Toll-like Receptor 4 Gene Research Articles

Genome-Wide Identification and Characterization of Toll-like Receptors (TLRs) in Diaphorina citri and Their Expression Patterns Induced by the Endophyte Beauveria bassiana.

Toll-like receptors (TLRs) are pathogen recognition receptors (PRRs), which play key roles in helping the host immune system fight pathogen invasions. Systematic information on TLRs at the genome-wide level and expression profiling in response to endophytic colonization is very important to understand their functions but is currently lacking in this field. Here, a total of two TLR genes were identified and characterized in Diaphorina citri. The TLR genes of D. citri were clustered into five families according to the phylogenetic analysis of different species’ TLRs. The domain organization analyses suggested that the TLRs were constituted of three important parts: a leucine-rich repeat (LRR) domain, a transmembrane region (TR) and a Toll/interleukin-1 receptor (TIR) domain. The mRNA expression levels of the two TLR genes (DcTOLL and DcTLR7) were highly regulated in both nymphs and adults of D. citri. These results elucidated the potentiated TLR gene expression in response to endophytically colonized plants. Furthermore, the 3D structures of the TIR domain were highly conserved during evolution. Collectively, these findings elucidate the crucial roles of TLRs in the immune response of D. citri to entomopathogens systematically established as endophytes, and provide fundamental knowledge for further understanding of the innate immunity of D. citri.

Glycoside oleandrin downregulates toll-like receptor pathway genes and associated miRNAs in human melanoma cells

Melanoma accounts for the majority of skin cancer-related deaths. Nerium oleander is a plant known to be toxic and consumed due to the cardiac glycosides it contains. Oleandrin is a cardiac glycoside obtained from of N. oleander. Beside capable of inhibiting proliferation and metastasis of cancer cells, cardiac glycoside derivative compounds cause cardiovascular side effects. Because of cardiovascular toxicity of clinically used cardiac glycosides, it is necessary to investigate cardiac glycoside derivative compounds capable of inhibiting proliferation and metastasis of cancer cells.It is known that oleandrin has anticarcinogenic effects in other cancers. Previous studies have shown that toll-like receptors (TLRs) and their related microRNAs (miRNAs) are associated with cancer. Therefore, aim was to investigate the effect of oleandrin on genes and miRNAs associated with TLRs in A375 melanoma cells in this study. The effects of oleandrin on cell viability, cytokines, apoptosis were evaluated using XTT, ELISA and TUNEL analyses, respectively. The effect of oleandrin on expression of TLR genes and 5 associated miRNAs in A375 cells has been determined by qRT-PCR. In addition, the levels of MyD88, TLR2 and TLR4 proteins were analyzed by western blot method.ELISA indicated that oleandrin treatment (47 nM at 48 h) reduced the level of proinflammatory cytokine IFNG. TUNEL analysis showed that apoptosis rate was significantly increased in the oleandrin dose group. According to qRT-PCR results, there was a significant decrease in IRAK1, IRAK4, MyD88, TLR2-TLR7 and TRAF3 expressions in the oleandrin treated group compared to the control (untreated cell). Also, a significant decrease in TLR4 protein expression has been observed. In addition, oleandrin significantly downregulated the levels of hsa-miRNA-146a-5p and hsa-miRNA-21-5p.In conclusion, it has been observed that oleandrin has an effect on TLR pathway-related genes and miRNAs in melanoma cells. We show that TLRs pathways and hsa-miR-146a-5p and hsa-miR-21-5p can participate in the oleandrin molecular mechanism of action.

Toll-like Receptor 7 (TLR7) Is Expressed in Adipocytes and the Pharmacological TLR7 Agonist Imiquimod and Adipocyte-Derived Cell-Free Nucleic Acids (cfDNA) Regulate Adipocyte Function.

Endosome-localized Toll-like receptors (TLRs) 3 and 9 are expressed and functionally active in adipocytes. The functionality and role of TLR7 in adipocyte biology and innate immunity of adipose tissue (AT) is poorly characterized. We analyzed TLR7 mRNA and protein expression in murine 3T3-L1 and primary adipocytes, in co-cultures of 3T3-L1 adipocytes with murine J774A.1 monocytes and in human AT. The effects of TLR7 agonists imiquimod (IMQ) and cell-free nucleic acids (cfDNA) on adipokine concentration in cell-culture supernatants and gene expression profile were investigated. We found that TLR7 expression is strongly induced during adipocyte differentiation. TLR7 gene expression in adipocytes and AT stroma-vascular cells (SVC) seems to be independent of TLR9. IMQ downregulates resistin concentration in adipocyte cell-culture supernatants and modulates gene expression of glucose transporter Glut4. Adipocyte-derived cfDNA reduces adiponectin and resistin in cell-culture supernatants and potentially inhibits Glut4 gene expression. The responsiveness of 3T3-L1 adipocytes to imiquimod is preserved in co-culture with J774A.1 monocytes. Obesity-related, adipocyte-derived cfDNA engages adipocytic pattern recognition receptors (PRRs), modulating AT immune and metabolic homeostasis during adipose inflammation.

