokines, such as IL-10, may help to prevent excessive oxidative stress. Marathon running is associated with alterations of DC subsets and TLR-expression independent of training level or body weight. Myeloid and plasmacytoid DCs are differently affected by the excessive physical stress. Immunomodulatory mechanisms seem to play a key role in the response and adaptation to acute excessive exercise.

Effect of creatine, creatinine, and creatine ethyl ester on TLR expression in macrophages

Despite the widespread availability and use of dietary supplements, minimal work has been performed to assess the potential dangers many of these supplements may have on the host's well-being, in particular the host's ability to respond to infection. One supplement extensively used by both adolescents and adults is creatine. Using Real-time PCR, we examined the impact of short-term exposure of a mouse macrophage cell line (RAW 264.7 cells) to two readily available forms of creatine used in supplements – creatine monohydrate (CR) and creatine ethyl ester (CEE) as well as the end product of creatine metabolism, creatinine (CRN), on expression of toll-like receptor-2 (TLR-2), TLR-3, TLR-4, and TLR-7. CR down-regulated TLR-2, TLR-3, TLR-4 and TLR-7 mRNA levels in RAW cells. Similar results were observed following exposure of RAW cells to CRN. Conversely CEE appears to possess immunostimulatory properties and increases expression of TLR-2, TLR-3, TLR-4, and TLR-7 in RAW cells. These data are supported by immunostaining using antibodies specific for the individual TLRs before and after exposure of RAW cells to CR, CRN, or CEE. To extend these findings, we isolated murine splenocytes and exposed the cells to CR, CEE, or CRN for 24 hours and performed immunofluorescent staining for TLR-2, TLR-3, TLR-4 and TLR-7. The results obtained from this study with primary splenocytes were consistent with the studies using RAW cells. Together, these data suggest that creatine and creatine derivatives may impact the ability of immune cells to sense a wide array of viral and bacterial pathogens. Of great interest, CRN - largely considered to be a waste product of the argenine biosynthesis pathway may also have immunosuppressive properties similar to those of CR.

Association of TLR7 copy number variation with susceptibility to childhood-onset systemic lupus erythematosus in Mexican population

ObjectiveVariations in gene copy number (CNV) have been recognised as a hereditable source of susceptibility in human complex diseases. Recent studies have shown that Tlr7 gene dosage has a significant...

Lack of SIGIRR/TIR8 aggravates hydrocarbon oil‐induced lupus nephritis

Multiple genetic factors contribute to the clinical variability of spontaneous systemic lupus erythematosus (SLE) but their role in drug-induced SLE remain largely unknown. Hydrocarbon oil-induced SLE depends on mesothelial cell apoptosis and Toll-like receptor (TLR)-7-mediated induction of type I interferons. Hence, we hypothesized that TIR8/SIGIRR, an endogenous TLR inhibitor, prevents oil-induced SLE. Sigirr-deficient dendritic cells expressed higher TLR7 mRNA levels and TLR7 activation resulted in increased IL-12 production in vitro. In vivo, lack of SIGIRR increased surface CD40 expression on spleen CD11c(+) dendritic cells and MX-1, TNF, IL-12, BAFF and BCL-2 mRNA expression 6 months after pristane injection. Spleen cell counts of CD4(-)/CD8(-) 'autoreactive' T cells and B220(+) B cells were also increased in Sigirr(-/-) mice. Serum autoantibody analysis revealed that Sigirr deficiency specifically enhanced the production of rheumatoid factor (from 4 months of age) and anti-snRNP IgG (from 5 months of age), while anti-Smith IgG or anti-dsDNA IgG were independent of the Sigirr genotype. This effect was sufficient to significantly aggravate lupus nephritis in Sigirr-deficient mice. Structure model prediction identified the BB loop of SIGIRR's intracellular TIR domain to interact with TLR7 and MyD88. BB loop deletion was sufficient to completely abrogate SIGIRR's inhibitory effect on TLR7 signalling. Thus, TIR8/SIGIRR protects from hydrocarbon oil-induced lupus by suppressing the TLR7-mediated activation of dendritic cells, via its intracellular BB loop.

