TIR Domain-containing Adaptor Protein Research Articles

Genetic Diversity of Plasmodium vivax Surface Ookinete Protein Pvs25 and Host Genes in Individuals Living along the Thai–Myanmar Border and Their Relationships with Parasite Density

Plasmodium vivax (Pv) accounts for over 50% of malaria cases in Latin America and Asia. Despite a significant reduction in Pv transmission in Thailand, the parasite remains endemic to the border areas. This study aimed to investigate the genetic diversity of the parasites and the host factors, as well as their relation to parasite density in Pvisolates, along the Thai–Myanmar border. Genetic variations in Pv markers, specifically the ookinete surface protein Pvs25, and host genes, including Toll-like receptor 6 (TLR6), TLR9, TIR Domain-containing adaptor protein (TIRAP), Toll-interacting protein (TOLLIP), Duffy antigen receptor for chemokines (DARC), and intercellular adhesion molecule 1 (ICAM-1), were investigated using polymerase chain reaction (PCR) with restriction fragment length polymorphism (RFLP). A total of 548 PCR-positive Pv samples collected from Tak and Kanchanaburi provinces during two periods (2006–2007 and 2014–2016) were included in the study. Pvs25 exhibited four haplotypes, with H1 (EGTKV) being the most prevalent in both provinces. Kanchanaburi isolates exhibited greater genetic diversity than Tak isolates. No significant deviations from neutrality were observed for Pvs25 in either area. ICAM-1 and TOLLIP s3750920 heterozygous carriers had greater median parasite densities than homozygous mutants. The TLR9 rs187084 T genotype had a significantly higher parasite density than the non-T genotype. The findings underscore the significant association between the rs3750920 C/T, rs5498 A/G, and rs187084 T genotypes and high parasite density in patients infected with Pv, highlighting their potentially critical role in malaria susceptibility.

A novel PD-1/PD-L1 pathway-related seven-gene signature for the development and validation of the prognosis prediction model for breast cancer.

Breast cancer (BC/BRCA) is the most common carcinoma in women. The average 5-year survival rate of BC patients with stage IV disease is 26%. A considerable proportion of patients still do not receive effective therapy. It is an unmet need to identify novel biomarkers for BC patients. Herein, we evaluated whether the programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) status is associated with the clinical outcomes of BC, based on data from The Cancer Genome Atlas (TCGA). Clinical and transcriptome data of BC patients were obtained from TCGA dataset, and prognostic genes in BC patients were identified, as well as the PD-1/PD-L1 pathway mainly associating with the BC patients. Following the execution of the consensus clustering algorithm, BC patients were segregated into two clusters, and subsequent investigation of the potential mechanisms between them was carried out. A comparison of ferroptosis and N6-methyladenosine (m6A) was conducted between the two groups with the greatest difference in prognosis. Based on least absolute shrinkage and selection operator (LASSO) analysis, a signature associated with the PD-1/PD-L1 pathway was developed, and the prognosis outcome and the predictive accuracy of the signature model were further assessed. Prognostic genes in BC patients were studied using TCGA data and it was found that the PD-1/PD-L1 pathway was most associated with the BC patients. Then, a low-risk (C1) group and a high-risk (C2) group of BC patients were constructed based on a PD-1/PD-L1 pathway-related signature. The functional analyses suggested that the underlying mechanisms between these groups were mainly associated with immune-related pathways. We found that ferroptosis and m6A were significantly different between the two groups. A PD-1/PD-L1 pathway-related gene signature was further developed to predict survival of BC patients, including 7 genes [mitogen-activated protein kinase kinase 6 (MAP2K6), NF-kappa-B inhibitor alpha (NFKBIA), NFKB Inhibitor Epsilon (NFKBIE), Interferon gamma (IFNG), Toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), IkappaB kinase (CHUK), and Casein kinase 2 alpha 3 gene (CSNK2A3)]. The receiver operating characteristic (ROC) curves were analyzed to further assess the prognostic values of these 7 genes. The 1-, 3-, and 5-year values of the areas under the curve (AUCs) for overall survival were 0.651, 0.658, and 0.653 in this seven gene signature model, respectively. PD-1/PD-L1 pathway-related subtypes of BC were identified, which were closely associated with the immune microenvironment, the ferroptosis status, and m6A in BC patients. The gene signature involved in the PD-1/PD-L1 pathway might help to make a distinction and predict prognosis in BC patients.