Comprehensive Bioinformatics Analysis of Toll-Like Receptors (TLRs) in Pan-Cancer.

Background To conduct a comprehensive bioinformatics analysis on the transcriptome signatures of Toll-like receptors (TLRs) in pan-cancer. Materials and methods. A total of 11,057 tissues consisting of 33 types of carcinoma in The Cancer Genome Atlas (TCGA) were retrieved, and then we further explored the correlation between TLRs' expression with tumorigenesis, immune infiltration, and drug sensitivity. We conducted a comprehensive bioinformatics analysis on TLR1 to 10 in pan-cancer, including differential expression analysis between normal and tumor tissues, differential immune subtype correlation, survival analysis, tumor immune infiltration estimating, stemness indices correlation, and drug responses correlation. Results TLR2 was highly expressed in most types of tumors. TLR9 was hardly expressed compared to other TLR genes, which lead to TLR9 showing less correlation with both immune-estimate scores and stromal-estimate scores. All the TLRs were related with immune subtype of tumor samples that all of them were differentially expressed in differential immune subtype samples. The expression of TLRs was positively related with immune-estimate scores and stromal-estimate scores in almost all types of tumor. The expression of TLRs was negatively correlated with mRNA expression-based stemness scores (RNAss) in nearly almost type of tumors except kidney renal clear cell carcinoma (KIRC) and also negatively correlated with DNA methylation-based stemness scores (DNAss) in many types of tumors except adrenocortical carcinoma (ACC), cholangiocarcinoma (CHOL), KIRC, acute myeloid leukemia (LAML), low-grade glioma (LGG), testicular germ cell tumors (TGCT), thyroid carcinoma (THCA), thymoma (THYM), and uveal melanoma (UVM). The expression of TLR9 was significantly positively correlated with the drug sensitivity of fluphenazine, alectinib, carmustine, and 7−hydroxystaurosporine. TLR7 was significantly positively correlated with the drug sensitivity of alectinib. Conclusions Our study reveals the significant role of TLRs family in pan-cancer and provides potential therapeutic strategies of cancer.

Association of Lactoferrin and Toll-like Receptor 2 Genotypes with Mastitis and Milk Components in Vietnamese Holstein Cattle.

Simply SummaryMastitis disease causes huge losses for the dairy industry worldwide. The molecular markers have been shown to be a promising tool for the diagnosis of mastitis infections and the selection of animals with better mastitis resistance. In this study, we surveyed the mastitis incidence in Vietnamese Holstein cows. We also examined the association of polymorphisms in Lactoferrin (LTF) and Toll-like receptor 2 (TLR2) genes with mastitis score and milk component traits. Our results indicated that the interaction between TLR2 genotypes and diseases has a significant impact on the fat and protein percentage of mink. Therefore, TLR2 polymorphism could be used for the selection of cows with better milk compositions.Mastitis is one of the most widespread diseases in dairy cows and causes huge losses for the dairy industry. Molecular markers can be used for the quick diagnosis of mastitis infection, consequently reducing the loss caused by this disease. Lactoferrin (LTF) and Toll-like receptor 2 (TLR2) have been suggested as candidate genes for mastitis; however, their associations with the mastitis incidence and milk components have not been reported in Vietnamese Holstein cows. This study examined the association of TLR2 and LTF polymorphisms with subclinical mastitis and milk components in the Holstein breed raised in Vietnam. Among 192 samples, we identified 44 mastitis-positive samples (22.92%). The mastitis significantly reduced the fat and lactose components in milk (p < 0.001) but increased the protein concentration in milk. A total of 94 (49%) and 98 (51%) cows had AA and AB genotypes for the LTF gene, respectively. No significant association was found between the LTF genotypes and the milk component traits or mastitis incidence (p > 0.05). The interaction between LTF and mastitis incidence was significantly associated with the protein percentage (p = 0.01). A total of 78, 76, and 38 cows had genotypes GG, GT, and TT for the TLR2 gene, respectively. TLR2 genotypes were not significantly associated with mastitis incidence (p > 0.05) but were significantly associated with pH value (p = 0.03). The interaction between TLR2 and mastitis incidence was significantly associated with the fat (p = 0.02) and protein percentage (p = 0.04). Further studies are required to confirm the roles of LTF and TFL2 in mastitis in the Holstein breed in Vietnam.

