TLR4 Gene Research Articles

Pharmacodynamic effects and exploratory efficacy of afimetoran, a TLR7/8 inhibitor, in patients with cutaneous lupus erythematosus

Background: Cutaneous lupus erythematosus (CLE) is a chronic inflammatory skin condition occurring alone or as a manifestation of systemic lupus erythematosus (SLE). There is an unmet need for safe and effective CLE-focused treatments. Afimetoran is an investigational, first-in-class, potent oral inhibitor of toll-like receptors (TLRs) 7/8. Given the involvement of TLRs 7/8 in SLE, afimetoran may have therapeutic potential for CLE. A phase 1b randomized, double-blind, placebo-controlled study (NCT04493541) demonstrated preliminary safety and efficacy of afimetoran in patients with active CLE. We assessed the pharmacodynamics, safety, and efficacy of afimetoran and its impact on CLE pathobiology. Methods: Patients aged 18–65 years with SLE and cutaneous manifestations by EULAR/ACR 2019 classification criteria or biopsy-proven CLE, and modified CLE Disease Area and Severity Index-Activity (CLASI-A) score ≥6, were randomized 2:1 to once-daily afimetoran (30 mg) or placebo for 16 weeks with a 4-week post-treatment follow-up period. The primary endpoints were safety and tolerability; efficacy was exploratory. Transcriptomics and cytokine analyses were performed using peripheral whole blood and serum, respectively, at baseline (pre-dose) and each study visit (weeks 1, 2, 4, 8, 12, 16, and 20 [post-dose]). Statistical analyses compared data from the 13 randomized patients with thirteen age-, ethnicity-, and gender-matched healthy volunteers. Treatment responses were evaluated longitudinally in each treatment arm. The dataset was analyzed using a linear regression model. Gene set variation analysis (GSVA) was performed for pathway enrichment. Results: 13 patients with CLE were randomized (afimetoran, n=8; placebo, n=5); 12 patients completed 16 weeks of treatment and 1 discontinued after 6 weeks due to COVID-19. Compared with placebo, afimetoran demonstrated a favorable safety profile with no serious adverse events, and showed a greater reduction in CLASI-A scores as early as week 4, which persisted to week 20. Improvement in the molecular disease profile was rapid (week 1), sustained through the 16-week treatment period, and the 4-week post-treatment follow-up period. Expression of interferon (IFN) pathway genes was significantly reduced with afimetoran compared with baseline (ΔGSVA enrichment score [ES]=1.57, P<0.0001) and placebo (ΔGSVA ES=0.56, P<0.01); this effect was maintained through week 16 and ≥4 weeks post-treatment. Expression of TLR7 and TLR8 pathway genes and key cytokines (interleukin [IL]-6, tumor necrosis factor α, IL-18, IFNγ, macrophage inflammatory protein-1 alpha [CCL3/MiP1α] or beta [CCL4/MiP1β]) were greatly reduced with afimetoran treatment. GSVA demonstrated robust pharmacodynamic activity on immune cell populations, including immature and activated dendritic cells, macrophages, and inflammatory pathway signatures. Conclusion: In patients with CLE, afimetoran was safe, well tolerated, and demonstrated durable efficacy beyond the treatment period. Afimetoran’s clinical effects and potent pharmacodynamic impact on the molecular disease profile suggest a substantial therapeutic benefit for patients with CLE.

Impact of Nano-Pomegranate Seed Oil on the Expression of TLR2 and TLR4 Genes in A549 Cells Sensitized with Alternaria alternata Cellular Extract