CaMKIIδ, Stabilized by RNA N6-Methyladenosine Reader IGF2BP2, Boosts Coxsackievirus B3-Induced Myocardial Inflammation via Interacting with TIRAP.

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to be aberrantly activated in viral myocarditis (VMC), but the role of its subtype CaMKIIδ in VMC remains unclear.VMC mice and cardiomyocytes models were induced by Coxsackievirus B3 (CVB3) treatment. Mice that underwent sham surgery and saline-treated cardiomyocytes served as controls. Body weight, survival, left ventricular ejection fraction (LVEF), and fractional shortening (LVFS) were measured, and HE staining was performed to evaluate heart function in VMC mice model and sham control. Inflammation factors in serum or cell supernatant were detected by ELISA. Expressions of CaMKIIδ, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), nuclear factor NF-kappaB (NF-κB) signals, and inflammation factors were examined by quantitative real time polymerase chain reaction (qRT-PCR) or western blot. CCK-8, EdU, and flow cytometry were used to evaluate cell behaviors. Co-immunoprecipitation (Co-IP), RNA immunoprecipitation (RIP), and RNA pull-down were utilized to validate molecule interaction. Methylated RNA immunoprecipitation (MeRIP) was performed to measure N6-methyladenosine (m6A) level of specific molecule.CaMKIIδ was upregulated in VMC mice and CVB3-treated primary cardiomyocytes, of which knockdown improved cell viability, proliferation, and suppressed cell apoptosis in vitro, thereby alleviating myocarditis in vivo. The stability of CaMKIIδ was attributed to the presence of IGF2BP2 through m6A modification. Loss of CaMKIIδ repressed NF-κB pathway via negatively and directly regulating TIRAP to be involved in inflammatory damage.CaMKIIδ, stabilized by m6A reader IGF2BP2, modulated NF-κB pathway via interacting with TIRAP to alter cell viability, proliferation, and apoptosis, thereby affecting VMC outcome.

Effectiveness of Narciclasine in Suppressing the Inflammatory Response in Sepsis: Molecular Docking and In Silico Studies.

Narciclasine is an alkaloid belonging to the Amaryllidaceae family which has been reported to have many beneficial properties. Especially its anticancer properties have been widely reported. Here, we have focused on its potential use in suppressing the inflammatory response in sepsis using in silico methods. Lipopolysaccharide (LPS) is an endotoxin which is present in the outer membrane of gram-negative bacteria and is a crucial player in the pathogenesis of gram-negative sepsis. Activation of toll-like receptor 4 (TLR4) signaling by LPS is an important event in the pathogenesis of gram-negative sepsis. This initiates a downstream signaling pathway comprising of several adaptor proteins such as toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), myeloid differentiation primary response protein 88 (MyD88), interleukin-1 receptor-associated kinase (IRAK)-1, IRAK-4, interferon regulatory factor 3 (IRF-3), tumor necrosis factor receptor-associated factor 6 (TRAF-6) leading to nuclear factor kappa B (NF-κβ) activation resulting in elevated production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6. S100 calcium binding proteins A8/A9 (S100A8/A9) have been found to be an agonist of TLR4, and it amplifies the inflammatory response in sepsis. Molecular docking studies of narciclasine with target proteins associated with the LPS-TLR4 pathway showed that it has good binding affinity and stable interactions with the targets studied. Molecular dynamics (MD) simulation studies over 100 ns showed that most of the ligand-target complexes were stable. The structures of all the targets except TRAF-6 were retrieved from the Protein Data Bank (PDB) database. Homology modeling was done to predict the 3-dimensional structure of TRAF-6. MD simulation of narciclasine-TRAF-6 complex showed that the structure is stable. Metapocket was used for active site prediction in the target proteins. Toxicity analysis by admetSAR revealed that narciclasine was readily biodegradable and exhibited minimum toxicity. These results indicate that narciclasine has effective anti-inflammatory properties which could be useful in suppressing the inflammatory response in sepsis.