Molecular Cloning and Characterization of tlr1, tlr5s, tlr5m, and tlr14 Genes in the Swamp Eel, Monopterus albus

Toll-like receptors (TLRs) are a critical family of pattern recognition receptors (PRRs) that are tightly regulated by complex mechanisms involving many molecules to ensure a good response to foreign invaders. In this study, we identified and analyzed the sequence characteristics and homology of four TLR genes (tlr1, tlr5s, tlr5m, and tlr14) from the swamp eel. Sequence similarity analysis, functional domain prediction, and phylogenetic analysis supported their annotation and orthologies. Their relative expression levels in different tissues were assessed. The four TLRs were expressed in all tested tissues. tlr5m was highly expressed in the spleen, whereas tlr5s was highly expressed in the kidney and spleen. High expression levels of tlr1 and tlr14 were detected in the spleen and muscle. These results indicated that the TLRs are related to host immunity. Moreover, the differential expression of TLRs was examined after Aeromonas veronii infection, which showed that all the TLR genes were induced with diverse patterns. tlr1 was significantly downregulated in the spleen after A. veronii challenge. In the kidneys and intestines, tlr1 expression decreased initially and then increased, with its lowest level at 4 h. tlr5s expression was upregulated significantly in three tissues at 1, 4, and 12 h, with the maximum expression at 1 h, indicating that tlr5s actively responded to bacterial invasion in the early stage of the challenge. tlr5m showed tissue specific expression: it was slightly upregulated in the intestines and spleen and downregulated in the kidneys. The expression pattern of tlr14 was similar to that of tlr5s, and both reached maximum expression at 1 h after infection. Collectively, our results indicated that TLRs might play important roles in the innate immune response against Gram negative bacteria in the swamp eel.

Clinical assessment and molecular mechanism of the upregulation of Toll-like receptor 2 (TLR2) in myocardial infarction

ObjectiveThe prevalence and mortality of cardiovascular diseases remain ranked first worldwide. Myocardial infarction (MI) is the central cause of death from cardiovascular diseases, seriously endangering human health. The clinical implication of toll-like receptor 2 (TLR2) remains contradictory, and its mechanism is still unknown. Hence, the objective of this study was to elucidate the clinical value and molecular mechanism of TLR2 in MI.MethodsAll high-throughput datasets and eligible literature were screened, and the expression levels of TLR2 were collected from the MI. The integrated expression level of TLR2 was displayed by calculating the standardized mean difference (SMD) and the area under the curve (AUC) of the summary receiver operating characteristic curve (sROC). The related TLR2 genes were sent for pathway analyses by gene ontology (GO), Kyoto encyclopedia of genes and genome (KEGG), and disease ontology (DO). Single-cell RNA-seq was applied to ascertain the molecular mechanism of TLR2 in MI.ResultsNine microarrays and four reported data were available to calculate the comprehensive expression level of TLR2 in MI, including 325 cases of MI and 306 cases of controls. The SMD was 2.55 (95% CI = 1.35–3.75), and the AUC was 0.76 (95% CI = 0.72–0.79), indicating the upregulation of TLR2 in MI. The related TLR2 genes were primarily enriched in the pathways of atherosclerosis, arteriosclerotic cardiovascular disease, and arteriosclerosis, suggesting the clinical role of TLR2 in the progression of MI. Afterward, TLR2 was upregulated in myeloid cells in MI.ConclusionsTLR2 may have a crucial role in progressing from coronary atherosclerosis to MI. The upregulation of TLR2 may have a favorable screening value for MI.

Role of cytokine and Toll-like receptor genes in pathogenesis of inborn heart disease

Sporadic congenital heart disease (CHD) may result from immune disorders in the mother – embryo system and/or constitutional disorders in regulatory systems, including those associated with TLR receptors, cytokines and their receptors. The aim of our study was to investigate associations between cytokine and TLR genes and sporadic congenital heart disease in children. In the main group, 188 children with sporadic (without family history) congenital heart defects were examined. Separate groups of CHD were identified: septal CHD – 98 children; valvular heart disease – 17 children; Fallot tetralogy – 15 children; aorta coarctation – 10 children; fetal drains – 32 children; single ventricle affection – 9 children, and anomalous drainage of v. pulmonalis was diagnosed in 7 children. The control group included 103 age- and sex-matched healthy children. We have determined gene polymorphisms of five genes encoding cytokines and their receptors (IL6 rs1800796, IL6 rs2069827, IL6R rs2228145, IL6R rs2229238, IL8 rs4073, IL10 rs1800871, IL10 rs1800896, IL10 rs1800872, TNF rs1800629, TNF rs361525, TNF rs1799964), four genes Toll-like receptors (TLR: TLR1 rs5743611, TLR1 rs5743551, TLR2 rs5743708, TLR2 rs3804099, TLR4 rs4986791, TLR4 rs4986790, TLR6 rs3775073, TLR6 rs5743810). The dbSNP, SNPinfo, SNPnexus databases were used to select and design test systems. Stepwise logistic regression was the main method of statistical analysis. Clinical diagnosis of congenital heart defects is associated with immune regulatory genes. In particular, the missense mutation TLR6 rs5743810, which was a predictor of congenital valvular heart disease, is of particular importance. Development of congenital heart valve defects and aortic coarctation is associated with intergenic interactions of TLR2 rs5743708 with TLR6 rs5743810, and TLR2 rs5743708 with TLR6 rs3775073, respectively. For congenital heart valve defects, such polymorphic regions are as follows: IL6 rs2069827, IL6R rs2229238, and IL8 rs4073, for aortic coarctation – IL6R rs2228145, IL8 rs4073. Development of septal congenital heart defects is associated with general contribution of polymorphic variants of the TLR genes and cytokines to this pathology. A missense mutation of the TLR4 rs4986790 gene and a TNF rs1799964 mutation leading to increased synthesis of the TNFα molecule, may have a combined effect on this process. In general, contribution of TLR and cytokine genes interactions to the CHD development seems to be not significant.