Background: Allergies impact nearly 30% of the world's population, with fungi being remarkable contributors to allergic sensitivity. Exposure to fungi allergens can trigger allergic reactions and severe asthma has been linked to hypersensitivity to fungi such as Aspergillus and Alternaria spp. Alternaria alternata, a common allergen in respiratory allergic diseases, releases proteases that initiate Th2 responses, causing inflammatory cytokine production. Given the anti-inflammatory properties of Pomegranate Seed Oil (PSO) and the potential of Nano-emulsions (NEs) to enhance drug delivery, this study investigates the impact of PSO-loaded NEs on TLR2 and TLR4 gene expression in A549 cells sensitized with A. alternata extract (ALT). Methods: A. alternata (ATCC 6663) was cultured and processed to obtain a cytosolic extract, with protein content measured using the Bradford method. Using a Soxhlet apparatus, PSO was extracted from cleaned, dried seeds and analyzed by gas chromatography. Alginate nanospheres containing PSO were prepared through a modified water-in-oil emulsification method and characterized for particle size, polydispersity index, and zeta potential. A549 cells were cultured and treated with various ALT, PSO, and PSO-loaded NEs combinations. Following treatment, RNA was extracted, and real-time RT- PCR was conducted to analyze TLR2 and TLR4 gene expression. Results: All treatment groups showed an increase in TLR2 gene expression compared to the control, with the ALT combined with PSO (P+ALT) causing the highest increase at 4.82-fold. Free PSO (P) and free NEs (NP) resulted in 3.92-fold and 2.93-fold increases, respectively, while the ALT and PSO- loaded NEs (NP+ALT) led to 2.26-fold and 2.50-fold increases. For TLR4 gene expression, the ALT treatment increased expression by 2.29-fold, but treatments containing PSO (P, P+ALT, NP+ALT) reduced TLR4 expression, with P+ALT and NP+ALT causing 0.45-fold and 0.61-fold decreases. Conclusion: The study confirms that herbal extracts like PSO selectively upregulate TLR2 and downregulate TLR4, suggesting targeted therapeutic potential in inflammation and immune modulation. PSO-loaded NEs demonstrated superior anti-inflammatory effects, supporting their development for treating inflammatory diseases and warranting further research into their molecular mechanisms and therapeutic applications.

The molecular and histopathological investigations of TLR2 rs5743708 and TLR4 (rs4986790 and rs4986791) polymorphisms effects on cutaneous leishmaniasis lesions

Cutaneous leishmaniasis (CL), a zoonotic and neglected disease, is prevalent in numerous regions, particularly in tropical and sub-tropical countries. In Iran, endemic foci of leishmaniasis exist in specific regions, with zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania major being common in most rural areas. Toll-like receptors (TLRs) play a crucial role in both innate and adaptive immunities, and the investigation of TLR2 rs5743708 and TLR4 (rs4986790 and rs4986791) polymorphisms in parasitic diseases can have significant implications for patient treatment. In the present study, a total of 88 leishmaniasis patients using the patients' lesions from Khuzestan province health-treatment centers, Iran, including 50 cases (56.8%; Central region) and 38 cases (43.2%; Western region) underwent examination between the years 2022 and 2023. Two direct smears from the lesions of each patient were prepared and one of the smears was stained with Giemsa for parasitological examination. Among the 88 patients, the highest frequency was observed in the 21–30 years' age group (35.2%), while the lowest was in the 11–20 years’ age group (10.2%). No statistically significant relationship was found between gender and age (P > 0.05). Following disease confirmation via microscopic examination, TLR2 rs5743708 and TLR4 (rs4986790 and rs4986791) polymorphisms in the patients were assessed using PCR-RFLP. Fragments of 264, 249, and 406 base pairs were successfully amplified, targeting the TLR2 and TLR4 genes, respectively. Out of the 88 leishmaniasis patients, 14 cases (15.9%) exhibited polymorphisms. Notably, all individuals in the polymorphism group carried both the TLR2 rs5743708 homozygous and the TLR4 rs4986791 heterozygous genotype combinations. There were no observations of TLR2 rs5743708 heterozygous, TLR4 rs4986790 heterozygous and homozygous and TLR4 rs4986791 homozygous genotypes within the polymorphism group. Biopsies from lesions for all contributors were prepared for histopathological examination. All patients with polymorphism showed larger lesions than patients without polymorphism (P < 0.05). Histophatological study showed abnormal cases in patients with polymorphism including mild hyperkeratosis, mild acanthosis, focal parakeratosis in the epithelium surface and mild hyperpigmentation of melanocytes in the basal layer. Furthermore, a strong infiltration of immune cells such as PMNs and a small number of lymphocytes was observed in the epidermal region of patients with polymorphisms. There was no statistically significant relationship between age and the quantity of lesions (P > 0.05). Additionally, some regions of the epidermal surface layer displayed pustule formation in patients with polymorphisms. No significant difference was discerned in the dermal layers of patients with polymorphisms compared to other patients. Considering that all patients with polymorphisms concurrently had TLR2 rs5743708 homozygous and TLR4 rs4986791 heterozygous genotype combinations, the anomalies observed in conjunction with histological changes in the skin lesions of patients with CL may plausibly be linked to the polymorphisms. Nonetheless, a more expansive dataset involving a larger population is imperative to comprehensively elucidate the pathogenesis of the Leishmania parasite and the potential impact of TLR2 rs5743708 and TLR4 (rs4986790 and rs4986791) gene polymorphisms in individuals afflicted with CL across diverse geographical locales.