Toll-like receptor 2 selectively modulates Ras isoforms expression in Leishmania major infection

Leishmania infection of macrophages results in altered Ras isoforms expression and Toll-like receptor-2 (TLR2) expression and functions. Therefore, we examined whether TLR2 would selectively alter Ras isoforms’ expression in macrophages. We observed that TLR2 ligands- Pam3CSK4, peptidoglycan (PGN), and FSL- selectively modulated the expression of Ras isoforms in BALB/c-derived elicited macrophages. Lentivirally-expressed TLR1-shRNA significantly reversed this Ras isoforms expression profile. TLR2-deficient L. major-infected macrophages and the lymph node cells from the L. major-infected mice showed similarly reversed Ras isoforms expression. Transfection of the macrophages with the siRNAs for the adaptors- Myeloid Differentiation factor 88 (MyD88) and Toll-Interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP)- or Interleukin-1 Receptor-Associated Kinases (IRAKs)- IRAK1 and IRAK4- significantly inhibited the L. major-induced down-regulation of K-Ras, and up-regulation of N-Ras and H-Ras, expression. The TLR1/TLR2-ligand Pam3CSK4 increased IL-10 and TGF-β expression in macrophages. Pam3CSK4 upregulated N-Ras and H-Ras, but down-regulated K-Ras, expression in C57BL/6 wild-type, but not in IL-10-deficient, macrophages. IL-10 or TGF-β signaling inhibition selectively regulated Ras isoforms expression. These observations indicate the specificity of the TLR2 regulation of Ras isoforms and their selective modulation by MyD88, TIRAP, and IRAKs, but not IL-10 or TGF-β, signaling.

ALKBH5-mediated m6 A demethylation of TIRAP mRNA promotes radiation-induced liver fibrosis and decreases radiosensitivity of hepatocellular carcinoma.

Radiation-induced hepatic stellate cell (HSC) activation promotes radiation-induced liver fibrosis (RILF), a complication for hepatocellular carcinoma (HCC) radiotherapy. The demethylase alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) decreases N6-methyladenylate methylation (m6 A) modification of RNA, while its role in regulating RILF pathogenesis and HCC radiosensitivity remains unknown. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-sequencing (RNA-seq) were used to screen target genes regulated by ALKBH5. HSC with altered ALKBH5 expression was used to assess irradiation-induced HSC activation and the effect of HSC on recruitment and polarisation of monocytes. Key cytokines in medium from irradiated HSC-educated monocytes were identified by cytokine array detection. The effects of blocking ALKBH5 and key cytokines on RILF and HCC radiosensitivity were also evaluated. Radiation-induced ALKBH5 expression in HSC mediated m6 A demethylation of toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) mRNA and activated its downstream NF-κB and JNK/Smad2 pathways to promote HSC activation. Additionally, ALKBH5 regulated CCL5 secretion by irradiated HSC to promote monocyte recruitment and M2 macrophage polarisation. Notably, polarised monocytes secreted CCL20 to up-regulate ALKBH5 expression in HSC, and reduce HCC radiosensitivity by activating ALKBH5/TIRAP axis in HCC cells. ALKBH5 knockdown-combined CCR6 (CCL20 receptor) inhibitor significantly alleviated RILF and improved HCC radiosensitivity in mice. HCC patients with high ALKBH5 and TIRAP expression were prone to radiation-induced liver injury and poor tumour response to radiotherapy. Collectively, irradiation up-regulates ALKBH5 in HSC to mediate monocyte recruitment and M2 polarisation and form positive feedback to promote RILF and reduce HCC radiosensitivity. The dual roles of ALKBH5 as a microenvironmental regulator and radiosensitisation target provide new ideas for RILF prevention and radiosensitisation of HCC.