Association of TLR3 gene 1377C/T (rs3775290) and TLR7 gene C/G (rs3853839) polymorphism with hand, foot, and mouth disease caused by human enterovirus 71 infection susceptibility and severity in the Chinese Han population: A meta-analysis of case-control studies.

Background:Several case-control studies have been conducted on the relationship between rs3775290 C/T and rs3853839 C/G single nucleotide polymorphisms of the Toll-like receptor (TLR) gene and hand, foot, and mouth disease (HFMD) susceptibility and severity. This meta-analysis aimed to offer a systemic review of HFMD susceptibility and severity among the Chinese Han population associated with the C/T (rs3775290) polymorphism of the TLR3 gene or C/G (rs3853839) polymorphism of the TLR7 gene.Methods:A computer search was conducted using PubMed, Web of Science, Embase, CNKI, CBM, VIP, and WanFang databases. The time ranges were from database establishment to 30/7/2021. Articles selected according to the inclusion and exclusion criteria underwent data extraction and methodological quality evaluation. RevMan 5.4 and Stata 16.0 were adopted for meta-analysis, and the incorporated odds ratio (OR) values and 95% confidence intervals (CIs) were calculated. Sensitivity and publication bias assessments were performed.Results:8 articles with 9 studies were selected. Among them, there were 858 cases and 577 controls in TLR3 rs3775290 studies as well as 2151 cases and 1554 controls in TLR7 rs3853839 studies. Regarding rs3775290 of TLR3, susceptibilities of the severe type of T-possessing individuals were larger than those of C-possessing individuals [OR = 1.34, 95%CI (1.10, 1.64), P = .004]. The susceptibility of individuals with the severe TT genotype was 1.61 times that of individuals with the CC genotype [95%CI (1.07, 2.43), P=0.02], while susceptibility to HFMD was not influenced by the genotype. In terms of the rs3853839 of the TLR7 gene, C allele carriers have a higher risk of developing HFMD than G allele carriers. The susceptibility to HFMD in CC+CG individuals was 1.24 times than that in GG individuals [95%CI (1.07, 1.43), P = .004]. However, no relationship was found between this polymorphism and severity of the severe type. No significant publication bias was observed in this study.Conclusions:rs3775290 (C/T) of TLR3 is associated with susceptibility to the severe type, whereas rs3853839 (C/G) of TLR7 is associated with susceptibility to HFMD. However, owing to the limited quantity and quality of the research, the aforementioned conclusions are yet to be justified by more high-quality research.

Association of polymorphous light eruption with NOD-2 and TLR-5 gene polymorphisms.

Polymorphous light eruption (PLE) is a common, immunologically mediated, photosensitive skin disease. After ultraviolet-B (UV-B) irradiation, patients with PLE show reduced Langerhans cell (LC) depletion in the epidermis, which results in a non-suppressive microenvironment in the skin. Interestingly, severe acute graft-versus-host disease (aGvHD) occurred in stem cell transplanted patients that showed no or incomplete depletion of LCs after UVB irradiation. Genetic variation in nucleotide-binding oligomerization domain 2 (NOD-2) and toll-like receptor 5 (TLR-5) genes also confers susceptibility to aGvHD. We hypothesized that PLE is associated with genetic variation in the NOD-2 and TLR-5 genes. We investigated single-nucleotid polymorphisms (SNPs) of NOD-2 (R702W, G908R, 3020Cins) and TLR-5 (A592S, P616L, N392STOP) in skin biopsies of patients with PLE (n = 143) and in healthy controls (n = 104) using restriction fragment length polymorphism analysis. The frequency of NOD-2 alleles with the SNP R702W was significantly higher in PLE than in controls (31.8% vs. 6.3%; P < 0.0001), and homozygous carriers of this mutation were more common in PLE (27.9% vs. 0%; P < 0.0001). For SNP 3020Cins, the allele frequency (7.3% vs. 0.7%; P = 0.0025) and the number of heterozygotes (14.7% vs. 1.3%; P = 0.0019) were higher in PLE. The frequency of alleles with the N392STOP SNP of the TLR5 gene, which is associated with a truncated, non-functional receptor, was significantly higher in PLE (21% vs. 5%; 7% vs. 1% homozygotes, 28% vs. 8% heterozygotes; P < 0.0001). The other SNPs did not differ significantly. This study yielded a high frequency of functional SNPs in the NOD-2 and TLR-5 genes in PLE. The same SNPs are associated with aGvHD and there are similarities in the reaction of LCs after UVB irradiation between aGvHD and PLE. This leads to the hypothesis that patients with PLE may be more susceptible to developing GvHD after stem cell transplantation, an assumption that needs to be investigated further.

