Toll-like Receptor 4 Gene Research Articles

СОДЕРЖАНИЕ КАЛЬПРОТЕКТИНА В СТУЛЕ И ЭОЗИНОФИЛЬНОГО ПРОТЕИНА Х В МОЧЕ У МЛАДЕНЦЕВ В ЗАВИСИМОСТИ ОТ ПОЛИМОРФИЗМА ГЕНОВ ИНТЕРЛЕЙКИНА-10 (G1082A, C592A) И ТОЛЛ-ПОДОБНОГО РЕЦЕПТОРА 4 (ASP299GLY)

Background. Cytokine and toll-like receptor gene polymorphisms may impact oral tolerance formation in infants. Objective: To determine the genotype distribution of IL-10 (G1082A, C592A) and TLR 4 (Asp299Gly) gene polymorphisms in children with different family history of allergy residing in Grodno region; to analyze the association between gene polymorphisms and concentrations of fecal calprotectin (FCP) and urine eosinophil protein X (UEoPX) in infants. Material and Methods. 92 infants were recruited and analyzed for IL-10 (G1082A, C592A) and TLR 4 (Asp299Gly) gene polymorphisms. Concentrations of FCP and UEoPX were examined in dynamics in children aged 1 and 3 months. Results. 80.4% and 48.9% of infants were carriers of the mutant allele of the G1082A and C592A polymorphisms respectively. 70.5% of children with negative family history of allergy were carriers of the wild G allele of G1082A polymorphism. Carriage of the mutant A allele of G1082A polymorphism is associated with lower FCP concentration in infants aged 1 month (AA: 1.9 ng/mL [1.9; 3.1], GA: 15.9 ng/mL [1.9; 93.8]) and GG: 88.3 ng/mL [2.4; 230.1]). The level of UEoPX in 3 months old infants with homozygote AA genotype was significantly (p=0.019) higher than in infants with heterozygote CA genotype (3.2 ng/ml [2.4; 4.5] and 2.3 ng/ml [1.3; 3.3] respectively). Conclusion. Carriage of the wild G allele of G1082A polymorphism is associated with higher fecal calprotectin concentration in one month old infants and significantly lower level of urine eosinophil protein X in 3 months old infants.

Protective Effect of Omega-3 and the Potential of Toll-like Receptor Gene Expression in Rats with Doxorubicin-induced Cardiac Toxicity

Despite the clinical signs of doxorubicin (DOX)-induced cardiomyopathy, the mechanisms underlying DOX-induced cardiac damage are unknown. Toll-like receptors (TLRs) allow cardiomyocytes to respond to either endogenous or external signals, both of which have the potential to alter the pathophysiological responses to dilated cardiomyopathy. Omega-3 (OMG-3) precursors are highly active metabolites with numerous therapeutic benefits in the prevention and/or treatment of a variety of diseases. Thus, this study was aimed at evaluating the preventive effect of omega-3 on rats suffering from acute DOX-induced cardiotoxicity associated with TLR gene expression. Thirty rats were divided into five equal groups; Group 1 received no treatment, Group 2 received doxorubicin (at a toxic dose of 20 mg/kg), and Groups 3-5 received doxorubicin (20 mg/kg) after receiving OMG-3 at various doses (100, 200, and 400 mg/kg/day, respectively) for 4 weeks. At the end of the experiment, blood samples were obtained from the heart. The real time polymerase chain reaction (RT-PCR) was used to assess TLR2 and TLR4 gene expression. TLR2 had a significantly (p<0.01) elevated fold change in the DOX alone group (28.74+4.95), whereas TLR2 expression was significantly reduced in the OMG-3 pretreated groups (1.633+0.51, 0.733+0.13, and 0.709+0.16, respectively). TLR4 fold change in the DOX alone group was 8.57+1.22, whereas TLR4 expression was significantly reduced in the OMG-3 pretreated groups (100, 200, and 400 mg/kg) with values of 0.809+0.32, 0.852+0.50, and 0.272+0.16, respectively. The findings of this study revealed that OMG-3 decreased doxorubicin-induced cardiotoxicity and showed a significant cardioprotective effect by lowering TLR2 and TLR4 gene expression.