Epigenetic biomarkers of cold and heat syndromes of rheumatoid arthritis: Combining DNA hydroxymethylation and mRNA-sequencing.

Epigenetic biomarkers of cold and heat syndromes of rheumatoid arthritis: Combining DNA hydroxymethylation and mRNA-sequencing.

Toll-like receptor 4 promotes bladder cancer progression upon S100A8/A9 binding, which requires TIRAP-mediated TPL2 activation

Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells.

The IRAK4 scaffold integrates TLR4-driven TRIF and MYD88 signaling pathways.

SUMMARYInterleukin-1 receptor-associated kinases (IRAKs) −4, −2, and −1 are involved in transducing signals from Toll-like receptors (TLRs) via the adaptor myeloid differentiation primary-response protein 88 (MYD88). How MYD88/IRAK4/2/1 complexes are formed, their redundancies, and potential non-enzymatic roles are subjects of debate. Here, we examine the hierarchical requirements for IRAK proteins in the context of TLR4 activation and confirmed that the kinase activity of IRAK4 is essential for MYD88 signaling. Surprisingly, the IRAK4 scaffold is required for activation of the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) by both MYD88 and TIR domain-containing adaptor protein inducing IFN-β (TRIF), a unique adaptation in the TLR4 response. IRAK4 scaffold is, therefore, essential in integrating MYD88 and TRIF in TLR4 signaling.

TIRAP/Mal Positively Regulates TLR8-Mediated Signaling via IRF5 in Human Cells

Toll-like receptor 8 (TLR8) recognizes single-stranded RNA of viral and bacterial origin as well as mediates the secretion of pro-inflammatory cytokines and type I interferons by human monocytes and macrophages. TLR8, as other endosomal TLRs, utilizes the MyD88 adaptor protein for initiation of signaling from endosomes. Here, we addressed the potential role of the Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) in the regulation of TLR8 signaling in human primary monocyte-derived macrophages (MDMs). To accomplish this, we performed TIRAP gene silencing, followed by the stimulation of cells with synthetic ligands or live bacteria. Cytokine-gene expression and secretion were analyzed by quantitative PCR or Bioplex assays, respectively, while nuclear translocation of transcription factors was addressed by immunofluorescence and imaging, as well as by cell fractionation and immunoblotting. Immunoprecipitation and Akt inhibitors were also used to dissect the signaling mechanisms. Overall, we show that TIRAP is recruited to the TLR8 Myddosome signaling complex, where TIRAP contributes to Akt-kinase activation and the nuclear translocation of interferon regulatory factor 5 (IRF5). Recruitment of TIRAP to the TLR8 signaling complex promotes the expression and secretion of the IRF5-dependent cytokines IFNβ and IL-12p70 as well as, to a lesser degree, TNF. These findings reveal a new and unconventional role of TIRAP in innate immune defense.

MiR-3150a-3p, miR-6883-3p and miR-627-5p participate in the phycocyanin-mediated growth diminishment of A549 cells, via regulating a common target toll/interleukin 1 receptor domain-containing adaptor protein

• Phycocyanin diminishes the viability of A549 cells. • Phycocyanin significantly promotes expressions of miR-3150a-3p, miR-6883-3p and miR-627-5p. • TIRAP is a shared directed target of miR-3150a-3p, miR-6883-3p and miR-627-5p. • Knockdown TIRAP expression resulted in similar cell phenotype as miRNAs overexpression. • Overexpression TIRAP restores the diminished A549 viability caused by miRNAs and phycocyanin. As a type of functional food additives, phycocyanin is well-known for its extraordinary antineoplastic activity in non-small cell lung cancer (NSCLC). Previously, a high-throughput miRNA sequencing was employed to excavate miRNAs altered by phycocyanin in A549 cells, while the in-depth regulatory mechanism was inadequate. In this work, three significant up-regulated miRNAs in phycocyanin-treated cells are identified for the first time, including miR-3150a-3p, miR-6883-3p and miR-627-5p. Overexpression of miRNAs obviously diminished the cell growth and colony formation capacity, respectively. Dual-luciferase reporter assay verified that toll/interleukin 1 receptor domain-containing adaptor protein (TIRAP) was a direct shared target of three miRNAs. Knockdown the expression of TIRAP resulted in similar cell growth inhibition phenotype as miRNAs overexpression. Strikingly, overexpression of TIRAP could significantly restore the diminished cell viability caused by miRNAs up-regulation, as well as phycocyanin treatment. Consequently, this study proposes that phycocyanin plays an inhibitory effect of NSCLC cells via a miR-3150a-3p/miR-6883-3p/miR-627-5p-TIRAP axis.