PRIMARY OPEN ANGLE GLAUCOMA: MECHANISMS OF PATHOGENESIS AND GENETIC PREDISPOSITION. Review

Relevance. Primary open-angle glaucoma (POAG) is a progressive optic neuropathy with loss of retinal ganglion cells (RGCs) and narrowing of the visual fields in the eyes with a gonioscopic open angle. The main mechanisms of this are increased intraocular pressure (IOP), circulatory disorders, trabecular meshwork (TM), ischemic metabolic disorders and chronic inflammation. However, questions about the role of POAG genetic predisposition remain open.&#x0D; Objective: analysis of current data on the mechanisms of pathogenesis of progressive neuropathy in POAG and the role of genetic predisposition.&#x0D; Methods. The analysis of scientific publications in open international electronic scientometric databases: Scopus, PubMed, Web of Science, Google Scholar, SID, MagIran, IranMedex, IranDoc, ScienceDirect, Embase by keywords (a total of 67 sources). Search depth – 10 years (2012-2022).&#x0D; Results. There are more than 60 million glaucoma patients in the world, 20% of whom have an incurable stage. By 2040, the number of patients is projected to increase to 112 million, with POAG accounting for 75% of cases. Among the main mechanisms of glaucoma, an important role belongs to chronic inflammation and immune damage, which occur in response to ischemic injury. Prolonged inflammatory process leads to hypersecretion of inflammatory mediators and infiltration of inflammatory cells into ischemic tissue, which aggravates the effects of increased IOP and ischemia. It is known that mutations in the gene of Toll-like receptor 4 (TLR4) are associated with both infectious and non-infectious diseases, including POAG: activation of TLR4 initiates TM fibrosis, causes increased IOP, activates RGCs apoptosis in the model of acute glaucoma. TLR4 ligands, such as heat shock proteins and lipopolysaccharides are candidate antigens for glaucoma. TLR4 overexpression at retinal microglia and astrocytes induce an innate immune response through NF-κB activation, which enhances the expression of proinflammatory cytokines.&#x0D; Conclusions. A promising direction is to study the contribution of TLR4 mutations to the POAG mechanisms, which will identify the mechanisms of immune disorders and establish the genetic risk of individual mutations in different ethnic groups.

The association of inflammatory status and immunological parameters with single-nucleotide polymorphisms of cytokine and Toll-like receptor genes in patients with schizophrenia

Schizophrenia is a multifactorial and clinically polymorphic disease with a significant hereditary component. The study of the influence of functionally significant polymorphisms of the genes regulating immune response and inflammation on the immune parameters in patients with schizophrenia is relevant for the development of new methods for predicting the course of the disease and finding new therapy targets. The aim of the study:To study the association of polymorphisms in cytokine and Toll-like receptor genes with the serum levels of immune mediators in patients with schizophrenia. Materials and methods: 95 patients diagnosed with schizophrenia and 99 healthy volunteers were recruited into the study. Polymorphisms of genes regulating the immune response (IL1B rs1143627, IL4 rs2243250, IL2 rs2069762, IL10 (rs1800896, rs1800872, rs1800871), IFNL rs8099917, IFNL rs12979860, TLR9 rs5743836, TLR9 rs352140, TLR3 rs3775291), the serum level of major cytokines and markers of systemic inflammation were determined. Results: The patients were found to have elevated levels of key pro-inflammatory cytokines IL-8 and IL-17A and other markers of systemic inflammation, as well as an increase in the content of antigen-antibody complexes in the bloodstream. Increased frequency of IL10 gene polymorphisms (rs1800896, rs1800872, rs1800871) was revealed in the patients with schizophrenia. A relationship between IL-6 and IL-8 levels and the carriage of IL1B T-511C (rs16944), IL10 1082A (rs1800896), IL10 592 C&gt;A (rs1800872) and TLR3 Leu412Phe (G/A) (rs3775291) single nucleotide polymorphisms was found. Conclusion: The revealed associations may indicate a role of SNPs in cytokine and Toll-like receptor genes in modulating the network genetic interactions underlying certain immunological endophenotypes in the patients. Based on the results of this work and literature data, it is important to further study the relationship of immunogenetic markers in schizophrenia with the immune profiles of the disease and its clinical manifestations, including in larger samples of patients.