Study of the association of toll like-4 receptor gene polymorphism (rs1927911) and susceptibility to urinary tract infection among Iraqi patients

Introduction and Aim: Toll-like receptors (TLRs) are cell innate immune receptors that explore microbial molecules. A quantity of single-nucleotide polymorphisms (SNPs) within the TLR genes have been related with altered susceptibility to infectious, inflammatory, and allergic diseases suggesting: This study was conducted to investigate the relationship between genetic polymorphism in TLR4 gene (rs1927911) with susceptibility to UTIs in a sample of Iraqi patients. Materials and Methods: Blood samples were collected from 220 patients who had UTIs and 69 healthy subjects as control group, during the period from October 2021 to March 2022, at the Rheumatology Department of Clinic Consultant at Al-Yarmouk Hospital. The genetic study included an extraction of genomic DNA and PCR technique, to detect the role of TLR4 gene in the variation site (rs1927911) as a risk factor for the incidence of UTIs. Results: The results of genotype distribution and allele frequency of rs1927911 indicated, CC (18.6 %), CT (70 %) and TT (11.4 %) among the patients while 57.9 % for CC and 26.1%; 16% for CT, TT genotypes respectively of healthy control. Heterozygous (CT) may be considered as a risk factor among UTIs patients. The results revealed significant differences between UTIs patients and healthy controls in serum levels TLR4, IL-6 IL-2, TNF-alpha and IL-10 at P 0.005. In addition, the data found a highly significant difference of serum level of TLR4 protein with patients who have a carrier CT genotype. Conclusion: our findings indicated that the carriers of CT genotype of TLR4 (rs1927911) were significantly more prone to UTIs.

Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells.

In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators, beneficial in controlling bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflammatory diseases. Since distinct and rapid induction of TLR4 signaling is difficult to achieve with LPS due to the specific and non-specific affinity to other surface molecules and receptors, we engineered new light-oxygen-voltage-sensing (LOV)-domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LECs and opto-TLR4-LOV HUVECs) that allow fast, precise temporal, and reversible activation of TLR4 signaling pathways. Using quantitative mass-spectrometry, RT-qPCR, and Western blot analysis, we show that pro-inflammatory proteins were not only expressed differently, but also had a different time course when the cells were stimulated with light or LPS. Additional functional assays demonstrated that light induction promoted chemotaxis of THP-1 cells, disruption of the EC monolayer and transmigration. In contrast, ECs incorporating a truncated version of the TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LECs) revealed high basal activity with fast depletion of the cell signaling system upon illumination. We conclude that the established optogenetic cell lines are well suited to induce rapid and precise photoactivation of TLR4, allowing receptor-specific studies.

IL-32 is induced by activation of toll-like receptors in multiple myeloma cells.

Multiple myeloma (MM) is a hematological cancer characterized by accumulation of malignant plasma cells in the bone marrow. The patients are immune suppressed and suffer from recurrent and chronic infections. Interleukin-32 is a non-conventional, pro-inflammatory cytokine expressed in a subgroup of MM patients with a poor prognosis. IL-32 has also been shown to promote proliferation and survival of the cancer cells. Here we show that activation of toll-like receptors (TLRs) promotes expression of IL-32 in MM cells through NFκB activation. In patient-derived primary MM cells, IL-32 expression is positively associated with expression of TLRs. Furthermore, we found that several TLR genes are upregulated from diagnosis to relapse in individual patients, predominantly TLRs sensing bacterial components. Interestingly, upregulation of these TLRs coincides with an increase in IL-32. Taken together, these results support a role for IL-32 in microbial sensing in MM cells and suggest that infections can induce expression of this pro-tumorigenic cytokine in MM patients.