Toll-like receptor 7 agonist induces apoptosis of human B-lineage acute lymphocytic leukemia cells in vitro and in vivo

3033 Background: Acute lymphocytic leukemia (ALL) is the most common childhood leukemia. In this study, we investigated the antitumor effects of TLR7 agonist (imiquimod) against human B-cell acute lymphocytic leukemia. Methods: Human B-ALL cell lines and purified CD19+CD10+ primary B-ALL cells were analyzed for TLR7 mRNA and protein expression by real-time RT-PCR and by Western blot. In vitro growth and apoptosis of B-ALL cells following TLR7 agonist treatment were determined by cell counts, TMRE staining, Western blot and flow cytometry of the activation/cleavage of caspases. Therapeutic effects of TLR7 agonist against human B-ALL were evaluated in xenograft mouse models, where luciferase-labeled RS4;11 or BLIN-1 cell line were injected into NOD/SCID IL2Rg mice. Cohorts of mice were injected with a lethal dose of RS4;11 or BLIN-1 cells with or without pre-incubation with TLR7 agonist to determine their leukemia development. Therapeutic efficacy of TLR7 agonist was evaluated by determining leukemia cell engraftment, tumor burden, the survival time and rate of the treated mice. Results: Human B-ALL cell lines (6/6 tested) and primary B-ALL cells (8/8 tested) express high levels of TLR7 mRNA and proteins. Triggering TLR7 on B-ALL cells with imiquimod strongly suppresses in vitro growth of B-ALL cell lines (RS4;11, BLIN-1) and induces profound apoptosis of primary B-ALL cells in culture in TLR7 agonist treatment time and dose-dependent manners. Injection of RS4;11 or BLIN-1 leukemia cells into NOD/SCID mice causes disseminated leukemia in multiple organs and resulting in uniform lethality of RS4;11 mice in 8 weeks and BLIN-1 mice in 12 weeks, respectively. Mice receiving TLR7 agonist pretreated B-ALL cells had a significant increase in long-term survival rate and a significant reduction in tumor burden. Administration of TLR7 agonist (daily i.p. injection for 5 days) in mice with B-ALL cells significantly reduced (8.2 to 11.4 fold) leukemia cell burdan and increased long-term survival time and rate of the treated mice. Conclusions: Our results demonstrate that TLR7-targeting directly inhibits the growth and induces apoptotic death of human B-ALL cells in vitro and in vivo, providing new insights into the biology and therapy of B-ALL. No significant financial relationships to disclose.

Toll-like receptor 7 and 9 expression in peripheral blood mononuclear cells from patients with chronic hepatitis B and related hepatocellular carcinoma

The aim of the present study was to investigate the expression of Toll-like receptors (TLR) 7 and 9 in peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B virus (HBV) infection and related hepatocellular carcinoma. The study group was comprised of 52 patients: 41 with chronic hepatitis B and 11 healthy controls. The protein and mRNA levels of TLR7 and TLR9 were evaluated using real-time PCR, Western blot analysis, and flow cytometry. We also detected the serum viral load of HBV in the patients and analyzed the correlation between HBV-DNA copies and the TLR expression. Our results demonstrated a lower TLR7 expression in all HBV infection groups compared to the controls. We found that HBV infection led to a decreased expression of TLR9 mRNA, but an increased expression of the TLR9 protein compared to the healthy group. The TLR protein levels are related to serum HBV-DNA (P<0.01). There are downregulations of TLR7 expression and TLR9 mRNA in PBMC of HBV-infected patients, but an increased TLR9 expression at the protein level.

P.048 Hepatitis B virus X protein mediates mitotic abnormalities and polyploidy via p53 deregulation and DNA re-replication

Despite the widespread availability and use of dietary supplements, minimal work has been performed to assess the potential dangers many of these supplements may have on the host's well-being, in particular the host's ability to respond to infection. One supplement extensively used by both adolescents and adults is creatine. Using Real-time PCR, we examined the impact of short-term exposure of a mouse macrophage cell line (RAW 264.7 cells) to two readily available forms of creatine used in supplements – creatine monohydrate (CR) and creatine ethyl ester (CEE) as well as the end product of creatine metabolism, creatinine (CRN), on expression of toll-like receptor-2 (TLR-2), TLR-3, TLR-4, and TLR-7. CR down-regulated TLR-2, TLR-3, TLR-4 and TLR-7 mRNA levels in RAW cells. Similar results were observed following exposure of RAW cells to CRN. Conversely CEE appears to possess immunostimulatory properties and increases expression of TLR-2, TLR-3, TLR-4, and TLR-7 in RAW cells. These data are supported by immunostaining using antibodies specific for the individual TLRs before and after exposure of RAW cells to CR, CRN, or CEE. To extend these findings, we isolated murine splenocytes and exposed the cells to CR, CEE, or CRN for 24 hours and performed immunofluorescent staining for TLR-2, TLR-3, TLR-4 and TLR-7. The results obtained from this study with primary splenocytes were consistent with the studies using RAW cells. Together, these data suggest that creatine and creatine derivatives may impact the ability of immune cells to sense a wide array of viral and bacterial pathogens. Of great interest, CRN - largely considered to be a waste product of the argenine biosynthesis pathway may also have immunosuppressive properties similar to those of CR.