Expression of mRNA-TLR-5 Gene in Patients with Endometriosis using Real-time PCR in Tehran, Iran.

Endometriosis is one of the common diseases of women, especially in reproductive age, and it is one of the most important causes of infertility in women. The aim of this study was to investigate the level of mRNA-TLR-5 expression in women with endometriosis. The present study was performed in Nikan Hospital, Tehran, Iran, in 2021. The samples of endometrial mucosa for the eutopic group and an ovarian endometriotic cyst for the ectopic group were obtained from the patients who underwent laparoscopic surgery at the Fetal Infertility Center and were diagnosed with endometriosis. Normal endometrial samples were also obtained from patients who had no history of infertility and underwent laparoscopic TL surgery for reasons other than endometriosis such as ovarian cysts (control group). After RNA extraction and cDNA synthesis, TLR-5 gene expression was evaluated by the Real-Time PCR method. Based on the results of the comparison of TLR-5 gene expression in all three ectopic, eutopic endometrium, and control groups by Real-Time PCR, it was found that the TLR-5 gene expression is significantly higher in ectopic samples than in the other two groups, but there is a significant difference between two utopic and control groups. The increase in TLR-5 expression in the ectopic group can probably be a reason for reducing the apoptosis of cells entered into the peritoneal cavity and creating an environment for the survival and proliferation of these cells.

Expression of Toll-like Receptor Genes and Antiviral Cytokines in Macrophage-like Cells in Response to Indole-3-carboxylic Acid Derivative

Ongoing outbreaks and often rapid spread of infections caused by coronaviruses, influenza, Nipah, Dengue, Marburg, monkeypox, and other viruses are a concern for health authorities in most countries. Therefore, the search for and study of new antiviral compounds are in great demand today. Since almost all viruses with pandemic potential have immunotoxic properties of various origins, particular attention is paid to the search and development of immunomodulatory drugs. We have synthesised a new compound related to indole-3-carboxylic acid derivatives (hereinafter referred to as the XXV) that has antiviral and interferon-inducing activity. The purpose of this work is to study the effect of the XXV on the stimulation of the expression of toll-like receptor genes, interferons, and immunoregulatory cytokines in a macrophage-like cell model. In this study, real-time PCR methods were used to obtain data on the transcriptional activity of genes in macrophage-like cells. Stimulation of the genes of toll-like receptors TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 was detected. A high-fold increase in stimulation (from 6.5 to 16,000) of the expression of the TLR3 and TLR4 genes was detected after 4 h of exposure to the XXV. Increased activity of interferon (IFNA1, IFNA2, IFNB1, IFNK, and IFNλ1) genes with simultaneous stimulation of the expression of interferon receptor (IFNAR1 and IFNAR2) genes and signalling molecule (JAK1 and ISG15) genes was detected. Increased fold stimulation of the expression of the cytokine genes IL6, TNFA, IL12A, and IL12B was also observed. Thus, it is shown that the XXV is an activator of TLR genes of innate immunity, which trigger signalling mechanisms of pathogen “recognition” and lead to stimulation of the expression of genes of proinflammatory cytokines and interferons.