α-Synuclein induces prodromal symptoms of Parkinson’s disease via activating TLR2/MyD88/NF-κB pathway in Schwann cells of vagus nerve in a rat model

BackgroundIncreasing evidence suggests that patients with Parkinson's disease (PD) present with peripheral autonomic dysfunction (AutD) that even precedes motor deficits, through which α-synuclein can spread to the central nervous system. However, the pathological mechanisms underlying AutD in prodromal PD remain unclear. Here, we investigated the role of α-synuclein and its interplay with the activation of Schwann cells (SCs) of the vagus nerve in AutD.MethodsRats were subjected to injection with adeno-associated viruses containing the human mutated A53T gene (AAV-A53T) or an empty vector into the left cervical vagus nerve and evaluated for gastrointestinal symptoms, locomotor functions, intestinal blood flow, and nerve electrophysiology. Further, we examined the impact of α-synucleinopathy on vagus nerves, SCs, and central nervous system neurons using electron microscopy, immunofluorescence, immunohistochemistry, and western blot. Finally, the role of Toll-like receptor 2 (TLR2) in regulating the neuroinflammation in the vagus nerve via MyD88 and NF-κB pathway was determined using genetic knockdown.ResultsWe found that rats injected with AAV-A53T in the vagus nerve exhibited prominent signs of AutD, preceding the onset of motor deficits and central dopaminergic abnormalities by at least 3 months, which could serve as a model for prodromal PD. In addition, reduced intestinal blood flow and decreased nerve conduction velocity were identified in AAV-A53T-injected rats, accompanied by disrupted myelin sheaths and swollen SCs in the vagus nerve. Furthermore, our data demonstrated that p-α-synuclein was deposited in SCs but not in axons, activating the TLR2/MyD88/NF-κB signaling pathway and leading to neuroinflammatory responses. In contrast, silencing the TLR2 gene not only reduced inflammatory cytokine expression but also ameliorated vagal demyelination and secondary axonal loss, consequently improving autonomic function in rats.ConclusionsThese observations suggest that overexpression of α-synuclein in the vagus nerve can induce symptoms of AutD in prodromal PD, and provide support for a deeper understanding of the pathological mechanisms underlying AutD and the emergence of effective therapeutic strategies for PD.

Paeoniflorin alleviates inflammation in bovine mammary epithelial cells induced by Staphylococcus haemolyticus through TLR2/NF-κB signaling pathways

Staphylococcus haemolyticus (S. haemolyticus) is one of the most common coagulase-negative staphylococci (CoNS) isolates from bovine mastitis. Paeoniflorin (PF) shows anti-inflammatory effects on different inflammatory diseases in vitro studies and in vivo animal experiments. In this study, the viability of bovine mammary epithelial cells (bMECs) was detected by the cell counting kit-8 experiment. Subsequently, bMECs were induced with S. haemolyticus, and the induction dosage was determined. The expression of pro-inflammatory cytokines and toll-like receptor (TLR2) and nuclear factor kappa-B (NF-κB) signaling pathway-related genes were investigated by quantitative real-time PCR. The critical pathway proteins were detected by western blot. The results showed that the multiplicity of infection (MOI; the ratio of bacteria to bMECs) 5:1 of S. haemolyticus for 12 h could cause cellular inflammation, which was selected to establish the inflammatory model. Incubation with 50 μg/ml PF for 12 h was the best intervention condition for cells stimulated by S. hemolyticus. Quantitative real-time PCR and western blot analysis showed that PF inhibited the activation of TLR2 and NF-κB pathway-related genes and the expression of related proteins. Western blot results showed that PF suppressed the expression of NF-κB unit p65, NF-κB unit p50, and MyD88 in bMECs stimulated by S. haemolyticus. The inflammatory response pathway and molecular mechanism caused by S. haemolyticus on bMECs are related to TLR2-mediated NF-κB signaling pathways. The anti-inflammatory mechanism of PF may also be through this pathway. Therefore, PF is expected to develop potential drugs against CoNS-induced bovine mastitis.

