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Schizophrenia (SZ) is a chronic disabling mental disorder with high heritability, and several immune-regulating genes have been implicated in its pathophysiology In this study, we investigated the expression of Toll-like receptors (TLRs) 1, 2, and 6 in peripheral blood monocytes from SZ patients and healthy control subjects (HCSs) in the Mexican population, focusing on specific SZ-associated gene variants. Gene expressions were assessed by qPCR, and protein expression was measured using flow cytometry. The secretory profiles of MALP2-stimulated monocytes were evaluated through immunoproteomic arrays. Our results indicate that patients with SZ carrying the rs4833093/TLR1 GG genotype exhibited significantly lower TLR1 gene expression compared to TT carriers. Notably, HCSs with the TT genotype showed markedly higher TLR1 protein expression, while all patients with SZ exhibited significantly reduced protein levels regardless of genotype. Furthermore, monocytes from patients with SZ displayed altered secretion profiles upon TLR stimulation, with significant elevations in IL-18, uPAR, angiopoietin-2, and serpin E1, alongside reductions in MCP-1, IL-17A, IL-24, MIF, and myeloperoxidase compared to HCSs. These findings suggest a dysfunctional TLR-mediated innate immune response in SZ. [ABSTRACT FROM AUTHOR]

Background & Objective: Acute appendicitis is the predominant etiology of emergency cases on a global scale, and it may have serious consequences. If there is no precise diagnosis. Toll-like receptors (TLRs) are a crucial group of receptors that form the initial barrier against infections. The study aimed to investigate the molecular structure of Toll-like receptor (TLRs) genes by conventional Polymerase Chain Reaction)PCR) and deoxyribonucleic acid (DNA) sequencing in acute appendicitis patients and investigate whether any alteration in this gene may act as a risk factor for appendicitis. Methodology: A case-control investigation was conducted at Al-Sadr Teaching Hospital and Al-Shifaa Teaching Hospital in Basrah, Iraq, from July to November 2023. The study involved 20 blood samples from 15 patients previously diagnosed with appendicitis; and 5 healthy individuals included as a control group. We randomly selected six samples from patients with appendicitis, with two samples for each primer, for DNA sequencing. Results: The results of conventional PCR and electrophoresis showed different molecular weights at different temperatures for TLR1, TLR4, and TLR5. DNA sequencing results showed several different mutations for each TLRs gene primer for both reverse and forward when compared by the Basic Local Alignment Search Tool (BLAST) to sequences from the National Centre for Biotechnology Information (NCBI). Six new mutations were recorded in the gene bank (NCBI), two mutations per TLR. The study found that TLR1's forward primer had two mutations, while the reverse primer had four. Four alterations were found in TLR4, while one in TLR5 was found in the reverse primer. Conclusion: Through altering the immune system's reaction to pathogenic stimuli, excessive immunological activation, and inflammation, genetic differences in TLRs may influence the pathophysiology or outcome of disease. These factors can also contribute to the development of appendicitis. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are expressed on both immune cells and tumor cells, triggering both anti-tumor and pro-tumor responses. Therefore, TLRs have potential as prognostic biomarkers and immunotherapeutic targets. The aim of this study was to investigate TLR1, TLR2, TLR4, TLR5, and TLR6 expression and association with clinicopathological variables and survival in gastric cancer. Immunohistochemical study on cancer specimens from 564 resected gastric cancer patients was performed using tissue microarrays. The association between patient survival and TLR expression was calculated with Cox regression adjusted for confounding factors. Patients with high cytoplasmic TLR2 expression had significantly poorer 5-year survival than the low cytoplasmic TLR2 expression group in multivariate analysis (adjusted HR 1.38, 95% CI 1.11–1.71), and this estimate was similar in intestinal type (adjusted HR 1.33, 95% CI 0.98–1.80) and diffuse type (adjusted HR 1.48, 95% CI 1.06–2.05) histology subgroups. Patients with high cytoplasmic TLR6 expression group had significantly better 5-year survival compared with low cytoplasmic TLR6 expression group in multivariate analysis (adjusted HR 0.74, 95% CI 0.60–0.91). In the subgroup analysis of diffuse type of histology, the 5-year survival was better in high cytoplasmic TLR6 expression group in multivariable analysis (HR 0.62, 95% CI 0.46–0.83). In the intestinal type of histology subgroup, no significant differences between the groups were present. TLR1, TLR4, and TLR5 expression were not associated with 5-year survival. In conclusion, cytoplasmic TLR2 and TLR6 expression seem to have independent prognostic impact in gastric cancer, while TLR1, TLR4, and TLR5 do not. [ABSTRACT FROM AUTHOR]

Abstract Background Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder of unknown etiology and unpredictable course. The aim of the work was to assess the levels of adropin, fibroblast growth factor-1 (FGF-1), and Toll-like receptor-1 (TLR1) biomarkers in IBD patients compared to controls and evaluate the gene expression of TNF-α as a marker of disease severity. Methods Adropin, fasting serum FGF-1 levels, TLR1, and TNF-α were measured in 60 IBD patients. They were also compared with 58 healthy controls matching age and gender. Moreover, the blood cells cDNA copy number of TNF-α were determined as a marker of severity. Results Adropin and TLR1 levels were significantly lower in patients than controls. FGF-1 was reduced but not statistically significant. The expression of TNF-α gene in the IBD patients was significantly increased (42%) in comparison with control samples (P

Objective: To investigate potential mechanism of Helicobacter pylori metabolites antagonizing host innate immunity. Methods: RNA sequencing and pathway enrichment analysis were used to analyze only LPS-stimulated gastric mucosal cells GES-1, GES-1 cells co-treated with LPS and Helicobacter pylori culture supernatant, and untreated GES-1 cells. The culture supernatant of Helicobacter pylori was filtered by a 3KD ultrafiltration tube, and the filtered filtrate (metabolite part) and the retained solution (protein part) were treated with LPS-stimulated GES-1 cells to detect activity of NF-κB pathway, phosphorylation level of NF-κB, secretion levels of NF-κB pathway effectors TNF-α, IL-6 and IL-8. Identification of key metabolites by untargeted metabolic mass spectrometry. Results: Compared with GES-1 cells stimulated only by LPS, after co-treated with LPS and Helicobacter pylori culture supernatant, expression levels of various genes were regulated and tended to the level of GES-1 in untreated gastric mucosal cells, mainly in the NF-κB pathway. After co-treatment with LPS and culture supernatant of Helicobacter pylori, activity of NF-κB pathway was inhibited (P< 0.05). Helicobacter pylori metabolites could inhibit the activity of NF-κ B pathway, inhibit phosphorylation of NF-κB, and inhibit the secretion of NF-κB pathway effectors TNF-α, IL-6 and IL-8 (P<0.05). 1, 5 and 25 µmol/L of Helicobacter pylori metabolite 2-D-Glu-copyranose (2DG) treatment inhibited activity of NF-κB pathway and phosphorylation of NF-κB in GES-1 cells, and secretion of NF-κB pathway effectors TNF-α, IL-6 and IL-8 were inhibited (P<0.05). After 2DG treatment, activity of NF-κB in GES-1 cells with TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10 knockout were significantly decreased (P<0.05); while there was no significant changes in activity of NF-κB in TLR1 and TLR2 knockout GES-1 cells. Both TLR1 and TLR2 interactions were attenuated in GES-1 cells after 2DG treatment. Molecular docking showed that 2DG could bind to TLR2 amino acid disabled R321, K347 and F349, the binding energy was -12 kcal/mol. TLR2 wild-type and mutant plasmids (R321K, K347R, F349A) were constructed, and TLR2-knockout GES-1 cells were respectively transfected. It was found that 2DG treatment did not reduce NF-κB activity in GES-1 cells transfected with TLR2 mutant. Conclusion: Helicobacter pylori metabolite 2DG can interact with TLR2, reduce the formation of heterodimers between TLR2 and TLR1, and inhibit the activity of innate immune NF-κB pathway. [ABSTRACT FROM AUTHOR]

AbstractNon-mammal models are gaining popularity due to their ease of maintenance, low cost and lack of ethical restrictions. As some processes taking place in insect hemocytes are mirrored in human immunocytes, many immunological studies are now turning to the use of insect models like Galleria mellonella. G. mellonella larvae are susceptible to infection by Conidiobolus coronatus: an entomopathogenic fungus causing rhinofacial mycosis in humans. The study examines G. mellonella larvae during C. coronatus infection for the presence of certain compounds known to be involved in the human immunological response (histamine, HSF1, Cysteinyl leukotriene TLR1 and TLR2). G. mellonella larvae were exposed to fully grown fungus, and the results were determined by ELISA tests, fluorescence microscopy and flow cytometry. CysLT showed no significant changes between the control group and the infected larvae. Both histamine and HSF1 levels increased significantly after exposure to C. coronatus. Immunolocalization and flow cytometry of TLR1 and TLR2 proves that both receptors increase their levels due to the fungus infection. Our findings show that the cellular response of the G. mellonella is similar to the human response, indicating that G. mellonella larvae may be considered as a human-like immunological model in further studies.