Exome Profiling Suggests Combined Effect of Myeloperoxidase, Toll-Like Receptors, and Metallopeptidase in Hidradenitis Suppurativa

Background: Hidradenitis suppurativa, also called acne inversa, is a chronic skin inflammatory condition involving hair follicles, sebaceous glands, and apocrine glands. Symptoms can be variable in intensity, ranging from mild to severe. The exact causes of hidradenitis suppurativa are not fully understood, but the etiology is presumed to be multifactorial, encompassing genetics and environmental factors. Methods: Two families presented with hidradenitis suppurativa with an autosomal dominant pattern. We performed whole-exome sequencing in two unrelated patients from the two families. Results: We identified two and three variants in the two families, respectively. Variants involved the TLR2 and MPO genes in the first family and the MMP2, GJB2, and TLR4 genes, some of which have already been previously reported as possible candidates for hidradenitis suppurativa. Conclusion: It is very likely that variants in a single gene only rarely cause the condition and that most cases, especially familial hidradenitis suppurativa cases, may more probably take the form of polygenic disorders.

Evaluation of the Relationship between TLR1(RS4833095) Gene Polymorphism and Disease in Patients with Ulcerative Colitis in the Turkish Population

Background: Ulcerative Colitis is a multifactorial disease which is characterized by recurrent periods of inflammation in the mucosal layer of the colon. Toll-like receptors (TLRs) are a class of transmembrane pattern recognition receptors that play a key role in the induction of pro/anti-inflammatory genes and in the control of adaptive immune responses. In this study, we aimed to evaluate the relation between TLR1(rs4833095) single nucleotide polymorphism and ulcerative colitis. Methods: The study included 90 patients with ulcerative colitis and a healthy control group consisting of 90 people. Taken medical treatment, laboratory data, colonoscopy findings, extraintestinal manifestations of patients included in this study were recorded. TLR1(rs4833095) single nucleotide polymorphism was studied with RT-PCR methods. Results: There was no increased risk for ulcerative colitis in patients with ulcerative colitis who has TLR1(rs4833095) single nucleotide polymorphism in Turkish population (p&amp;gt;0.05). There was no association between TLR 1(rs4833095) single nucleotide polymorphism and the spread of the disease in the colon, severity of disease and treatment required for remission in our study(p&amp;gt;0.05). Conclusion: In the Turkish population, TLR1 (rs4833095) single nucleotide polymorphism was evaluated and no significant difference was found between the patients with ulcerative colitis and the control group.

Profiling of Toll-like Receptors and Related Signaling Mediators in the Pathogenesis of Morphea

Introduction: Morphea, also known as localized scleroderma, is a rare fibrosing inflammatory disease of unknown pathogenesis. Objectives: Although the genetic basis for morphea is important, the evaluation of Toll-like receptors (TLR) in this disease is quite limited. We aimed to evaluate TLR expression levels and serum IL-6, IL-17A, TGF-b1, FGF, and VEGF levels in patients with morphea, and compare these results with healthy controls. Methods: The expression levels of TLRs in the lesional and non-lesional adjacent skin of patients with morphea, and normal skin of healthy controls were evaluated by RT-PCR whereas serum levels of IL-6, IL-17A, TGF-b1, FGF, and VEGF were evaluated by ELISA. Results: Based on our findings, TLR1 gene expression increased 34.3-fold in the lesional skin of patients with morphea. In addition, IL-6, IL-17A, TGF-β, FGF and VEGF were found to be higher in the blood samples of the patient group than in the healthy group. Conclusion: TLRs are important parts of the pathogenesis of morphea, and a better understanding of it will lead to more directed and effective treatments. We believe that this study will be important for pioneering TLR-targeted therapeutic approaches in the treatment of morphea in the future.

Alleviating effects of Gracilaria verrucosa supplement on non-specific immunity, antioxidant capacity and immune-related genes of pacific white shrimp (Litopenaeus vannamei) provoked with white spot syndrome virus