Identification of novel inhibitors targeting TIRAP interactions with BTK and PKCδ in inflammation through an in silico approach

ABSTRACT Advanced computational tools focusing on protein–protein interaction (PPI) based drug development is a powerful platform to accelerate the therapeutic development of small lead molecules and repurposed drugs. Toll/interleukin-1 receptor (TIR) domain-containing adapter protein (TIRAP) and its interactions with other proteins in macrophages signalling are crucial components of severe or persistent inflammation. TIRAP activation through Bruton’s tyrosine kinase (BTK) and Protein Kinase C delta (PKCδ) is essential for downstream inflammatory signalling. We created homology-based structural models of BTK and PKCδ in MODELLER 9.24. TIRAP interactions with BTK and PKCδ in its non-phosphorylated and phosphorylated states were determined by multiple docking tools including HADDOCK 2.4, pyDockWEB and ClusPro 2.0. Food and Drug Administration (FDA)-approved drugs were virtually screened through Discovery Studio LibDock and Autodock Vina tools to target the common TIR domain residues of TIRAP, which interact with both BTK and PKC at the identified interfacial sites of the complexes. Four FDA-approved drugs were identified and found to have stable interactions over a range of 100 ns MD simulation timescales. These drugs block the interactions of both kinases with TIRAP in silico. Hence, these drugs have the potential to dampen downstream inflammatory signalling and inflammation-mediated disease.

TIRAP drives myelosuppression through an Ifnγ-Hmgb1 axis that disrupts the endothelial niche in mice.

Inflammation is associated with bone marrow failure syndromes, but how specific molecules impact the bone marrow microenvironment is not well elucidated. We report a novel role for the miR-145 target, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), in driving bone marrow failure. We show that TIRAP is overexpressed in various types of myelodysplastic syndromes (MDS) and suppresses all three major hematopoietic lineages. TIRAP expression promotes up-regulation of Ifnγ, leading to myelosuppression through Ifnγ-Ifnγr-mediated release of the alarmin, Hmgb1, which disrupts the bone marrow endothelial niche. Deletion of Ifnγ blocks Hmgb1 release and is sufficient to reverse the endothelial defect and restore myelopoiesis. Contrary to current dogma, TIRAP-activated Ifnγ-driven bone marrow suppression is independent of T cell function or pyroptosis. In the absence of Ifnγ, TIRAP drives myeloproliferation, implicating Ifnγ in suppressing the transformation of MDS to acute leukemia. These findings reveal novel, noncanonical roles of TIRAP, Hmgb1, and Ifnγ in the bone marrow microenvironment and provide insight into the pathophysiology of preleukemic syndromes.

DOP52 Development of a host-microbe interaction workflow to reveal the cell- and condition-specific effects of a commensal bacteria upon IBD