Expressions of Toll Like Receptor (TLR) Genes in Paralichthys olivaceus After Induced by Different Extracts of Edwardsiella tarda.

Toll like receptors (TLRs) are the main innate immune ‘pattern recognition receptors’ of animals, which play a central role in host cell recognition and responses to invasive pathogens, particularly common structures of microbial pathogens. In this study, the gene expression profiles of TLRs in the spleen, head kidney, gill, small intestine, liver, muscle, and heart of healthy Paralichthys olivaceus were detected by real-time quantitative PCR (qPCR). The TLR family members were widely expressed in different tissues with different basic expression profiles. The highest expressions of TLR1, 5m, 7, 8, 9, 14, and 21 were found in the spleen; the highest expressions of TLR3 and TLR21 were found in the gill; the highest expressions of TLR2 and 5s were found in the small intestine. The second highest expressions of TLR3, 7, and 8 were found in small intestine. The gene expression profiles of TLRs stimulated with Edwardsiella tarda DNA, RNA, and lipopolysaccharide (LPS) were also detected in spleen, head kidney and gill. TLR9 and TLR21 were sensitive to E. tarda DNA; TLR 8 and TLR21 were sensitive to E. tarda RNA; and TLR1 and TLR14 were sensitive to E. tarda LPS. The expressions of the other TLR genes showed no significant changes. The results imply that the expressions of these TLR genes in P. olivaceus are differently regulated in the whole body and play important roles in the immune response against E. tarda infection.

Peptidomimetic Lipid-Nanoparticle-Mediated Knockdown of TLR4 in CNS Protects against Cerebral Ischemia/Reperfusion Injury in Mice

Ischemic stroke activates toll-like receptor 4 (TLR4) signaling, resulting in proinflammatory polarization of microglia and secondary neuronal damage. Herein, we report a novel lipid-nanoparticle (LNP)-mediated knockdown of TLR4 in microglia and amelioration of neuroinflammation in a mouse model of transient middle cerebral artery occlusion (tMCAO). siRNA against TLR4 (siTLR4) complexed to the novel LNP (siTLR4/DoGo310), which was based on a dioleoyl-conjugated short peptidomimetic (denote DoGo310), was readily internalized by the oxygen–glucose-deprived (OGD) mouse primary microglia, knocked-down TLR4, and polarized the cell to the anti-inflammatory phenotype in vitro. Systemic administration of siTLR4/DoGo310 LNPs in the tMCAO mice model resulted in the accumulation of siRNA mainly in the Iba1 positive cells in the peri-infarct. Analysis of the peri-infarct brain tissue revealed that a single injection of siTLR4/DoGo310 LNPs led to significant knockdown of TLR4 gene expression, reversing the pattern of cytokines expression, and improving the neurological functions in tMCAO model mice. Our data demonstrate that DoGo310 LNPs could be a promising nanocarrier for CNS-targeted siRNA delivery for the treatment of CNS disorders.

Abstract CT205: A phase I/Ib trial of intratumoral Poly-ICLC in resectable malignant pleural mesothelioma