Our work evaluated the possible underlying roles of dietary dried seaweed (Gracilaria verrucosa; GV) on the inherent immune response, antioxidant capacity, immune-related gene expression, and protection of whiteleg shrimp (Litopenaeus vannamei) contra white spot syndrome virus (WSSV). Three hundred and sixty healthy L. vannamei (15.26 g ± 1.29 g) were graded into four supplemental groups ( Triplicate/group) and fed with diets including 0 (control), 2, 4, and 8 g GV (kg diet) −1 for 21 days. Following the feeding period, each group of shrimp received an intramuscular WSSV injection (1.4 × 106 copies/ml). Hemolymph and gills samples were collected before and after the challenge with WSSV. Notably, the administration of dietary GV significantly enhanced the innate immune parameters of pacific white shrimp including total hemocyte count (THC), phagocytosis, phenoloxidase activity, reactive oxygen species (ROS) production, and lysozyme activity before and after challenge with WSSV. Additionally, dietary supplementation of 4, and 8 g of GV (kg diet)−1 remarkably elevated ACP, AKP, SOD, GPx, and catalase activities along with a decrease in the MDA level in gills of shrimp before and post-WSSV challenge. In response to the GV supplement, significant upregulation of expression of ALF1, CRU1, PEN4, and CTL with downregulation of TRAF6, STAT, TLR1, and NOS genes was recorded in the gills tissue before and post-challenge with WSSV, especially at a dose of 8.0 GV g kg − 1. Dietary inoculated shrimp with GV revealed notably higher survival percentages after being challenged with WSSV. Conclusively, these data indicate that Gracilaria verrucosa can be recommended as a valuable supplemented seaweed to stimulate the innate immunity and enhance the health of Litopenaeus vannamei against viral infection.

Alterations in expression of TLR2 and TLR4 during successful and pathological aging

The generally accepted theory of aging is the theory of inflammaging. The leading role in its development is played by the mechanisms of innate immunity. Key receptors of innate immunity are TLRs. The patterns of age-related changes in the functioning of the TLR system remain the subject of study. The purpose of the work is to study the expression of TLR2 and TLR4 receptors in peripheral blood cells of centenarians and elderly people at the level of genes and surface molecules. The study analyzed the expression of genes and molecules TLR2 and TLR4 in young donors (n = 50), elderly people (n = 50) and centenarians (n = 100). Analysis of TLR2 and TLR4 gene expression was carried out using RT-PCR. Determination of surface expression of TLR2 and TLR4 was carried out using flow cytofluorimetry. This study is the first to analyze the age-related dynamics of expression of genes and molecules TLR2 and TLR4. It was revealed that in groups of elderly patients and long-livers, TLR2 expression is increased both at the gene and on the cell surface, compared to young donors. In contrast, TLR4 expression decreased with age. Studied groups of patients were divided depending on the aging phenotype into two subgroups: successful and pathological aging. In elderly individuals, TLR2 is increased in both phenotypes, while TLR4 is decreased in the pathological aging. In the group of centenarians with a pathological aging, a decrease in both TLR2 and TLR4 is observed, compared with young. In long-livers with successful aging, an increase in TLR2 expression was observed at the gene and protein level and TLR4 expression was comparable to the group of young individuals. Identified changes in the expression of TLR2 and TLR4, observed in different age groups, can be considered as markers of various aging phenotypes.

Clopidogrel ameliorates high-fat diet-induced hepatic steatosis in mice through activation of the AMPK signaling pathway and beyond.

Metabolic dysfunction-associated steatotic liver disease (MASLD) frequently confers an increased risk of vascular thrombosis; however, the marketed antiplatelet drugs are investigated for the prevention and treatment of MASLD in patients with these coexisting diseases. To determine whether clopidogrel could ameliorate high-fat diet (HFD)-induced hepatic steatosis in mice and how it works, mice were fed on normal diet or HFD alone or in combination with or without clopidogrel for 14weeks, and primary mouse hepatocytes were treated with palmitate/oleate alone or in combination with the compounds examined for 24h. Body weight, liver weight, insulin resistance, triglyceride and total cholesterol content in serum and liver, histological morphology, transcriptomic analysis of mouse liver, and multiple key MASLD-associated genes and proteins were measured, respectively. Clopidogrel mitigated HFD-induced hepatic steatosis (as measured with oil red O staining and triglyceride kit assay) and reduced elevations in serum aminotransferases, liver weight, and the ratio of liver to body weight. Clopidogrel downregulated the expression of multiple critical lipogenic (Acaca/Acacb, Fasn, Scd1, Elovl6, Mogat1, Pparg, Cd36, and Fabp4), profibrotic (Col1a1, Col1a2, Col3a1, Col4a1, Acta2, and Mmp2), and proinflammatory (Ccl2, Cxcl2, Cxcl10, Il1a, Tlr4, and Nlrp3) genes, and enhanced phosphorylation of AMPK and ACC. However, compound C (an AMPK inhibitor) reversed enhanced phosphorylation of AMPK and ACC in clopidogrel-treated primary mouse hepatocytes and alleviated accumulation of intracellular lipids. We concluded that clopidogrel may prevent and/or reverse HFD-induced hepatic steatosis in mice, suggesting that clopidogrel could be repurposed to fight fatty liver in patients.