Carlo E Sotelo-Ramírez,1,2 Beatriz Camarena,2 Marco Antonio Sanabrais-Jiménez,2 Julio Uriel Zaragoza-Hoyos,2 Bruno Ordoñez-Martínez,2 Raul I Escamilla-Orozco,3 Beatriz Gómez-González4 1Posgrado en Biología Experimental, Universidad Autónoma Metropolitana-Iztapalapa, México City, México; 2Departamento de Farmacogenética, Instituto Nacional de Psiquiatría Ramon de la Fuente Muñiz, Mexico City, Mexico; 3Dirección de Servicios Clínicos, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Ciudad de México, México; 4Área de Neurociencias, Departamento de Biología de la Reproducción, Universidad Autónoma Metropolitana-Iztapalapa, Ciudad de México, MéxicoCorrespondence: Beatriz Camarena, Departamento de Farmacogenética, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Calz México-Xochimilco, 101, Col. San Lorenzo Huipulco, México City, 14370, Mexico, Tel +52554160-5075, Email bcamarenam@gmail.comIntroduction: Schizophrenia is a complex psychiatric disorder with an important genetic contribution. Immunological abnormalities have been reported in schizophrenia. Toll-like receptor (TLR) genes play an important role in the activation of the innate immune response, which may help to explain the presence of inflammation in people with this disorder. The aim of this study was to analyze the association of TLR1, TLR2, and TLR6 gene polymorphisms in the etiology of schizophrenia.Methods: We included 582 patients with schizophrenia and 525 healthy controls. Genetic analysis was performed using allelic discrimination with TaqMan probes.Results: We observed significant differences between patients and controls in the genotype and allele frequencies of TLR1/rs4833093 (χ 2 = 17.3, p = 0.0002; χ 2 = 15.9, p = 0.0001, respectively) and TLR2/rs5743709 (χ 2 = 29.5, p = 0.00001; χ 2 = 7.785, p = 0.0053, respectively), and in the allele frequencies of TLR6/rs3775073 (χ 2 = 31.1, p = 0.00001). Finally, we found an interaction between the TLR1/rs4833093 and TLR2/rs5743709 genes, which increased the risk of developing schizophrenia (OR = 2.29, 95% CI [1.75, 3.01]).Discussion: Our findings add to the evidence suggesting that the activation of innate immune response might play an important role in the development of schizophrenia.Keywords: TLR1, TLR2, TLR6, gene–gene interaction, schizophrenia, immune response

Hui Han,1 Penggang Lian,2 Haiyuan Chen,3 Bilal Haider Shamsi,4 Yonglin Liu,4 Yongliang Niu5 1Clinical Laboratory, Shenmu Hospital, the Affiliated Shenmu Hospital of Northwest University, Shenmu, 719300, People’s Republic of China; 2Otorhinolaryngologic Department, Shenmu Hospital, the Affiliated Shenmu Hospital of Northwest University, Shenmu, 719300, People’s Republic of China; 3Information Department, Shenmu Hospital, the Affiliated Shenmu Hospital of Northwest University, Shenmu, 719300, People’s Republic of China; 4Department of Science and Education, Shenmu Hospital, the Affiliated Shenmu Hospital of Northwest University, Shenmu, 719300, People’s Republic of China; 5Department of Respiratory and Critical Care Medicine, Shenmu Hospital, the Affiliated Shenmu Hospital of Northwest University, Shenmu, 719300, People’s Republic of ChinaCorrespondence: Yongliang Niu, Tel/Fax +86-13892234287, Email niuyl7312@126.comBackground: Environmental factors and genetic predisposition can influence the occurrence and development of AR. Toll-like receptor 1 (TLR1) belongs to the TLR receptor family, which plays a fundamental role in the activation of innate immunity. This study aimed to explore the association between TLR1 genetic loci and AR susceptibility in the Han Chinese from northern China.Methods: Genotyping of three SNPs in the TLR1 has proceeded using the Agena MassARRAY platform. Odds ratio (OR) and 95% confidence interval (CI) were used to assess the correlation between candidate SNPs and AR susceptibility. Using FPRP (false-positive report probability analysis) to detect whether the positive results are noteworthy findings. The SNP-SNP interactions were detected by multifactor dimensionality reduction (MDR).Results: TLR1-rs72493538 (Allele “G”: OR=0.77, p = 0.034) and -rs76600635 (Allele “G”: OR=0.75, p = 0.024) were associated with reducing the risk of AR among Han Chinese in northern China. In addition, we found evidence that TLR1-rs72493538 (males, participants with aging > 43 years, or coming from the wind-blown sand region) and -rs76600635 (males, participants with BMI ≤ 24 kg/m2, or coming from the wind-blown sand region) were associated with AR risk in stratified analyses. FPRP showed that all positive results are noteworthy findings. MDR analysis showed that a two-loci genetic model composed of rs72493538 and rs76600635 can be chosen as the best genetic model to predict the risk of AR.Conclusion: TLR1-rs72493538 and -rs76600635 have a close association with reducing the risk of AR.Keywords: allergic rhinitis, TLR1, susceptibility, case-control study

Non-mammal models are gaining popularity due to their ease of maintenance, low cost and lack of ethical restrictions. As some processes taking place in insect hemocytes are mirrored in human immunocytes, many immunological studies are now turning to the use of insect models like Galleria mellonella. G. mellonella larvae are susceptible to infection by Conidiobolus coronatus: an entomopathogenic fungus causing rhinofacial mycosis in humans. The study examines G. mellonella larvae during C. coronatus infection for the presence of certain compounds known to be involved in the human immunological response (histamine, HSF1, Cysteinyl leukotriene TLR1 and TLR2). G. mellonella larvae were exposed to fully grown fungus, and the results were determined by ELISA tests, fluorescence microscopy and flow cytometry. CysLT showed no significant changes between the control group and the infected larvae. Both histamine and HSF1 levels increased significantly after exposure to C. coronatus. Immunolocalization and flow cytometry of TLR1 and TLR2 proves that both receptors increase their levels due to the fungus infection. Our findings show that the cellular response of the G. mellonella is similar to the human response, indicating that G. mellonella larvae may be considered as a human-like immunological model in further studies. [ABSTRACT FROM AUTHOR]

Background: Bipolar disorder (BD) is a complex neuropsychiatric disease that has been strongly linked to immune dysregulation. In particular, an abnormal inflammatory response mediated by toll-like receptor 2 − 1/6 (TLR2-1/6) was described in BD. Nevertheless, genetic factors' contribution is still unknown. Thus, we suggested that functional polymorphisms of TLR2, 1 and 6 could be involved in BD predisposition. Methods and results: TLR2, 1 and 6 polymorphisms were genotyped by PCR-RFLP in 292 controls and 131 patients from a Tunisian population. Polymorphisms and haplotype associations were explored in BD and binary logistic regression analysis was performed for more powerful associations. In dominant model, we found a significantly higher genotype and minor allele frequencies in healthy females compared to patients for TLR2-196-174Ins/Del (p = 0.04; OR = 0.3, p = 0.04; OR = 0.3, respectively) and for TLR6-S249P only with minor allele (p = 0.03; OR = 0.2). In contrast, TLR2-R677W CT + TT and T allele frequencies were significantly higher in BD (padjusted<10− 4; ORadjusted =46.6, p < 10− 4; OR = 6.3, respectively), specifically in females (CT + TT: 100%). Similarly, TLR1-R80T showed significantly increased GC + CC and C allele frequencies in patients compared to controls (padjusted=0.04; ORadjusted=4, p = 0.009; OR = 4.3, respectively). Moreover, haplotype investigation demonstrated that InsGTCGT (p < 10− 4, OR = 275) and delGCCGT (p = 0.03, OR = 18.5) were significantly overrepresented in BD patients compared to controls. Conclusions: We suggest that TLR2-196-174Ins/Del and TLR6-S249P could be protective factors of females against BD. However, TLR2-R677W and TLR1-R80T could be strongly associated with higher risk of BD. Interestingly, TLR2-R677W could be a genetic marker for BD in females. However, further studies with larger groups are needed to confirm these findings. [ABSTRACT FROM AUTHOR]