The Impact of Variation in the Toll-like Receptor 3 Gene on Epizootic Hemorrhagic Disease in Illinois Wild White-Tailed Deer (Odocoileus virginianus).

Epizootic hemorrhagic disease (EHD) leads to high mortality in white-tailed deer (Odocoileus virginianus) and is caused by a double-stranded RNA (dsRNA) virus. Toll-like receptor 3 (TLR3) plays a role in host immune detection and response to dsRNA viruses. We, therefore, examined the role of genetic variation within the TLR3 gene in EHD among 84 Illinois wild white-tailed deer (26 EHD-positive deer and 58 EHD-negative controls). The entire coding region of the TLR3 gene was sequenced: 2715 base pairs encoding 904 amino acids. We identified 85 haplotypes with 77 single nucleotide polymorphisms (SNPs), of which 45 were synonymous mutations and 32 were non-synonymous. Two non-synonymous SNPs differed significantly in frequency between EHD-positive and EHD-negative deer. In the EHD-positive deer, phenylalanine was relatively less likely to be encoded at codon positions 59 and 116, whereas leucine and serine (respectively) were detected less frequently in EHD-negative deer. Both amino acid substitutions were predicted to impact protein structure or function. Understanding associations between TLR3 polymorphisms and EHD provides insights into the role of host genetics in outbreaks of EHD in deer, which may allow wildlife agencies to better understand the severity of outbreaks.

Development of immortalized feline respiratory epithelial cells in an air-liquid-interface culture system for feline herpesvirus-1 study

Feline herpesvirus-1 (FHV-1) is responsible for approximately 50% of diagnosed viral upper respiratory tract disease in cats. The virus infects and replicates in the epithelial cells located in upper respiratory tract. Commercial vaccines do not protect cats from the infection itself or development of latency. Previously, our lab developed a cell culture model using primary feline respiratory epithelial cells (pFRECs) to study respiratory innate immunity to FHV-1 and FHV-1 deletion mutants. However, the numbers of pFRECs that can be obtained per cat is limited. To improve the usage of respiratory epithelial 3D cultures in FHV-1 research, the present study immortalized feline respiratory epithelial cells (iFRECs) and characterized them morphologically and immunologically and evaluated the response to FHV-1 infection. Immortalization was achieved by transduction with Lenti-SV40T and Lenti-HPV E6/E7. Immortalized FRECs could be successfully subcultured for >20 passages, with positive gene expression of SV40T and HPV E6/E7. Immortalized FRECs expressed similar innate immunity-associated genes compared to pFRECs, including genes of Toll-like receptors (TLR1–9), interferon induced genes (OAS1, OAS3, IFI44, IFITM1, IFIT1), chemokines (CCL2, CCL3, CXCL8), pro-inflammatory and regulatory cytokines (IL-6, IL-4, IL-5, IL-12, and IL-18), and antimicrobials (DEFβ10, DEFβ4B). Finally, FHV-1 inoculation resulted in characteristic cytopathic effects starting at 24 hpi, with more than 80% cells detached and lysed by 72 hpi. Overall FHV-1 growth kinetics in iFRECs resembled the kinetics observed in pFRECs. In conclusion, we demonstrated that iFRECs are a useful tool to study feline respiratory disease including but not limited to FHV-1.