Influence of polymorphisms on the phenotype of TLR1, TLR4 and TLR9 genes and their association with cervical cancer: Bioinformatics prediction analysis and a case-control study

Susceptibility to cervical cancer has been associated with Toll-like receptors (TLRs), which is an important component of innate immunity. According to previous studies, polymorphisms in TLRs genes can affect immune response pathways and lead to the development of cervical cancer. The present study aims to evaluate the functionality of polymorphisms in TLR1, TLR4 and TLR9 genes and their associations with cervical cancer. To identify the functionality of polymorphisms, we used the following tools: MUpro, ChimeraX, SNP2TFBS and GTEx. A case-control study including 57 cases (11 High-grade Intraepithelial Lesion - HSIL and 46 cervical cancer) and 67 clinically healthy controls was conducted in the Brazilian population. Polymorphisms genotyping was performed by real-time PCR, using TaqMan probes, using the allelic discrimination method. Bioinformatics prediction showed that the TLR1 rs4833095 [NM_003263.4 (TLR1):c.743T>C (p.Asn248Ser)] and TLR4 rs4986790 [NM_138554.5 (TLR4):c.896A>G (p.Asp299Gly)] polymorphisms alter the structure and stability of their respective proteins. TLR9 rs187084 [NM_017442.3(TLR9):c.-1486A>G] polymorphism seems to affect the THAP1 binding site and modify gene expression. In the case-control study, the c.743TC heterozygous genotype of the rs4833095 SNP in the TLR1 gene was associated with an increased risk for HSIL/cervical cancer. No association of TLR4 rs4986790 and TLR9 rs187084 SNPs with HSIL/cervical cancer was found in the studied population. Allelic combination CAG (rs4833095/ rs4986790/ rs187084) increased the risk of cervical cancer. In conclusion, the present study identified that polymorphisms in TLRs genes can affect the phenotype of their respective genes and contribute to the development of HSIL or cervical cancer.

Fentanyl enhances immune cell response through TLR4/MD-2 complex.

Opioids have been shown to induce neuroinflammation and immune cell activation, that might contribute to some of the opioid side effects, such as opioid-induced tolerance and paradoxical hyperalgesia. In this context, TLR4/MD-2 complex has been proposed as an off-target site for opioid action. This study was aimed at investigating the effect of fentanyl on lipopolysaccharide (LPS)-induced TLR4/MD-2 activation in rat primary microglia and human monocyte-derived macrophages (MDM). The effect of fentanyl was first explored by measuring the expression and release of different proinflammatory mediators in primary rat microglia and human MDM by real-time PCR and ELISA. Then, the involvement of TLR4/MD-2 signaling was investigated studying NF-κB activation in HEK293 cells stably transfected with human TLR4, MD-2, and CD14 genes (HEK-Blue hTLR4 cells) and in human MDM. Fentanyl increased mRNA levels, as well as the LPS-induced secretion of proinflammatory mediators in primary microglia and MDM. Two inhibitors of TLR4/MD-2 signaling, namely the oxazoline derivative of N-palmitoylethanolamine (PEA-OXA) and CLI-095, blocked the production and release of proinflammatory cytokines by microglia stimulated with LPS and fentanyl, suggesting that TLR4/MD-2 could be the target of the proinflammatory activity of fentanyl. Finally, we showed that fentanyl in combination with LPS activated NF-κB signaling in human MDM and in HEK-Blue hTLR4 cells and this effect was blocked by inhibitors of TLR4/MD-2 complex. These results provide new insight into the mechanism of the proinflammatory activity of fentanyl, which involves the activation of TLR4/MD-2 signaling. Our findings might facilitate the development of novel inhibitors of TLR4/MD-2 signaling to combine with opioid-based analgesics for effective and safe pain management.