Objective: Sepsis-induced cardiomyopathy is the leading cause of death in sepsis and is characterized by reversible myocardial depression. However, the specific mechanisms responsible for myocardial injury in sepsis are not known. The present study used bioinformatic analysis to explore the possible mechanisms of sepsis-induced myocardial injury and the therapeutic potential of curcumin. Methods: The GSE125042 microarray gene expression matrix was obtained from the Gene Expression Omnibus database, which includes 10 septic cardiomyocyte samples from cecum ligation perforation constructs and 10 sham-operated groups cardiomyocyte samples. Background correction and matrix data normalization were performed using the robust multiarray average algorithm. Differentially expressed genes (DEGs) screening was performed using the Limma R package expression matrix, and whole gene analysis was performed using the weighted gene co-expression network analysis R package to construct gene networks and identify modules. Enrichment analysis and gene set enrichment analysis was performed on the genes to be selected. Construct cellular and animal models of myocardial injury in sepsis were assessed and the effects of curcumin on a rat or cardiac myocytes were observed. Results: A total of 2876 DEGs were screened based on the GSE125042 chip, of which 1424 genes were upregulated and 1452 genes were down regulated. WGCNA analysis of the whole genes was also performed and a total of 20 gene modules were generated. Among them, the selected TLR1 gene was present in the most strongly correlated Brown module. Enrichment analysis of the upregulated DEGs with the Brown module showed that they were significantly enriched in biological processes related to ribosomal protein complex generation, cellular components related to phagocytic vesicles and molecular functions related to Toll-like receptor binding, affecting cardiomyocyte survival as a target for molecular intervention in septic cardiomyopathy. Animal experiments showed that curcumin reduced inflammation levels, improved cardiac function and increased survival in rats with septic myocardial injury. Cellular experiments showed that curcumin increased the survival rate of lipopolysaccharide-treated cardiomyocytes and down regulated TLR1 expression and inhibited NF-κB phosphorylation in cells in a dose-dependent manner. Molecular docking analysis revealed that curcumin interacted with TLR1 by hydrogen bonding and could be stably bound to inhibit the biological function of TLR1. Conclusion: Our study shows that curcumin attenuates myocardial injury in sepsis by inhibiting TLR1 expression, which provides a molecular theoretical basis for clinical treatment. Resumo: Objetivos: A miocardiopatia induzida por sepsis é a principal causa de morte por sepsis e é caracterizada por uma depressão reversível do miocárdio. No entanto, os mecanismos através dos quais a sepsis induz lesão do miocárdio não são conhecidos. O presente estudo utilizou uma análise bioinformática para identificar os possíveis mecanismos de lesão do miocárdio induzida por sepsis e avaliar o potencial terapêutico da curcumina. Métodos: O microarray de expressão génica GSE125042 foi obtida na base de dados Gene Expression Omnibus (GEO), que inclui 10 amostras de cardiomiócitos de animais com sepsis, induzida por perfuração e ligação do ceco (CLP), e 10 amostras de cardiomiócitos de animais controlo (Sham). A correção e a normalização dos dados da matriz foram realizadas recorrendo ao algoritmo robusto de média multiarray (RMA). A identificação dos genes diferencialmente expressos (DEGs) foi realizada usando a matriz de expressão Limma R e a análise de genes inteiros foi realizada através da análise ponderada de rede de coexpressão de genes (WGCNA) R para construir redes de genes e identificar módulos. A análise de enriquecimento e a análise de enriquecimento do conjunto genético (GSEA) foram realizadas nos genes a selecionar. Foi avaliado o efeito da curcumina na lesão miocárdica, em modelos celulares e animais de lesão sepsis. Resultados: Foram avaliados 2876 DEGs com base no chip GSE125042, dos quais 1424 genes estavam sobrerregulados e 1452 genes estavam subregulados. Foi também realizada uma análise WGCNA de todos os genes e foram gerados 20 módulos de genes. Entre eles, o gene selecionado TLR1 estava presente no módulo de Brown mais fortemente correlacionado. A análise de enrriquecimento dos DEGs sobrerregulados com o módulo Brown mostrou que estes foram significativamente enriquecidos em processos biológicos relacionados com a geração do complexo proteico ribossómico (BP), componentes celulares relacionados com vesículas fagocitárias (CC) e funções moleculares relacionadas os Toll-like receptors (MF), que podem constituir alvos moleculares envolvidos na sobrevivência dos cardiomiócitos na cardiomiopatia séptica. Experiências com animais mostraram que a curcumina reduziu os níveis de inflamação, melhorou a função cardíaca e aumentou a sobrevivência em ratos com lesão séptica do miocárdio. Experiências celulares mostraram que a curcumina aumentou a taxa de sobrevivência de cardiomiócitos tratados com LPS e a expressão de TLR1 desregulada e inibiu a fosforilação NF-κB nas células, de uma forma dependente da dose. A análise da acoplagem molecular revelou que a curcumina interagia com TLR1 através de ligação de hidrogénio, o que poderia inibir a função biológica de TLR1. Conclusão: O nosso estudo demonstrou que a curcumina atenua a lesão do miocárdio por sepsis ao inibir a expressão do TLR1, constituindo uma base teórica molecular para novas abordagens terapêuticas.

The aim of the work was to evaluate the diagnostic value of TLR2 and TLR4 expression on periodontal and peripheral blood lymphoid cells by immunofluorescence microscopy in patients with chronic periodontitis associated with key periodontal pathogenic species Filifactor alocis, Porphyromonas gingivalis. Materials and methods. The study included 150 patients 88 (59%) women and 62 (41%) men aged 18 to 73 years with chronic periodontitis in the acute phase (CP) and 32 people without signs of chronic periodontal inflammation. To confirm the diagnosis of periodontitis, the Multident-5 PCR kit was used (detection of P. gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans), as well as rt-PCR for F. alocis and P. gingivalis in the contents of the periodontal pocket (NPF GenLab, Russia). To evaluate cells carrying CD282 and CD284 markers, gingival fluid flushes from the periodontal pocket with Hanks' solution were used. The isolated cells were stained with antibodies to CD282 markers (corresponding to TLR2 receptor) or CD284 (corresponding to TLR4 receptor) labeled with FITC, and fixed with paraformaldehyde for subsequent immunofluorescence microscopy. Results. The expression of TLR2 and TLR4 on peripheral blood and gingival fluid leukocytes was studied in individuals with healthy periodontitis and patients with chronic periodontitis associated with F. alocis, P. gingivalis. According to the results of PCR, the detection rate of F. alocis and P. gingivalis was 64 and 62.7%, respectively, which confirmed their dominance in the microbial association. It was found that the expression of TLR2 and TLR4 on peripheral blood lymphoid cells varied in humans. The possible diagnostic significance of this phenomenon in assessing the progression of chronic periodontitis is discussed. Conclusion. In patients with chronic periodontitis associated with the dominance of periodontopathogenic species F. alocis, P. gingivalis, the multidirectional expression of TLR2 and TLR4 on peripheral blood cells was observed, which may have diagnostic significance in assessing the progression of periodontal diseases.