TLR4 involved in immune response against Vibrio Parahaemolyticus by MyD88-dependent pathway in Crassostrea hongkongensis

Vibrio parahaemolyticus (V. parahaemolyticus) is a salt-loving gram-negative bacterium, and is the leading cause of mortality in cultured shellfish in recent years. Toll-like Receptor 4 (TLR4) is a classical pattern recognition receptor (PRRs) that recognizes pathogen-associated molecular patterns (PAMPs) of pathogenic microorganism and activates the immune response. However, the function and signal pathway of TLR4 in oyster are still unknown. In this study, a new TLR4 gene was identified from the Crassostrea hongkongensis (C. hongkongensis). The ChTLR4 contained an open reading frame of 2643 bp, encoding 880 amino acids with seven leucine-rich repeat (LRR) domains and a Toll/IL-1R (TIR) domain. The ChTLR4 shared the highest sequence identity (83.0%) with TLR4 of Crassostrea gigas. Tissue expression analysis revealed that ChTLR4 showed the highest constitutive expression in the gill and hepatopancreas, and was significantly upregulated in immune tissues post V. parahaemolyticus infection, especially in gill and hemocytes. Moreover, TLR4 silencing significantly inhibited the immune-enzyme activities, including SOD, CAT, ACP, AKP in gill and LZM in hemolymph supernatant, and increased MDA content in hemolymph supernatant. Meanwhile, the antimicrobial activities of the hemolymph supernatant were also significantly inhibited by TLR4 silencing. These data demonstrated that the ChTLR4 involved in innate immune response of C. hongkongensis against V. parahaemolyticus challenge. Finally, qRT-PCR analysis showed that ChTLR4 silencing clearly inhibited the expression of genes in TLR4-MyD88 pathway, indicating that MyD88-dependent pathway played a crucial role in ChTLR4-mediated immune response against V. parahaemolyticus.

Toll-like Receptor 3 in the Hybrid Yellow Catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂): Protein Structure, Evolution and Immune Response to Exogenous Aeromonas hydrophila and Poly (I:C) Stimuli.

As a major mediator of cellular response to viral infection in mammals, Toll-like receptor 3 (TLR3) was proved to respond to double-stranded RNA (dsRNA). However, the molecular mechanism by which TLR3 functions in the viral infection response in teleosts remains to be investigated. In this study, the Toll-like receptor 3 gene of the hybrid yellow catfish was identified and characterized by comparative genomics. Furthermore, multiple sequence alignment, genomic synteny and phylogenetic analysis suggested that the homologous TLR3 genes were unique to teleosts. Gene structure analysis showed that five exons and four introns were common components of TLR3s in the 12 examined species, and interestingly the third exon in teleosts was the same length of 194 bp. Genomic synteny analysis indicated that TLR3s were highly conserved in various teleosts, with similar organizations of gene arrangement. De novo predictions showed that TLR3s were horseshoe-shaped in multiple taxa except for avian (with a round-shaped structure). Phylogenetic topology showed that the evolution of TLR3 was consistent with the evolution of the studied species. Selection analysis showed that the evolution rates of TLR3 proteins were usually higher than those of TLR3-TIR domains, indicating that the latter were more conserved. Tissue distribution analysis showed that TLR3s were widely distributed in the 12 tested tissues, with the highest transcriptions in liver and intestine. In addition, the transcription levels of TLR3 were significantly increased in immune-related tissues after infection of exogenous Aeromonas hydrophila and poly (I:C). Molecular docking showed that TLR3 in teleosts forms a complex with poly (I:C). In summary, our present results suggest that TLR3 is a pattern recognition receptor (PRR) gene in the immune response to pathogen infections in hybrid yellow catfish.