Abstract Background Humans are colonized by complex microbial communities which contribute to physiological processes in the host. The communication between microbes and host is crucial to maintain the homeostasis and gut health. Disruption in the microbiome composition leads to increased inflammation and appearance of diseases, such as inflammatory bowel disease (IBD). Interspecies interaction prediction combined with gene expression patterns on individual cell level by single-cell omics data reveals a new insight into the molecular background of cell-type specific host-microbe interactions. Methods Previously we developed the MicrobioLink pipeline (Andrighetti et al, Cells, 2020), an in silico microbe-host protein-protein interaction prediction algorithm. Here, we implemented a computational workflow based on MicrobioLink to predict and compare the cell-specific effects of a commensal bacteria in healthy and diseased conditions using a publicly available single-cell RNAseq dataset (Smilie et al, Cell, 2019) from colon biopsies describing 51 cell types - including fibroblasts, epithelial and immune cells - in healthy, non-inflamed and inflamed ulcerative colitis (UC). With functional analysis, microbe-affected processes have been discovered, while reliable network biology resources, such as OmniPath and Reactome, were used to identify the direct mechanism of action of the bacterial molecules. Results Demonstrating the applicability of the new computational workflow, we analysed the effect of a common gut commensal bacteria - Bacteroides thetaiotaomicron (Bt) - on human immune cells focusing on the Toll-like receptor (TLR) signalling. We found that extracellular vesicles (EVs) secreted by Bt may be able to modulate the TLR pathway intracellularly. The analysis highlighted that Bt targets differ among cells and between the same cells in healthy versus UC conditions. The in silico findings were validated in EV-monocyte co-cultures demonstrating the requirement for TLR4 and Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) in EV-elicited NF-kB activation. Conclusion The current pipeline offers potentially interesting connection points and detailed mechanistic insight containing mechanistic information about microbe-host interactions. This information can be tested and harnessed to understand better how microbial proteins may be of therapeutic value in inflammatory diseases, such as IBD.

Extracellular vesicles produced by the human commensal gut bacterium Bacteroides thetaiotaomicron affect host immune pathways in a cell-type specific manner that are altered in inflammatory bowel disease.

The gastrointestinal (GI) tract harbours a complex microbial community, which contributes to its homeostasis. A disrupted microbiome can cause GI‐related diseases, including inflammatory bowel disease (IBD), therefore identifying host‐microbe interactions is crucial for better understanding gut health. Bacterial extracellular vesicles (BEVs), released into the gut lumen, can cross the mucus layer and access underlying immune cells. To study BEV‐host interactions, we examined the influence of BEVs generated by the gut commensal bacterium, Bacteroides thetaiotaomicron, on host immune cells. Single‐cell RNA sequencing data and host‐microbe protein‐protein interaction networks were used to predict the effect of BEVs on dendritic cells, macrophages and monocytes focusing on the Toll‐like receptor (TLR) pathway. We identified biological processes affected in each immune cell type and cell‐type specific processes including myeloid cell differentiation. TLR pathway analysis highlighted that BEV targets differ among cells and between the same cells in healthy versus disease (ulcerative colitis) conditions. The in silico findings were validated in BEV‐monocyte co‐cultures demonstrating the requirement for TLR4 and Toll‐interleukin‐1 receptor domain‐containing adaptor protein (TIRAP) in BEV‐elicited NF‐kB activation. This study demonstrates that both cell‐type and health status influence BEV‐host communication. The results and the pipeline could facilitate BEV‐based therapies for the treatment of IBD.

Toll-like receptor 4 activation in platelets from myocardial infarction patients

IntroductionPlatelet toll-like receptor 4 (TLR4) is overexpressed in patients with myocardial infarction (MI) but it remains to elucidate if it is activated and the potential trigger. MethodsSerum levels of lipopolysaccharides (LPS) and platelet aggregation (PA) by collagen alone or in combination with a TLR4 inhibitor (TLR4i) were studied ex vivo in platelets from 40 MI patients and 40 controls matched for age, sex and atherosclerotic risk factors; platelet TIR domain-containing adaptor protein (TIRAP) and TIRAP-MyD88 interaction were also investigated by western blot and co-immunoprecipitation, respectively. In vitro experiments were conducted to see if LPS triggers platelet TIRAP phosphorylation. ResultsSerum LPS was significantly higher in patients compared to controls (29.5±7.1 vs 16.2±3.8 pg/mL; p<0.001). Collagen-stimulated platelets from MI pre-treated with TLR4i showed a significant decrease of PA compared to platelets stimulated with collagen. Ex vivo study showed that TIRAP phosphorylation as well as TIRAP-MyD88 co-immunoprecipitation were higher in patients compared to controls. In vitro study showed that LPS, at concentrations like those found in MI, dose-dependently activated TIRAP and amplified the platelet response to the agonist, an effect blunted by TLR4i. ConclusionThe study provides evidence that in MI patients platelet TLR4 is activated and suggests circulating LPS as potential trigger.