Background: Single nucleotide variants in toll-like receptor genes play a crucial role in leprosy susceptibility or resistance. Methods: With an epidemiology case–control study, associations between SNVs rs5743618 in TLR1, rs5743708 in TLR2, and rs5743810 in TLR6 and overall susceptibility for leprosy were estimated in 114 cases and 456 controls. Following that, stratified analysis was performed. DNA was extracted from peripheral blood. Genotyping was performed using predesigned TaqMan probes. Results: The A/G genotype of rs5743810 behaved as a protective factor for the development of leprosy in the codominant (OR= 0.37; 95% CI = 016–0.86, p = 0.049) and over-dominant (OR = 0.38; 95% CI = 0.16–0.88, p = 0.019) inheritance models. The A/G and A/A genotypes behaved as a protective factor (OR = 0.39; 95% CI = 0.17–0.87, p = 0.016) in the dominant model. The SNVs rs5743618 and rs5743708 showed no association with any of the models. The CGG haplotype (rs5743618–rs5743708–rs5743810) behaved as a susceptibility factor for developing leprosy (OR = 1.86; 95% CI = 1.11–3.10, p = 0.019). The latter haplotype behaved as a susceptibility factor for leprosy development in women (OR = 2.39; 95% CI = 1.21–4.82, p = 0.013). Conclusions: The identified variants in the genes encoding TLRs, specifically rs5743810 in TLR6 and CGG (rs5743618–rs5743708–rs5743810) haplotypes, may somehow explain leprosy susceptibility in the studied population in a leprosy endemic region in Colombia.

Leprosy is a chronic disease and also a global health issue, with a high number of new cases per year. Toll-like receptors can respond to mycobacterial molecules in the early stage of infection. As important components of the innate immune response, alterations in genes coding for these receptors may contribute to susceptibility/protection against diseases. In this context, we used a casecontrol study model (183 leprosy cases vs. 185 controls) to investigate whether leprosy patients and the control group, in southern Brazil, have different frequencies in TLR1 (TLR1 G>T; rs5743618), TLR2 (TLR2 T>C, rs1816702 and rs4696483), and TLR4 (TLR4 A>G, rs1927911) polymorphisms. Analysis of the TLR1 1805G>T polymorphism presented the G/G genotype more frequently in the control group. TLR2 T>C rs1816702 and TLR2 T>C rs4696483, the T/T and C/T genotype, respectively, were more frequent in the control group than in leprosy patients, suggesting protection from leprosy when the T allele is present (rs4696483). Haplotype analyses between TLR1 (rs5743618) and TLR2 (rs1816702 and rs4696483) polymorphisms suggest risk for the presence of the TCC haplotype and protection in the presence of the TCT haplotype. This study suggests that polymorphisms in TLR1 and TLR2 are factors that may contribute to development/resistance of leprosy. [ABSTRACT FROM AUTHOR]

Leprosy is a chronic disease and also a global health issue, with a high number of new cases per year. Toll-like receptors can respond to mycobacterial molecules in the early stage of infection. As important components of the innate immune response, alterations in genes coding for these receptors may contribute to susceptibility/protection against diseases. In this context, we used a case-control study model (183 leprosy cases vs. 185 controls) to investigate whether leprosy patients and the control group, in southern Brazil, have different frequencies in TLR1 (TLR1 G>T; rs5743618), TLR2 (TLR2 T>C, rs1816702 and rs4696483), and TLR4 (TLR4 A>G, rs1927911) polymorphisms. Analysis of the TLR1 1805G>T polymorphism presented the G/G genotype more frequently in the control group. TLR2 T>C rs1816702 and TLR2 T>C rs4696483, the T/T and C/T genotype, respectively, were more frequent in the control group than in leprosy patients, suggesting protection from leprosy when the T allele is present (rs4696483). Haplotype analyses between TLR1 (rs5743618) and TLR2 (rs1816702 and rs4696483) polymorphisms suggest risk for the presence of the TCC haplotype and protection in the presence of the TCT haplotype. This study suggests that polymorphisms in TLR1 and TLR2 are factors that may contribute to development/resistance of leprosy.

CD71+ erythroid cells (CECs) were only known as erythrocyte progenitors not so long ago. In present times, however, they have been shown to be active players in immune regulation, especially in immunosuppression by the means of ROS, arginase-1 and arginase-2 production. Here, we uncover organ-of-origin differences in cytokine gene expression using NanoString and protein production using Bio-Plex between CECs from healthy human adult bone marrow and from human fetal liver parenchyma. Namely, healthy human adult bone marrow CECs both expressed and produced IFN-a, IL-1b, IL-8, IL-18 and MIF mRNA and protein, while human fetal liver parenchyma CECs expressed and produced IFN-a, IL15, IL18 and TNF-b mRNA and protein. We also detected TLR2 and TLR9 gene expression in both varieties of CECs and TLR1 and NOD2 gene expression in human fetal liver parenchyma CECs only. These observations suggest that there might be undiscovered roles in immune response for CECs. [ABSTRACT FROM AUTHOR]

Alopecia areata (AA) is a common non-scarring hair loss disease of defined patterns with varied patches size and body sites. The etiology of AA has a complex basis of autoimmunity, environment, and genetic variations. The latter factor is found to play a crucial role in AA risk. Thus, this study aimed to investigate the potential impact of specific immune-related gene polymorphisms among a cohort of Jordanian patients, which was previously reported in other populations. Blood samples of AA patients and control subjects were collected for genomic DNA (gDNA) extraction. Targeted single nucleotide polymorphisms (SNPs) of MASP2, TLR1, CTLA4, and C11orf30 were genotyped in duplicate using the Sequenom MassARRAY® system (iPLEX GOLD). Genotype and allele analysis reveals statistical differences in TLR1 rs4833095 (allele C, P = 0.044), MASP2 rs2273346 (genotype AA, P = 0.0026), and C11orf30 rs2155219 (genotype GG, P = 0.0069) distribution. These findings present the significant contribution of genetic variations in AA susceptibility in the Jordanian population, which is infrequently studied.

TLRs are the first and best-characterized pattern recognition receptors conserved across all the species. Different from mammals, the TLRs in teleost fishes are very diversified due to various evolutionary mechanisms. Here, we characterized one TLR1 gene in turbot, with a 2,415 bp open reading frame (ORF), that encoding 804 amino acid residues, and have the highest similarity and identity both to Paralichthys olivaceus with 88.9% and 79.9%. In phylogenetic analysis, it was firstly clustered with P. olivaceus , and then clustered with Takifugu rubripes. TLR1 was widely expressed in all the examined healthy tissues with the highest expression level in spleen, followed by head-kidney. In addition, it was significantly regulated in gill, skin and intestine following Edwardsiella tarda and Vibrio anguillarum challenge with different expression patterns. In in vitro stimulation with pathogen-associated molecular patterns, TLR1 showed significantly strong and elevated responses to LPS, but only responded to LTA and Poly(I:C) at the highest evaluated concentration, while no response was detected using PGN stimulation. Moreover, in subcellular localization analysis, TLR1 was distributed in the cytoplasm, membrane and nucleus. Taken together, TLR1 played vital roles for host immune response to bacterial infection, only with strong binding ability to LPS and involved in the production of inflammatory cytokines. However, the specific ligand for TLR1 and its functional association with other TLRs should be further characterized in fish species. • The TLR1 gene from turbot exhibited highly similarities with that in other teleosts. • Turbot TLR1 was widely expressed in all the examined tissues and significantly regulated in mucosal tissues following different bacteria challenge. • Turbot TLR1 showed much more significantly strong responses to LPS than LTA, poly I:C and PGN stimulation.. • Turbot TLR1 was distributed in the cytoplasm, membrane and nucleus. [ABSTRACT FROM AUTHOR]

ABSTRACTMelioidosis, an infectious disease caused by the bacterium Burkholderia pseudomallei, is a common cause of sepsis in Southeast Asia. We investigated whether novel TLR1 coding variants are associated with outcome in Thai patients with melioidosis. We performed exonic sequencing on a discovery set of patients with extreme phenotypes (mild vs. severe) of bacteremic melioidosis. We analysed the association of missense variants in TLR1 with severe melioidosis in a by-gene analysis. We then genotyped key variants and tested the association with death in two additional sets of melioidosis patients. Using a by-gene analysis, TLR1 was associated with severe bacteremic melioidosis (P = 0.016). One of the eight TLR1 variants identified, rs76600635, a common variant in East Asians, was associated with in-hospital mortality in a replication set of melioidosis patients (adjusted odds ratio 1.71, 95% CI 1.01–2.88, P = 0.04.) In a validation set of patients, the point estimate of effect of the association of rs76600635 with 28-day mortality was similar but not statistically significant (adjusted odds ratio 1.81, 95% CI 0.96–3.44, P = 0.07). Restricting the validation set analysis to patients recruited in a comparable fashion to the discovery and replication sets, rs76600635 was significantly associated with 28-day mortality (adjusted odds ratio 3.88, 95% CI 1.43–10.56, P = 0.01). Exonic sequencing identifies TLR1 as a gene associated with a severe phenotype of bacteremic melioidosis. The TLR1 variant rs76600635, common in East Asian populations, may be associated with poor outcomes from melioidosis. This variant has not been previously associated with outcomes in sepsis and requires further study.