Role of intergenic interactions between cytokine and Toll-like receptor genes in the etiology of congenital heart defects

The incidence of congenital heart defects (CHDs) reaches 1% of all newborns. Of particular importance is the group of heart defects without a family history and chromosomal disorders, which makes up>80% of all CHDs. These heart defects can be designated as sporadic CHDs, and their etiology and pathogenesis continue to be studied.Aim. To study the association of cytokine and Toll-like receptor (TLR) genes with CHDs in children.Material and methods. We examined 188 children with sporadic (without family history) CHDs (main group) and 103 healthy children without CHDs (control group). Genotyping was performed by real-time polymerase chain reaction (PCR).Results. Sporadic CHDs, in general, and septal CHDs, in particular, are formed on similar intergenic associations that determine the dysregulation of the proliferation and differentiation of progenitor cells of the cardiovascular system. Probably, one of the molecular mechanisms for sporadic septal CHDs may be the inefficiency of intracellular signaling pathways for Nuclear Factor kappa B (NF-κB) due to the primary deficiency of membrane heterodimers TLR1/TLR6, TLR2/TLR6, and TLR1/TLR2.Conclusion. Data on altered proinflammatory potential in the group of sporadic septal CHDs should be taken into account in the postnatal period when conducting cardiac surgery.

Role of TLRs in HIV-1 Infection and Potential of TLR Agonists in HIV-1 Vaccine Development and Treatment Strategies.

Toll-like receptors (TLRs), as a family of pattern recognition receptors, play an important role in the recognition of HIV-1 molecular structures by various cells of the innate immune system, but also provide a functional association with subsequent mechanisms of adaptive immunity. TLR7 and TLR8 play a particularly important role in the innate immune response to RNA viruses due to their ability to recognise GU-rich single-stranded RNA molecules and subsequently activate intracellular signalling pathways resulting in expression of genes coding for various biological response modifiers (interferons, proinflammatory cytokines, chemokines). The aim of this review is to summarise the most recent knowledge on the role of TLRs in the innate immune response to HIV-1 and the role of TLR gene polymorphisms in the biology and in the clinical aspects of HIV infections. In addition, the role of TLR agonists as latency reversing agents in research to treat HIV infections and as immunomodulators in HIV vaccine research will be discussed.

Toll-like receptor 9 polymorphisms in brazilian patients with systemic lupus erythematosus: a pilot study.

Toll-like receptor 9 (TLR9) is an important component of the innate immune system and have been associated with several autoimmune diseases, such as Systemic Lupus Erythematosus (SLE). The aim of this study was to investigate polymorphisms in TLR9 gene in a Brazilian SLE patients group and their association with clinical manifestation, particularly Jaccoud's arthropathy (JA). We analyzed DNA samples from 204 SLE patients, having a subgroup of them presenting JA (n=24). A control group (n=133) from the same city was also included. TLR9 single nucleotide polymorphisms (SNPs) (-1237 C>T and +2848 G>A) were identified by sequencing analysis. The TLR9 gene genotype frequency was similar both in SLE patients and the control group. In the whole SLE population, an association between the homozygosis of allele C at position -1237 with psychosis and anemia (p < 0.01) was found. Likewise, the homozygosis of allele G at position +2848 was associated with a discoid rash (p < 0.05). There was no association between JA and TLR9 polymorphisms. These data show that TLR9 polymorphisms do not seem to be a predisposing factor for SLE in the Brazilian population, and that SNPs are not associated with JA.

Toll-like receptor 9 is involved in the induction of galectin-9 protein by dietary anti-allergic compound fucoidan