Toll-Like Receptors and Mannose Binding Lectin Gene Polymorphisms Associated with Cryptosporidial Diarrhea in Children in Southern India.

ABSTRACT.In low-resource settings, Cryptosporidium spp. is a common cause of diarrheal disease in children under the age of 3 years. In addition to diarrhea, these children also experience subclinical episodes that have been shown to affect growth and cognitive function. In this study, we screened polymorphisms in the promoter and exon1 regions of the mannose binding lectin 2 (MBL2) gene, as well as single nucleotide polymorphisms (SNPs) described in toll-like receptors (TLR) TLR1, TLR2, TLR4, and TLR9 and TIR domain-containing adaptor protein (TIRAP) genes among children with cryptosporidial diarrhea (cases) and children who only experienced asymptomatic (subclinical) cryptosporidiosis (controls). Among the polymorphisms screened, the variant allele B at codon 54 (rs1800450) of the MBL2 gene was associated with susceptibility to cryptosporidial diarrhea (odds ratio [OR] = 2.2, 95% confidence interval [CI] 1.1–4.5). When plasma MBL levels were compared, 72% of cases were found to be deficient compared with 32% among controls (OR = 5.09). Among TLR polymorphisms screened, multivariate analysis showed that heterozygous genotypes of TLR4 896A/G (rs4986790, OR = 0.33, 95% CI: 0.11–0.98) and TIRAP 539 C/T (rs8177374, OR = 0.19, 95% CI: 0.06–0.64) SNPs were associated with protection from cryptosporidial diarrhea. Although not statistically significant, these findings suggest that polymorphisms of MBL2 and TLR genes influence susceptibility to symptomatic cryptosporidial diarrhea even in settings with high exposure levels. Further studies to validate these findings in a larger cohort and to understand the role of these polymorphisms in mediating innate and adaptive immune responses to cryptosporidial infection are necessary.

Case report: hepatitis in a child infected with SARS-CoV-2 presenting toll-like receptor 7 Gln11Leu single nucleotide polymorphism

BackgroundCovid-19 has the respiratory tract as the main target of infection, and patients present mainly dyspnea, pneumonia, dry cough, and fever. Nevertheless, organs outside the respiratory tract had been reported in recent studies, including the gastrointestinal tract and liver. The host innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through their pattern recognition receptor (PRRs). Toll-like receptor 7 (TLR-7) is a pattern recognition receptor recognizing ssRNA (SARS-CoV-2 is an ssRNA). Polymorphisms are characterized by two or more alternative forms of a distinct phenotype in the same population. Polymorphisms in tlrs genes can negatively influence the immune response to infectious diseases. There are several references in the literature to non-synonymous single nucleotide (rs) polymorphisms related to several genes. Some of them are important for the innate immunity, as rs 179008 (tlr-7), rs3775291 (tlr3), rs8177374 (tir domain-containing adaptor protein, tirap), rs1024611 (monocyte chemoattractant protein-1, mcp-1) and rs61942233 (2′-5′-oligoadenylate synthase-3, oas-3).Case presentationWe identified a 5-year-old-male child with gastrointestinal symptoms and fever presenting acholic stool and jaundice, who was positive for SARS-CoV-2 IgM, IgA, and IgG and presenting the Gln11Leu rs 179008 in tlr-7. The child presented high levels of aspartate aminotransferase, alanine aminotransferase, bilirubin, C-reactive protein, D-dimer, gamma-glutamyl transferase, alkaline phosphatase, and was negative for serological tests for hepatitis A, B, C, E, HIV 1 and 2, herpes virus, cytomegalovirus, Epstein–Barr virus, and negative for RTqPCR for Influenza A and B, RSV and SARS-CoV-2. We also investigated other SNPs in the tlr-3 (rs3775291), tirap (rs8177374), mcp-1 (rs1024611), and oas-3 (rs61942233) genes, and no mutation was detected. After an interview with the child's caregivers, any possible accidental ingestion of drugs or hepatotoxic substances was ruled out.ConclusionTo our knowledge, this is the first report of a SARS-CoV-2 caused hepatitis in a male child that has the tlr-7 Gln11Leu rs 179008, which could impair an efficient initial immune response. The knowledge of the patient's immune deficiency could improve the treatment to correct this deficiency with specific medications.