Identification of genetic predispositions to susceptibility towards bovine tuberculosis (bTB) for the purpose of improving herd resistance to bTB through marker/genomic selection has been one of the thrust areas of research in animal production. Immune response genes, such as Toll Like Receptors (TLRs) which are involved in mycobacterial recognition and activation of innate and adaptive immune responses and Tumor Necrosis Factor alpha (TNFa) which is a vital pro-inflammatory cytokine with pleiotropic roles in immune response that enables mounting of a strong microbicidal action, are potential strong candidates for exploring genetic basis of resistance. Present investigation was aimed at exploring the association of three SNPs (rs43702940, rs68343175 and rs55617317) in TLR1 gene and one SNP (rs109967811) in TNFa gene with susceptibility to bTB infection in cattle. In a case-control population of bTB established using single intradermal tuberculin test, three of the investigated SNPs were found to be polymorphic while rs43702940 revealed monomorphism. SNP loci rs109967811 in TNFa gene was found to be significantly (P < 0.01) associated with susceptibility to bTB in the study population. These findings suggest that SNPs rs109967811 located in exonic region of TNFa can likely serve as a potential marker against bTB infection in cattle upon validation in independent, larger resource population. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Bone marrow mesenchymal stem cells (BMSCs) promote bone tissue repair. MiR-1 regulates myogenic and osteogenic differentiation of human adipose tissue stem cells. However, miR-1's effect on BMSCs osteogenesis is unclear. MATERIALS AND METHODS: Rat BMSCs were isolated and divided into control group, miR-1 group, and si-miR-1 group respectively transfected with miR-1 plasmid and miR-1 siRNA followed by analysis of cell proliferation by MTT assay and Caspase 3 activity. The expression of osteogenic genes Runx2 and OPN was measured by Real Time-PCR. Healthy male Sprague-Dawley rats were separated into fracture group, NC group, and si-miR-1 group followed by analysis of bone mineral density, miR- 1 level by Real Time-PCR, type I collagen, and BMP-2 by enzyme-linked immunosorbent assay (ELISA), and TLR1 expression by Western blot. RESULTS: Transfection of miR-1 siRNA into BMSCs significantly downregulated miR-1 expression, promoted BMSCs cell proliferation, inhibited Caspase 3 activity, as well as promoted osteogenic genes Runx2 and OPN expression and decreased TLR1 expression (p<0.05). The upregulation of miR-1 expression significantly reversed the above changes. TLR1 is a target of miR-1. Downregulation of miR-1 expression in BMSCs of fractured rats significantly increased bone density and ALP activity, promoted type I collagen and BMP-2 expression, and decreased TLR1 expression (p<0.05). CONCLUSIONS: The downregulation of miR-1 promotes BMSCs osteogenic differentiation via targeting TLR1, which promotes osteogenic differentiation and bone healing. [ABSTRACT FROM AUTHOR]

The extraordinary success of Mycobacterium tuberculosis (M. tb) has been attributed to its ability to modulate host immune responses, and its genome encodes multiple immunomodulatory factors, including several proteins of the multigenic PE_PPE family. To understand its role in M. tb pathophysiology we have characterised the PPE50 (Rv3135) - PPE51 (Rv3136) gene cluster, one of nine PPE–PPE clusters in the genome. We demonstrate here that this cluster is operonic, and that PPE50 and PPE51 interact - the first demonstration of PPE–PPE interaction. THP-1 macrophages infected with recombinant Mycobacterium smegmatis strains expressing PPE50 and PPE51 showed lower intracellular viability than the control, which correlated with an increase in transcript levels of iNOS2. Infected macrophages also exhibited an upregulation in levels of IL-10 , indicating an immunomodulatory role for these proteins. Using pull-downs and signalling assays, we identified TLR1 to be the cognate receptor for PPE50 - all the phenotypes observed on infection of THP-1 macrophages were reversed on pre-treatment with an anti-TLR1 antibody, validating the functional outcome of PPE50-TLR1 interaction. Our data reveals a TLR1 dependent role for the PPE50-PPE51 cluster in promoting bacillary persistence, via CFU reduction and concomitant upregulation of the anti-inflammatory response - a two-pronged strategy to circumvent host immune surveillance. [ABSTRACT FROM AUTHOR]

Background: Patients receiving home parenteral nutrition (HPN) have an increased risk for central line-associated bloodstream infections (CLABSIs), including candidemia. Recently, 7 single-nucleotide polymorphisms (SNPs) in TLR1, CD58, LCE4A-Clorf68, and TAGAP have been associated with the development of candidemia. Identification of host-genetic as well as clinical risk factors may help to identify patients who have an increased susceptibility to such infections. The aim of this study was to investigate the relevance of the reported SNPs in patients receiving HPN, and to explore clinical risk factors associated with candidemia.Methods: We analyzed blood samples of adult patients who started HPN between 1976 and 2017 at our referral center for intestinal failure. Primary outcome was the association between TLR1, CD58, LCE4A-Clorf68, or TAGAP SNPs and candidemia. Secondary outcomes included the relation between severity of infection and these SNPs, and clinical risk factors for candidemia.Results: Of 341 included patients, 42 (12%) experienced a candidemia (range 1-6). None of the 7 SNPs were associated with candidemia or the severity of infection. The rate of non-Candida-related CLABSIs was significantly associated with candidemia (rate ratio, 1.29; 95% CI, 1.14-1.46; P < 0.001).Conclusions: None of 7 known SNPs in TLR1, CD58, LCE4A-Clorf68, or TAGAP were associated with candidemia or severity of infection in patients receiving HPN. The rate of non-Candida-related CLABSIs was significantly associated with the development of candidemia. The latter supports the key role of aseptic catheter handling with respect to Candida susceptibility in patients receiving HPN. [ABSTRACT FROM AUTHOR]

Toll‐like receptors are membrane‐bound proteins which plays a vital role in the regulation of innate immune response which is involved in various inflammatory disorders. 3,6‐Dihydroxyflavone (3,6‐DHF) is a known chemo preventive agent which is used in the treatment of various cancers including breast cancer and an effective JNK kinase inhibitor which can efficiently block the TLR2‐mediated signaling pathways. In this current study, we have explored inhibition of 3,6‐DHF in TLR2/TLR1 heterodimerization. We stimulated HEK cells by Pam3CSK4 which specifically induced inflammation through TLR2/TLR1 binding. Secretion of the inflammatory cytokine in Pam3CSK4‐induced HEK293‐hTLR2 cells was considerably reduced by 3,6‐DHF, implying that 3,6‐DHF inhibited Pam3CSK4‐induced TLR2/TLR1 signaling specifically. In addition, 3,6‐DHF did not cause severe cytotoxicity against human embryonic kidney (HEK) cells at high concentration up to 100 μM. The binding affinity of 3,6‐DHF to TLR2 and TLR1 was explored with 10−5 affinity using bio‐layer interferometry and molecular docking studies identified the important active site residues that participate in the inhibition of TLR2/TLR1 heterodimerization. Our results showed that 3,6‐DHF inhibits TLR2‐mediated inflammatory signaling by direct binding and the insights in designing more potent drug candidates by targeting the interacting crucial active site residues in TLR2/TLR1. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) of teleost innate immune system. However, information about TLRs is absent in Dabry's sturgeon ( Acipenser dabryanus ), one of the most primitive actinopterygii species. In the present study, the full lengths of adaTLR1 and adaTLR4 were cloned from Dabry's sturgeon using RT-PCR and RACE-PCR. The obtained adaTLR1 was 2957 bp in length, encoding a putative protein of 767 amino acids and adaTLR4 cDNA was 2902 bp in length, encoding a putative protein of 830 aa. Both adaTLR1 and adaTLR4 possessed several typical TLRs motifs, including signal peptides, leucine-rich repeat (LRR) motifs, a transmembrane domain and a TIR motifs. In addition, adaTLR4 contained three conserved boxes in its TIR motif, involving in TLRs signal transduction. A proline, important for LPS recognition of mammalian TLR4, was also found in adaTLR4. Physicochemical features of adaTLR1 and adaTRL4 were also analyzed. Quantitative realtime PCR showed that both transcripts were ubiquitously expressed in all 11 normal tissues selected, but they exhibited different expression patterns, with adaTLR1 highly expression in heart and adaTLR4 highly in skin. Further, adaTLR1 and adaTLR4 were up-regulated in the primary head-kidney leucocytes following LPS and polyI:C stimulation, indicating that both genes involved in the sturgeon immune response to LPS and polyI:C. To our best knowledge, this was the first report of these genes in sturgeon and these results provided the basis for further elucidating the ligand specificity and signaling pathway of fish TLRs. [ABSTRACT FROM AUTHOR]