&lt;abstract&gt; &lt;p&gt;Dietary intervention of fucoidan extracted from &lt;italic&gt;Saccharina japonica&lt;/italic&gt; brown seaweed has been ascertained to favor an increase of galectin-9 protein in the intestine of allergic mice, resulting in the attenuation of the food allergy symptoms. The molecular mechanism underpinning that galectin-9 secretion remains unclear. Recently, some evidence has suggested an implication of Toll-like receptor 9 (TLR9) in galectin-9 secretion. However, no investigation has been done. For this study, we aimed to understand the relationship between galectin-9 production and fucoidan intake, which will improve the therapeutic use of fucoidan in allergy treatment. Intestinal epithelial cells (IECs) were cultured in solid or transwell plates and apically exposed to fucoidan solutions and/or synthetic TLR9 agonist (CpG-ODN). The transcriptional response of the cells to galectin-9 (lgals9) and the TLR9 gene was evaluated by using q-RTPCR, and the protein expression of galectin-9 was analyzed by conducting an ELISA test. Knockdown of TLR9 in IECs was performed by targeting TLR9 siRNA, and its effect on galectin-9 release was assessed. We found that the interaction of fucoidan and IECs resulted in the upregulation of galectin-9 released in a dose- and time-dependent manner. The increase was further potentiated in combination with the TLR9 agonist. Fucoidan exposure to IECs tended to increase the mRNA expression of TLR9 in a way similar to that of the TLR9 agonist effect, and knockdown of TLR9 in IECs resulted in a decreased tendency of fucoidan-induced galectin-9 protein. TLR9 activation is therefore involved in the increased release of galectin-9 protein observed in IECs upon fucoidan exposure.&lt;/p&gt; &lt;/abstract&gt;

Effects of the Co-Administration of Morphine and Lipopolysaccharide on Toll-Like Receptor-4/Nuclear Factor Kappa β Signaling Pathway of MDA-MB-231 Breast Cancer Cells.

The Toll-like receptor 4 (TLR4) gene promotes migration in adenocarcinoma cells. Morphine is an agonist for TLR4 that has a dual role in cancer development. The promoter or inhibitor role of morphine in cancer progression remains controversial. This study aims to evaluate the effects of morphine on the TLR4, myeloid differentiation primary response protein 88-dependent (MyD88), and nuclear factor-kappa B (NF-κB) expressions in the human MDA-MB-231 breast cancer cell line. The cells were examined after 24 hours of incubation with morphine using the Boyden chamber system. TLR4, MyD88, and NF-κB mRNA expressions were assessed using quantitative real-time polymerase chain reaction (RT-PCR). The concentration of interleukin-2 beta was also measured using the ELISA assay. According to the findings, three doses of morphine (0.25, 1.25, and 0.025 μM) increased the expression of the TLR4 and NF-κB genes, whereas no significant change was observed in the mRNA expression of MyD88. Furthermore, treatment with morphine and lipopolysaccharide (LPS) significantly decreased the expression of TLR4, MyD88, and NF-κB. However, no significant change was observed in interleukin 2 beta concentration. These findings confirmed the excitatory effects of morphine on TRL4 expression and the MYD88 signaling pathway in vitro.

Evaluation of toll-like receptors 2 and 4 polymorphism and intestinal microbiota in children with food allergies.

Mutual regulation between immune system and gut microbiota is achieved through several mechanisms including the engagement of toll-like receptors (TLRs) which is expressed on numerous cell types. In this study we aimed to explore the association between food allergies and TLR gene polymorphisms in association with gut microbiota. Toll-like receptors polymorphism frequencies and some bacteria in the gut microbiota in 130 infants aged 1-24 months with egg and/or milk allergy in a prospective cohort were compared with 110 non-food allergic controls. Four candidate polymorphisms (TLR2 rs1898830/rs5743708 and TLR4 rs4986790/rs4986791) were genotyped by allelic discrimination polymerase chain rection (PCR) method. Gut microbiota analysis was achieved by using high-throughput sequencing. The TLR4 rs4986790 (Asp299Gly) single nucleotide polymorphism (SNP) major/minor allele frequency was 0.788/0.212 in food allergy patients and 0.719/0.280 in controls (p=0.017). There was a statistically significant difference between groups in terms of genotype frequencies (AA, AG, GG). Gut microbiota analysis revealed increased Firmicutes phylum in stool of the patients with food allergy. Except for TLR4 rs4986791 (Thr399lle) allele, the other TLR polymorphisms were not associated with food allergies in children. When the bacteria in the intestinal microbiota and TLR2 and TLR4 gene polymorphisms were compared; we determined a statistically significant increase in Bifidobacterium concentration in the intestinal microbiota in TLR4 rs4986791 CT heterozygous genotype (p=0.004). This study demonstrated a partial role of TLR4 gene polymorphism and gut microbiota in the development of food allergies. Future work in this area will be required to clarify the roles of different microbial strains that modulate gut microbiota composition and function in conjunction with TLR transcription pathways.