Abstract Background: Malignant pleural mesothelioma (MPM) is usually fatal, though multimodality therapy— now including immunotherapy— has improved survival. Recurrence after surgery is close to 100%, even with adjuvant chemotherapy and radiation. Our collaborators have performed deep immunophenotyping of treatment-naïve MPM lesions using mass cytometry (CyTOF) and single-cell RNA sequencing (scRNAseq) to define the tumor microenvironment. A population of rare CD141+ dendritic cells (DC1) is disproportionately represented in some MPM lesions analyzed. These DC1 cells— which express high levels of Toll-like receptor 3 (TLR3)— are among the most potent cross-presenters of antigen and are key to priming anti-tumor CD4+ and CD8+ T cell responses. Polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC), is a double-stranded RNA host-targeted therapeutic viral-mimic. Poly-ICLC activates multiple innate immune receptors including TLR3 and melanoma differentiation-associated gene 5 (MDA5), leading to cross-presentation of antigen to T cells and induction of strong Th1 response. We hypothesize that injection of poly-ICLC prior to surgical resection may activate intratumoral (IT) DC1s, increase tumor antigen presentation to cytotoxic T cells, and induce tumor-specific immune surveillance. Methods: This is a phase I/Ib study to evaluate the safety of IT poly-ICLC prior to surgical resection for patients with MPM (NCT04525859). The primary endpoint is safety as assessed by frequency and severity of toxicities by CTCAE 5.0. Secondary endpoints are objective response as measured by RECIST 1.1 and recurrence free survival measured from the time of first poly-ICLC injection. Exploratory endpoints include evaluation of circulating immune cells (including regulatory T cells and NK cells), evaluation of immune cell infiltration in pre-injection tumor biopsy and surgically resected tissue, as well as characterization of immune parameters such as local B cell specificity. The protocol features a Simon’s two-stage design, with six patients enrolled in a phase I safety cohort, proceeding to a phase Ib expansion cohort (additional 13 patients) if no more than 1 dose limiting toxicity occurs. Eligible patients must have MPM deemed operable by the treating thoracic surgeon. Eligible subjects may not have uncontrolled immunocompromised states or autoimmune disorders. After enrollment, patients undergo biopsies at which time 2mg poly-ICLC is injected across two sites in the tumor. Patients then undergo resection of the tumor (pleurectomy/decortication or extra pleural pneumonectomy per standard of care) at day 21+/- 7 after poly-ICLC injection. Blood is drawn at three points (prior to poly-ICLC injection, at time of surgery, and at a post-operative visit) for immune profiling. At the time of submission six patients have been treated and phase Ib accrual is continuing as planned. Interim analysis of phase I safety and exploratory endpoints will be reported in late 2022. Citation Format: Bailey G. Fitzgerald, Thomas U. Marron, Robert Sweeney, Jorge Gomez, Nicole Hall, Daniel O'Grady, Christian Rolfo, Raj Veluswamy, Deborah Doroshow, John Mandeli, David Yankelevitz, Nina Bhardwaj, Sacha Gnjatic, Fred R. Hirsch, Miriam Merad, Alexander Tsankov, Raja Flores, Andrea Wolf. A phase I/Ib trial of intratumoral Poly-ICLC in resectable malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT205.

Leveraging an existing whole-genome resequencing population data set to characterize toll-like receptor gene diversity in a threatened bird.

Species recovery programs are increasingly using genomic data to measure neutral genetic diversity and calculate metrics like relatedness. While these measures can inform conservation management, determining the mechanisms underlying inbreeding depression requires information about functional genes associated with adaptive or maladaptive traits. Toll‐like receptors (TLRs) are one family of functional genes, which play a crucial role in recognition of pathogens and activation of the immune system. Previously, these genes have been analysed using species‐specific primers and PCR. Here, we leverage an existing short‐read reference genome, whole‐genome resequencing population data set, and bioinformatic tools to characterize TLR gene diversity in captive and wild tchūriwat’/tūturuatu/shore plover (Thinornis novaeseelandiae), a threatened bird endemic to Aotearoa New Zealand. Our results show that TLR gene diversity in tchūriwat’/tūturuatu is low, and forms two distinct captive and wild genetic clusters. The bioinformatic approach presented here has broad applicability to other threatened species with existing genomic resources in Aotearoa New Zealand and beyond.

Comparison of Selected Immune Parameters in a Single Infection and Co-Infection with Infectious Pancreatic Necrosis Virus with Other Viruses in Rainbow Trout.

Infectious pancreatic necrosis virus (IPNV) often occurs in an aquatic environment in co-infection with other viruses. In this study, we wanted to investigate the effect of this virus on the course of co-infection with other viruses in rainbow trout. For co-infection we used viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV) and salmonid alphavirus (SAV) field strains and infected rainbow trout divided into eight groups; I; IPNV, II; IHNV, III; VHSV, I; SAV, V; IPNV+IHNV, VI; IPNV+VHSV, VII; IPNV+SAV, and the control group. We assessed apoptosis in white blood cells and used a real time RT-PCR to analyze RNA obtained from the internal organs of the fish. During single infection and co-infection the level of expression of immune genes such as interferon and toll-like receptor 3 (TLR-3) was assessed. The highest mortality during the experiment was observed in group III infected by VHSV. The average percentage of apoptotic cells was higher in groups without co-infection, especially in groups II and III. Interferon expression was higher in singly infected groups, the highest being in the heart in group III, while expression of the TLR-3 gene was generally raised in all tested organs in all groups. We found that co-infection with IPNV had a positive impact on the course of infection with the viruses listed because it lowered mortality, reduced apoptosis in co-infected cells, and positively affected fish health.