Abstract: Toll‐like receptors (TLRs) play an indispensable role in fish immunity, being involved in pathogen recognition and the triggering of immune reactions. Here, a member of the TLR family, TLR1, from Lateolabrax japonicus was characterized and its expression pattern and intracellular localization were analysed. The full‐length LjTLR1 cDNA (2,755 bp) was found to encode a polypeptide of 827 amino acids. The deduced amino acid sequence contained three main structural domains: an extracellular leucine‐rich repeat domain, a transmembrane domain and a Toll/IL‐1 receptor domain. Tissue distribution analysis indicated that LjTLR1 was expressed in all of the examined tissues to varying degrees, with the highest levels being measured in the head kidney. In order to assess the antibacterial functions of LjTLR1 during infection, the pathogenic bacteria Vibrio harveyi and Streptococcus agalactiae were used. LjTLR1 was significantly upregulated in the three immune organs (the head kidney, spleen and liver) following bacterial stimulation, and its expression was detected 6 hr after initial exposure. In mRNA in situ hybridization experiments, positive signals were more numerous in the treatment group than the control group, verifying the expression patterns observed. Assessment of the intracellular localization of LjTLR1 revealed it to be present in the cytoplasm. These results indicate the potential role of LjTLR1 in immune responses to bacterial infection. This study enriches our knowledge of L. japonicus immune genes and provides a theoretical basis for further research concerning the antibacterial functions of fish TLRs during infection. [ABSTRACT FROM AUTHOR]

Background Toll like receptors (TLRs) are an important and evolutionary conserved class of pattern recognition receptors associated with innate immunity. The recognition of Gram-positive cell wall constituents strongly depends on TLR2. In order to be functional, TLR2 predominantly forms a heterodimer with TLR1 or TLR6 within specialized membrane microdomains, the lipid rafts. The membrane lipid composition and the physicochemical properties of lipid rafts are subject to modification by exogenous fatty acids. Previous investigations of our group provide evidence that macrophage enrichment with polyunsaturated fatty acids (PUFA) induces a reordering of lipid rafts and non-rafts based on the incorporation of supplemented PUFA as well as their elongation and desaturation products. Methods In the present study we investigated potential constraining effects of membrane microdomain reorganization on the clustering of TLR2 with its co-receptors TLR1 and TLR6 within lipid rafts. To this end, RAW264.7 macrophages were supplemented with either docosahexaenoic acid (DHA) or arachidonic acid (AA) and analyzed for receptor expression and microdomain localization in context of TLR stimulation. Results and Conclusions Our analyses showed that receptor levels and microdomain localization were unchanged by PUFA supplementation. The TLR2 pathway, in contrast to the TLR4 signaling cascade, is not affected by exogenous PUFA at the membrane level.

Background and Objectives: This study aimed to investigate the possible association between exclusive breastfeeding duration during early infancy and susceptibility to allergy and influenza in adulthood. Furthermore, we also investigated the association of breastfeeding duration with DNA methylation at two sites in the promoter of the toll-like receptor-1 (TLR1) gene, as well as the association between DNA methylation of the toll-like receptor-1 (TLR1) gene and susceptibility to different diseases. Materials and Methods: Blood samples were collected from 100 adults and classified into two groups according to breastfeeding duration (TLR1 gene was then amplified by polymerase chain reaction (PCR) and sequenced. Results: We found a significant association between increased breastfeeding duration and a reduction in susceptibility to influenza and allergy, as well asa significant reduction in DNA methylation within the promoter of the TLR1 gene. No association was found between DNA methylation and susceptibility to different diseases. Conclusions: The findings demonstrate the significance of increased breastfeeding duration for improved health outcomes at the gene level.

Background: Toll like receptors (TLRs) are an important and evolutionary conserved class of pattern recognition receptors associated with innate immunity. The recognition of Gram-positive cell wall constituents strongly depends on TLR2. In order to be functional, TLR2 predominantly forms a heterodimer with TLR1 or TLR6 within specialized membrane microdomains, the lipid rafts. The membrane lipid composition and the physicochemical properties of lipid rafts are subject to modification by exogenous fatty acids. Previous investigations of our group provide evidence that macrophage enrichment with polyunsaturated fatty acids (PUFA) induces a reordering of lipid rafts and non-rafts based on the incorporation of supplemented PUFA as well as their elongation and desaturation products. Methods: In the present study we investigated potential constraining effects of membrane microdomain reorganization on the clustering of TLR2 with its coreceptors TLR1 and TLR6 within lipid rafts. To this end, RAW264.7 macrophages were supplemented with either docosahexaenoic acid (DHA) or arachidonic acid (AA) and analyzed for receptor expression and microdomain localization in context of TLR stimulation. Results and Conclusions: Our analyses showed that receptor levels and microdomain localization were unchanged by PUFA supplementation. The TLR2 pathway, in contrast to the TLR4 signaling cascade, is not affected by exogenous PUFA at the membrane level. [ABSTRACT FROM AUTHOR]

Psoriasis is a common chronic inflammatory skin disease. RIPK1 plays an important role in inflammatory diseases. At present, the clinical efficacy of the RIPK1 inhibitor is limited and the regulatory mechanism is unclear in the treatment of psoriasis. Therefore, our team developed a new RIPK1 inhibitor, NHWD-1062, which showed a slightly lower IC50 in U937 cells than that of GSK'772 (a RIPK1 inhibitor in clinical trials) (11 nM vs. 14 nM), indicating that the new RIPK1 inhibitor was no less inhibitory than GSK'772. In this study, we evaluated the therapeutic effects of NHWD-1062 using an IMQ-induced mouse model of psoriasis and explored the precise regulatory mechanism involved. We found that gavage of NHWD-1062 significantly ameliorated the inflammatory response and inhibited the abnormal proliferation of the epidermis in IMQ-induced psoriatic mice. We then elucidated the mechanism of NHWD-1062, which was that suppressed the proliferation and inflammation of keratinocytes in vitro and in vivo through the RIPK1/NF-κB/TLR1 axis. Dual-luciferase reporter assay indicated that P65 can directly target the TLR1 promoter region and activate TLR1 expression, leading to inflammation. In summary, our study demonstrates that NHWD-1062 alleviates psoriasis-like inflammation by inhibiting the activation of the RIPK1/NF-κB/TLR1 axis, which has not been previously reported and further provides evidence for the clinical translation of NHWD-1062 in the treatment of psoriasis. [Display omitted] • The new RIPK1 inhibitor, NHWD-1062, can effectively inhibit the activity of RPIK1 kinase. • NHWD-1062 alleviates IMQ-induced ear inflammation and epidermal proliferation in mice. • The expression of psoriasis-related cytokines can be inhibited by NHWD-1062 via the RIPK1/NF-κB/TLR1 pathway. • NHWD-1062 is expected to be a new oral drug for the treatment of psoriasis. [ABSTRACT FROM AUTHOR]