Use of CRISPR/CAS9 Technologies to Study the Role of TLR in Dendritic Cell Subsets.

Dendritic cells (DCs) have a significant role in coordinating both innate and adaptive immunity by serving as sentinels that detect invaders and initiate immune responses to eliminate them, as well as presenting antigens to activate adaptive immune responses that are specific to the antigen and the context in which it was detected. The regulation of DC functions is complex and involves intracellular drivers such as transcription factors and signaling pathways, as well as intercellular interactions with adhesion molecules, chemokines, and their receptors in the microenvironment. Toll-like receptors (TLRs) are crucial for DCs to detect pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways that lead to DC maturation and education in bridging with adaptive immunity, including the upregulation of MHC class II expression, induction of CD80, CD86, and CD40, and production of innate cytokines. Understanding the TLR pathways that DCs use to respond to innate immune stimuli and convert them into adaptive responses is important for new therapeutic targets identification.We present a novel platform that offers a fast and affordable CRISPR-Cas9 screening of genes that are involved in dendritic cells' TLR-dependent activation. Using CRISPR/Cas9 screening to target individual TLR genes in different dendritic cell subsets allows the identification of TLR-dependent pathways that regulate dendritic cell activation and cytokine production. This approach offers the efficient targeting of TLR driver genes to modulate the immune response and identify novel immune response regulators, establishing a causal link between these regulators and functional phenotypes based on genotypes.

Polymorphism study of TLR4 gene in crossbred cattle of Kerala

The present study was conducted to identify single nucleotide variations in exon 3 region of the bovine Toll-Like Receptor gene (TLR4) associated with somatic cell score (SCS) in crossbred cattle of Kerala using polymerase chain reaction-single stranded conformational polymorphisms (PCR-SSCP) technique. Blood samples were collected from 200 lactating crossbred cattle in different farms of Kerala Veterinary and Animal Sciences University (KVASU) and field centres of ICAR- FPT scheme, KVASU. Genomic DNA was isolated and polymorphisms were identified by SSCP analysis. The 231 bp fragment of exon 3 in TLR4 gene was found to polymorphic and two genotypes viz. CC and CD were obtained with frequencies 77 per cent and 23 per cent respectively. The frequencies of C and D allele in crossbred were found to be 0.88 and 0.12. On sequencing, C to T transition had led to one non-synonymous change at 2021th position of ORF. Cattle with CC genotype were associated with significantly lower SCS (p≤0.05) as compared to CD genotype. The association between TLR4 polymorphism and SCS described in this study suggests the possible role of SNP during mastitis infection. Thus, could be employed as a useful marker for genetic selection to promote mastitis resistance in dairy cattle.

Determining Endosomal Toll-Like Receptors Gene Expression in NK Cells After Stimulation with Specific Agonists.

Poor knowledge is currently available about the biology of Toll-like receptors (TLRs) in natural killer (NK) cells. This is particularly due to the old belief that NK cells are unable to specifically eliminate microbes without presensitization. On the contrary, it has been clearly demonstrated that not only they can be activated through the engagement of Toll-like receptors (TLRs) by microbial molecules, but also that this interaction induces NK cells to release cytokines that, in turn, activate other cells of both innate and adaptive immunity. For this reason, immunotherapy based on local infusion of TLRs ligands is currently considered as a novel potential strategy to treat solid tumors. Here, we provide a protocol to efficiently stimulate NK cells via endosomal TLRs agonists and to determine endosomal TLRs gene expression level. This protocol can be used for in vitro investigation into endosomal TLRs function in NK cells under different conditions.