TLR-2 Germ Line Variants as a Risk for Obesity in Local Pakistani Population

Obesity is increasing rapidly affecting half billion adult's population. Pathophysiology of obesity involves low grade inflammation sustained by Toll like receptor 2 (TLR-2) the innate immune adapters. This study was conducted for detection and association of TLR-2 gene mutations with obesity. In this case-control study 228 individuals with obesity and 228 controls were enrolled based on Body Mass Index (BMI) ≥25 and 18-24 kg/m2 respectively. The variations in TLR-2 gene were detected by Sanger sequencing. These identified TLR-2 variants were further analyzed in silico for change in miRNA binding and mRNA strucutre. Four novel single base substitutions (153688371 T >C, 153702295 T >C, 153703504 T >C and 153705074 C >A) were identified in exon 3 and 4 of TLR-2 gene affecting splice site and poly-A tail. The genotypic and allelic frequencies of the variants were strongly associated with increasing obesity susceptibility. Only variant 153703504 T >C was significantly associated with preobesity. Despite variations in gene sequence, no change in miRNA binding except for variant 153688371 T >C of Exon 3 where a novel binding site for hsa-miR-4523 was created. Furthermore, mRNA stability and secondary structure were also compromised in identified variants. All detected variants of TLR-2 gene were significantly associated with and posed risk for development of obesity. Furthermore, in silico analysis revealed generation of new miRNA (hsa-miR-4523) binding site and change in mRNA structure/stability which needs to be further investigated for possible role in altering TLR-2 gene regulation/expression in obesity.

TLR4 regulatory region variants reduce the susceptibility of small-cell lung cancer in Chinese population.

Toll-like receptors (TLRs) participate in the induction and regulation of immune responses and are closely related to the occurrence and development of small-cell lung cancer (SCLC). This study aimed to investigate the impact of polymorphisms in the regulatory regions of TLRs on the susceptibility of SCLC. The case-control study included 304 SCLC patients and 304 healthy controls. TLRs gene polymorphisms were genotyped by PCR-restrictive fragment length polymorphism analysis and TaqMan assay. Unconditional logistic regression was used to estimate odds ratio (OR) and its 95% confidence interval (95% CI). Our results showed that TLR4 rs1927914 GG genotype and TLR4 rs7869402 TT genotype reduced the risk of SCLC with OR (95% CI) of 0.54 (0.32-0.90) and 0.47 (0.28-0.80), respectively. Stratified analysis suggested that TLR4 rs1927914 GG genotypes significantly reduced the risk of SCLC among male (OR = 0.35; 95% CI, 0.18-0.69; P < 0.01), the younger patients (OR = 0.49; 95% CI, 0.26-0.94; P = 0.03) and non-drinkers (OR = 0.47; 95% CI, 0.24-0.89; P = 0.02). TLR4 rs7869402 CT or TT genotype significantly reduced the susceptibility to SCLC among male patients (OR = 0.37; 95% CI, 0.19-0.71, P < 0.01), the younger patients (OR = 0.41; 95% CI, 0.22-0.79; P < 0.01), smokers (OR = 0.25; 95% CI, 0.10-0.60; P < 0.01) and drinkers (OR = 0.31; 95% CI, 0.11-0.88; P = 0.03). TLR3 rs5743303, TLR4 rs11536891, TLR5 rs1640816 and TLR7 rs3853839 had no significant correlation with the risk of SCLC. These findings emphasized the important role of TLR4 in the development of SCLC.

Association of X-linked TLR-7 gene polymorphism with the risk of knee osteoarthritis: a case–control study

Knee osteoarthritis (OA) is the most prevalent type of OA, and Toll-like receptor 7 (TLR7) may lead to the pathogenesis of OA. Recently, X-linked TLR7 polymorphism has been confirmed to be associated with arthritis. However, there is a lack of studies on TLR7 gene polymorphism associated with knee OA susceptibility. The current study aimed to determine whether TLR7 gene polymorphism is associated with the risk of knee OA. Genotyping of two polymorphic sites (rs3853839 and rs179010) in the TLR7 gene was performed in 252 OA patients, and 265 healthy controls using the SNaPshot sequencing technique. Data were analyzed statistically by Chi-square tests and logistic regression. Rs3853839-C allele showed frequencies of 28% and 27% in the healthy control and female knee OA groups, respectively. The differences were not statistically significant (P > 0.05). The rs3853839-CG genotype frequency was significantly lower in the female knee OA group as compared to the healthy control group (OR 0.60; 95%CI 0.36–0.99; P = 0.044). In the male hemizygote population, the rs3853839-CC showed significantly lower frequencies in the male knee OA group as compared to the healthy control group (OR 0.35; 95%CI 0.17–0.71; P = 0.0025). Regarding rs179010, there were no differences in the genotype distribution and allele frequencies between OA patients and healthy subjects under any models (P > 0.05). Stratified analysis showed that the frequency of the rs3853839-CG genotypes was lower in high Kellgren-Lawrence grades (KLG) (OR 0.48; 95%CI 0.21–1.08; P = 0.066), and significantly lower in OA patients with effusion synovitis (OR 0.38; 95%CI 0.17–0.88; P = 0.013). TLR7 rs3853839 polymorphism may play a role in the susceptibility of knee OA in the Chinese Han Population and may be associated with OA severity and the risk of effusion synovitis in Knee OA.