Background: Toll-like receptors (TLRs) are the major pattern recognition receptors that mediate sensing of a wide range of microorganisms. TLR2 forms heterodimers with either TLR1 or TLR6, broadening its ligand diversity against pathogens. TLR1, TLR2 and TLR6 have been implicated in the recognition of Candida albicans, an opportunistic fungal pathogen that colonizes the gastrointestinal tract. In this study, we explored whether the deficiency in TLR1, TLR2 or TLR6 impacts C. albicans colonization and inflammation-associated colonic injury in the dextran sulfate sodium (DSS)-induced colitis in mice. Results: DSS treatment and C. albicans challenge induced greater weight loss, worse clinical signs of inflammation, higher histopathologic scores, and increased mortality rates in TLR1-/- and TLR2-/- mice when compared to TLR6-/- and wild-type mice. The number of C. albicans colonies in the stomach, colon and feces was decreased in TLR6-/- mice as compared to TLR2-/-, TLR1-/- and wild-type mice. Interestingly, the population of E. coli in colonic luminal contents, intestinal permeability to FITC-dextran and cytokine expression were significantly increased in TLR1-/- and TLR2-/- mice, while they were decreased in TLR6-/- mice. Conclusion: In contrast to TLR6, both TLR1 and TLR2 deficiencies increased intestinal inflammation, and the overgrowth of C. albicans and E. coli populations in the colitis model, suggesting the involvement of TLR1 and TLR2 in epithelial homeostasis, and a role of TLR6 in increasing intestinal inflammation in response to pathogen-sensing. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are a group of pattern-recognition receptors which play vital roles in ligand recognition and activation of the innate immune response. As an important member of TLRs family, TLR1 is mainly responsible for PAMPs from bacteria and play a pivotal role in sensing microbial products. Recent studies revealed that TLR1 could perceive LPS stimulation and transfer signals to activate the NF-κB pathway, whereas ligands and signaling pathway of TLR1 are still unclear in fish. Growing evidence has shown that miRNAs (microRNAs) play as negative regulators in controlling the diverse of biophysical and biochemical processes at the post-transcriptional level. In this study, we used a combination of bioinformatics and experimental techniques to exhibit that both miR-8159-5p and miR-217-5p were the direct negative regulators of TLR1 in miiuy croaker. Furthermore, dual-luciferase reporter assays showed that combining miR-8159-5p and miR-217-5p exhibited a greater negative regulatory effect on TLR1 than only miR-8159-5p or miR-217-5p. Additionally, we also demonstrated that the expression of both the two miRNAs could be up-regulated by LPS stimulation in either LPS-stimulation spleen tissue or LPS-treated cultured macrophage, which indicating that miR-8159-5p and miR-217-5p could be induced by LPS and may be as the negative regulators of TLR1 involved in the immune response to LPS stimulation. These results would enhance our understanding of the miRNA regulation in fish TLR signaling pathways. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs which post-transcriptionally regulate various biological processes by repressing mRNA translation or degradating mRNA. It has been demonstrated that miRNAs play crucial roles in regulating the immune system. In this study, we explored the potential roles of miR-200a-3p in regulating TLR signaling pathway in miiuy croaker. Bioinformatics analysis showed that miiuy croaker TLR1 (mmiTLR1) was a putative target of miR-200a-3p. Negative expression profiles in spleen of Vibrio anguillarum challenged miiuy croaker and in lipopolysaccharide (LPS) stimulated miiuy croaker leukocytes further validated the prediction. Luciferase reporter assays showed that the dual-luciferase reporter fused to the 3′UTR of wild type mmiTLR1 cotransfected with miR-200a-3p mimics exhibited a reduction in luciferase activity compared with the controls. All of the present data provide direct evidence that miR-200a-3p is involved in TLR1 expression modulation in miiuy croaker, which will offer a basis for better understanding of miRNA regulation in fish TLR signaling pathways. [ABSTRACT FROM AUTHOR]

TLR2 heterodimers with TLR1 or TLR6 recognize distinct pathogen-associated molecules such as tri- and di-acylated lipopeptides. The activated TLR2 heterodimers recruit Toll-IL-1R domain- (TIR-) containing adapter proteins, TIRAP and MyD88, through the receptor TIR domains. Molecular recognition mechanisms responsible for agonist-driven, TIR domain-mediated receptor-adapter interactions as well as the structure of resultant signaling complexes remain unknown. We previously reported that the cell-permeable peptide derived from helix D of TLR2 TIR (2R9) specifically binds TIRAP in vitro and in cells and thereby inhibits TIRAP-dependent TLR signaling. This study demonstrates that cell-permeable peptides from D helix of TLR1 or TLR6, peptides 1R9 and 6R9 respectively, inhibit signaling mediated by cognate TLR2 co-receptors. Interestingly, 1R9 and 6R9 bind different TLR2 adapters, as they selectively bind MyD88 and TIRAP TIR, respectively. Both peptides block the agonist-induced co-immunoprecipitation (co-IP) of TLR2 with TIRAP or MyD88, but not TLR2 co-IP with co-receptors. Our data suggest that D helices of TLR1 and TLR6 TIR domains are adapter recruitment sites in both co-receptors; yet the sites recruit different adapters. The D helix in TLR1 is the MyD88 docking site, whereas in TLR6 this site recruits TIRAP. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are well-described pattern recognition receptors that recognize different pathogen-associated molecular patterns (PAMPs) to activate innate immune responses. TLR1 forms a heterodimer with TLR2 that works to identify PAMPs including bacterial components. In the present study, we identified a TLR1 homolog from rock bream Oplegnathus fasciatus ( RbTLR1) and characterized it at the genomic level. The complete coding sequence of RbTLR1 was 2406 bp in length and encoded an 802 amino acid protein possessing a calculated molecular mass of 90.7 kDa and typical TLR domain architecture. A single exon spanned the genomic length (2931 bp) of RbTLR1. Phylogenetic analysis showed a close evolutionary relationship between RbTLR1 and its fish orthologs. Using quantitative real time polymerase chain reaction, we detected RbTLR1 mRNA in all the tissues examined, suggesting its merely universal tissue expression. RbTLR1 was expressed strongly in the spleen, kidney, and liver. Following immune stimulation by bacterial pathogens and mitogens, RbTLR1 transcript levels were elevated at various time points post stimulation. Moreover, injection of a viral stimulant, polyinosinic:polycytidylic acid, and rock bream iridovirus altered the basal transcription of RbTLR1. Collectively, these data showed that RbTLR1 is transcriptionally regulated in response to bacterial and viral stimulation, suggesting its putative importance in host immune defense. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) may contribute to the process of autoimmune attacks on hair follicles. To investigate whether the TLR1 gene polymorphisms are associated with the development and clinical features of alopecia areata (AA), a case-control comparison of two single nucleotide polymorphisms (SNPs) (rs4833095, Asn248Ser and rs5743557, −414C > T) of TLR1 were studied in 239 AA patients and 248 controls. Using multiple logistic regression model, odds ratios, 95% confidence intervals and corresponding p values were estimated. Clinical features were analyzed based on the age of onset, family history, type of AA, nail involvement and body hair involvement. The missense SNP rs4833095 was significantly associated with the development of AA (codominant2, p = 0.002; recessive, p = 0.001; log-additive, p = 0.0071; and allele frequency, p = 0.0066). The promoter SNP rs5743557 was weakly associated with the development of AA (codominant2, p = 0.019; recessive, p = 0.032; log-additive, p = 0.020; and allele frequency, p = 0.03). In the clinical features, rs4833095 was only weakly associated with age of onset between 15 and 50 years (codominant2, p = 0.043 and recessive, p = 0.022). The results suggest that rs4833095 of TLR1 may be associated with the susceptibility for AA in the Korean population. [ABSTRACT FROM AUTHOR]

Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function. [ABSTRACT FROM AUTHOR]