A 192\xa0bp ERV fragment insertion in the first intron of porcine TLR6 may act as an enhancer associated with the increased expressions of TLR6 and TLR1

BackgroundToll-like receptors (TLRs) play important roles in building innate immune and inducing adaptive immune responses. Associations of the TLR genes polymorphisms with disease susceptibility, which are the basis of molecular breeding for disease resistant animals, have been reported extensively. Retrotransposon insertion polymorphisms (RIPs), as a new type of molecular markers developed recently, have great potential in population genetics and quantitative trait locus mapping. In this study, bioinformatic prediction combined with PCR-based amplification was employed to screen for RIPs in porcine TLR genes. Their population distribution was examined, and for one RIP the impact on gene activity and phenotype was further evaluated.ResultsFive RIPs, located at the 3' flank of TLR3, 5' flank of TLR5, intron 1 of TLR6, intron 1 of TLR7, and 3' flank of TLR8 respectively, were identified. These RIPs were detected in different breeds with an uneven distribution among them. By using the dual luciferase activity assay a 192 bp endogenous retrovirus (ERV) in the intron 1 of TLR6 was shown to act as an enhancer increasing the activities of TLR6 putative promoter and two mini-promoters. Furthermore, real-time quantitative polymerase chain reaction (qPCR) analysis revealed significant association (p < 0.05) of the ERV insertion with increased mRNA expression of TLR6, the neighboring gene TLR1, and genes downstream in the TLR signaling pathway such as MyD88 (Myeloid differentiation factor 88), Rac1 (Rac family small GTPase 1), TIRAP (TIR domain containing adaptor protein), Tollip (Toll interacting protein) as well as the inflammatory factors IL6 (Interleukin 6), IL8 (Interleukin 8), and TNFα (Tumor necrosis factor alpha) in tissues of 30 day-old piglet. In addition, serum IL6 and TNFα concentrations were also significantly upregulated by the ERV insertion (p < 0.05).ConclusionsA total of five RIPs were identified in five different TLR loci. The 192 bp ERV insertion in the first intron of TLR6 was associated with higher expression of TLR6, TLR1, and several genes downstream in the signaling cascade. Thus, the ERV insertion may act as an enhancer affecting regulation of the TLR signaling pathways, and can be potentially applied in breeding of disease resistant animals.

TIRAP in the Mechanism of Inflammation.

The Toll-interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP) represents a key intracellular signalling molecule regulating diverse immune responses. Its capacity to function as an adaptor molecule has been widely investigated in relation to Toll-like Receptor (TLR)-mediated innate immune signalling. Since the discovery of TIRAP in 2001, initial studies were mainly focused on its role as an adaptor protein that couples Myeloid differentiation factor 88 (MyD88) with TLRs, to activate MyD88-dependent TLRs signalling. Subsequent studies delineated TIRAP’s role as a transducer of signalling events through its interaction with non-TLR signalling mediators. Indeed, the ability of TIRAP to interact with an array of intracellular signalling mediators suggests its central role in various immune responses. Therefore, continued studies that elucidate the molecular basis of various TIRAP-protein interactions and how they affect the signalling magnitude, should provide key information on the inflammatory disease mechanisms. This review summarizes the TIRAP recruitment to activated receptors and discusses the mechanism of interactions in relation to the signalling that precede acute and chronic inflammatory diseases. Furthermore, we highlighted the significance of TIRAP-TIR domain containing binding sites for several intracellular inflammatory signalling molecules. Collectively, we discuss the importance of the TIR domain in TIRAP as a key interface involved in protein interactions which could hence serve as a therapeutic target to dampen the extent of acute and chronic inflammatory conditions.