Background: Advanced age results in crucial alterations of the innate and adaptive immune system leading to functional defects resulting in infection and chronic diseases. Toll-like receptors (TLR) recognize pathogenic structures and are important in the immune response to infections and vaccination. However, the role of TLR single nucleotide polymorphisms (SNP) is poorly understood in the setting of human ageing. This study investigated the impact of the TLR1 SNPs A743G and T1805G on ageing in different age groups from two European populations. Results: The TLR1 genotypes 743AA/1805GG (TLR1neg) are associated with a TLR1 negative phenotype, impaired function and susceptibility to tuberculosis. Carriers of heterozygous 743AG/1805TG and homozygous 743GG/1805TT genotypes (TLR1pos) have a TLR1 positive phenotype. By comparing healthy young and old German donors, the old group showed a tendency to carry more TLR1neg and less homozygous TLR1pos genotypes. Anti-inflammatory Interleukin (IL)-1 receptor antagonist (Ra) was significantly elevated in supernatants of mononuclear cells from old German subjects with a TLR1pos genotype in contrast to those with the 743AA genotype. Healthy old individuals and nonagenarians from Italy displayed significantly higher frequencies of TLR1pos genotypes than the old group from Germany. The data show that tumor-necrosis-factor (TNF)α, CXCL8 and CCL2 levels were higher in old donors from Germany than in plasma levels from old Italian donors. TNFa and CCL2 levels were significantly raised in old German individuals compared to Italian nonagenarians. German and Italian donors with the TLR1neg genotype basically produced more CCL2 than older European donors with TLR1pos genotypes. Conclusion: The higher frequency of the TLR1pos genotype in elderly Italian subjects may result from different ethnic populations. Lower inflammatory mediator release of aged Italian individuals is probably due to different background in nutrition, diet, genetics and to psychological aspects. Elderly donors carrying TLR1pos genotypes basically release more anti-inflammatory IL-1Ra and less inflammatory CCL2 suggesting a decline of the pro-inflammatory status found in ageing and, therefore, this may define an anti-inflammatory phenotype. Future studies are needed to elucidate the association of a TLRpos genotype with decreased susceptibility to infections and reduced risk to develop artherosclerosis. [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptors (TLRs) are a class of pattern recognition receptors that play a pivotal role in the innate and adaptive immune systems. Studies have shown that TLR variants play roles in various human infectious diseases. The aim of the present study was to investigate whether the functional genetic variations at positions C632T, G1409A, A1475C, G1550A and G1596A in TLR1 and at A2700G and A3156G in TLR9 confer susceptibility or resistance to bovine tuberculosis (bTB). Genotyping of the TLR1 and TLR9 gene polymorphisms was performed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and single-strand conformation polymorphism (PCR-SSCP) in 586 Chinese Holstein cows (154 infected with bTB, 432 non-infected). The frequencies of the GH and HH genotypes at TLR1-G1596A differed significantly between bTB-infected and non-infected animals [p =0.001 for both GH and HH; GH odds ratio (OR)=2.43 95% confidence interval (CI) (1.47–4.03); and HH OR=1.49 95% CI (0.85–2.62)]. There was a trend toward an increased relative risk of tuberculosis (TB) incidence in the CD genotype at the TLR1-A1475C locus [p =0.056, OR=1.59 95% CI (0.98–2.58)]. The present study suggests that variants in the TLR1 gene are associated with susceptibility to bTB, whereas no significant association can be inferred from the polymorphisms in the TLR9 gene. [Copyright &y& Elsevier]

Abstract: Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5′ UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/6 and TLR2/1 agonists diacylated lipoprotein (Pam2CSK4) and triacylated lipoprotein (Pam3CSK4). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling are critical for future elucidation of omTLR1 functions. [Copyright &y& Elsevier]

Abstract: Placentae from newborns with congenital human cytomegalovirus (HCMV) infection often display shallow implantation, chronic villitis and disruptions of the syncytiotrophoblast. Little is known about how HCMV infection induces inflammation in the placenta and loss of the trophoblast. We propose that the inflammation is initiated with innate defense responses of mature syncytiotrophoblast (ST) to virus. Previously we have shown that ultraviolet irradiation-inactivated (UV-) HCMV interacts with toll-like receptor 2 (TLR2) on primary placental cytotrophoblasts (CT) differentiated into ST-like cells thereby stimulating the release of tumor necrosis factor alpha (TNFα) and inducing apoptosis of neighboring cells (Chan et al, J. Pathol. 210: 111, 2006). We now determine whether known co-factors of the interaction of HCMV and TLR2 (TLR1 and CD14) bind to UV-HCMV to stimulate expression of TNFα and apoptosis in ST-like cells but not CT. We show that CT both fail to express detectable TLR1 and express much less CD14 than ST and that ST express CD14 but not TLR1 both in vivo and in cultured cells. The interaction of UV-HCMV and HCMV with CD14 on the surface of ST-like cells increases TNFα expression and induces apoptosis in the population. Antibody to CD14 also inhibits infectious HCMV induction of HCMV immediate early (HCMV IE) expressing ST-like cells. We conclude that primary villous CT express low levels of CD14 and no TLR1 but that ST strongly expresses CD14 which acts upstream of TLR2 to collect even transcriptionally inactive virus particles to stimulate TNFα expression and villous trophoblast damage. [Copyright &y& Elsevier]

Abstract: Toll-like receptors (TLRs) are key sensors of pathogen-associated molecular patterns (PAMPs). Their role in immunity is difficult to examine in species of veterinary interest, due to restricted access to the knockout technology and TLR-specific antibodies. An alternative approach is to generate cell lines transfected with various TLRs and to examine the recognition of PAMPs or relevant bacteria. In this report, we examined whether recognition of various PAMPs and mastitis-causing bacteria is achieved by transfection of recombinant bovine TLR2 (boTLR2). Therefore, human embryonic kidney (HEK) 293 cells were transfected by whole boTLR2. A clonal analysis of stably transfected cells disclosed variable recognition of several putative TLR2 agonists although expressing similar amounts of the transgene and endogenous TLR6. One clone (clone 25) reacted by copious interleukin-8 (IL-8) production to several stimulants of TLR2 such as di-palmitoylated cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam2), a biochemical preparation of lipoteichoic acid from Staphylococcus aureus, a commercial preparation of peptidoglycan from S. aureus, and heat-killed Listeria monocytogenes (HKLM). TLR2-dependent induction of IL-8 release was stronger in medium containing human serum albumin than in medium containing fetal calf serum. Clone 25 cells responded to high concentrations of S. aureus and to Escherichia coli causing mastitis, but not to Streptococcus uberis and to Streptococcus agalactiae which also cause mastitis. Stimulation by S. aureus was relatively weak when compared (i) with stimulation of the same cells by HKLM and PAMPs derived from S. aureus, (ii) with a clone stably transfected with TLR4 and MD-2 and stimulated by E. coli causing mastitis, and (iii) with interferon-γ-costimulated bovine macrophages stimulated by S. aureus and S. agalactiae. Thus, clone 25 is suitable for studying the interaction of putative TLR2 agonists with bovine TLR2-transfected cells, provides a cell to search for TLR2-specific antibodies, and is a tool for studying the interaction of TLR2 with bacteria causing disease, e.g. mastitis, in cattle. [Copyright &y& Elsevier]

Toll-like receptor-1 (TLR1) and TLR6 are receptors of the TLR family that form heterodimers with TLR2. The role of TLR1 and TLR6 for the recognition of the fungal pathogen Candida albicans was investigated. TLR1 is not involved in the recognition of C. albicans, and TLR1 knock-out (TLR1−/−) mice showed a normal susceptibility to disseminated candidiasis. In contrast, recognition of C. albicans by TLR6 modulated the balance between Th1 and Th2 cytokines, and TLR6 knock-out mice displayed a defective production of IL-10 and an increased IFN-γ release. Production of the monocyte-derived cytokines tumor necrosis factor, IL-1, and IL-6 was normal in TLR6−/− mice, and this was accompanied by a normal susceptibility to disseminated candidiasis. In conclusion, TLR6 is involved in the recognition of C. albicans and modulates the Th1/Th2 cytokine balance, but this results in a mild phenotype with a normal susceptibility of TLR6−/− mice to Candida infection. [ABSTRACT FROM AUTHOR]

The last decade has revealed interesting insights into the initiation and pathophysiology of the innate immune system. Toll-like receptors are of key importance for this process and they are a family of receptors expressed mainly on leukocytes that recognize a variety of microbial products derived from bacteria, viruses, protozoa and fungi. As key players of innate immunity, TLRs and downstream signalling components are important target candidates for drug development. In this review, we focus on TLR2, which recognizes bacterial lipopeptide. TLR2 forms dimers with TLR1 or TLR6. The TLR2/TLR1 dimer recognizes triacylated lipopeptides, whilst the TLR2/TLR6 dimer recognizes diacylated lipopeptides. TLR2 has been implicated in several auto-immune and inflammatory conditions, and its role in disease pathogenesis has been supported by numerous reports of TLR2 polymorphisms in humans linked to disease. Here we discuss the potential of TLR2 as a drug target in autoimmune and inflammatory disease. [ABSTRACT FROM AUTHOR]