Association of the Single Nucleotide Polymorphism 19216T/C in the TLR2 Gene (rs3804099) with Entamoeba histolytica/Entamoeba dispar/Entamoeba moshkovskii Infection Among Lebanese Children.

Toll-like receptors (TLRs), particularly the TLR2, take part in the elicitation of immune responses against Entamoeba histolytica. This study aimed to investigate the relationship between a specific polymorphism called rs3804099 in the TLR2 gene and E. histolytica/E. dispar/E. moshkovskii infection among Lebanese children. A case-control study encompassed 180 participants including 68 children with amebiasis and 112 matched controls. Blood samples were collected, and genomic DNA was extracted using the classical proteinase K digestion and phenol-chloroform extraction method. The variant rs3804099 was examined using the Amplification Refractory Mutation System Polymerase Chain Reaction. The accuracy of the genotyping was supported by sequencing 5% of samples. The TLR2 rs3804099 polymorphism was identified in the studied population, and the observed genotypic distributions were consistent with Hardy-Weinberg equilibrium (P > 0.05). The frequency of the rare CC genotype was significantly higher in patients compared to the noninfected group (P < 0.01). In controls, the homozygous TT genotype was less frequent than the heterozygous CT genotype. The rare CC genotype was associated with a higher risk of amebiasis among children (odds ratios = 3.27, P = 0.002). These findings provide evidence supporting the association between the rs3804099 SNP in the TLR2 gene and E. histolytica/E. dispar/E. moshkovskii infection among Lebanese children.

S1839 Frameshift Mutations in the TLR4 Gene with Potential Protective Effect on Progression to Fibrosis in MASLD Patients: A Pilot Study

S1839 Frameshift Mutations in the TLR4 Gene with Potential Protective Effect on Progression to Fibrosis in MASLD Patients: A Pilot Study

TLR-2 and TLR-4 mRNA expression in different grades of histopathological lesions of equine endometrium from follicular phase.

Increased synthesis and deposition of collagen(COL) in the extracellular matrix (ECM) of equine endometrium contributes to endometrosis. Toll-like receptors (TLRs) are transmembrane receptors involved in the innate immune response, recognized for their role in antigen recognition and previously associated with equine endometritis. The TLRs not only recognize pathogen-associated molecular patterns but also regulate inflammations, fibrosis and cancer. The aim of this study was to explore the relationship between TLR expression at different stages of Kenney and Doig's (K-D) grading and COL1 expression during the follicular phase of the oestrous cycle. Forty samples of endometrial tissues were collected post-mortem from mares on the follicular phase of the oestrous cycle (10 samples of each K-D category). Relative mRNA transcription of TLR-2, TLR-4 and COL1A2 genes was assessed using qPCR, and COL1 protein expression by Western blot analysis. The COL1A2 transcription increased in category IIB when compared to categories I, IIA and III endometria (p < .01). The relative protein abundance of COL1 showed no significant differences between endometrial categories (p > .05). As for the TLRs mRNA transcription, TLR-2/-4 transcripts increased in IIA when compared to the other K-D endometria categories (p < .05). Our findings suggest that TLRs may be involved in the initiation of the endometrial inflammatory response. Additional studies are needed to explore TLRs' potential role as diagnostic markers for monitoring inflammation progression and fibrosis development, as well as their involvement in the mechanisms underlying fibrotic pathways.

TLR4 and TNF-α single nucleotide polymorphisms in patients with brucellosis: Association with infection complications

ObjectivesTo investigate associations of the carriage of single nucleotide polymorphisms (SNPs) of proteins involved in the immune response of patients with brucellosis. MethodsA case control study of patients with brucellosis upon WHO criteria. Blood genomic analysis was performed by RFLP- PCR for the detection of SNPs: i) at promoters -376 G > A (rs1800750); -308 G > A (rs 1,800,629); -238 G > A (rs361525) of the TNF gene, ii) at -896 A > G Asp299Gly (rs4986790) and -1196 C > T Thr399Ile (rs4986791) positions of the TLR-4 gene. Logistic regression analysis of factors related to brucellar spondylodiscitis was performed. ResultsPatients with brucellosis (n = 105) were male (n = 67, 63.8 %); mean age (SD): 49.51(18.31); spondylodiscitis (n = 30), sacral osteomyelitis (n = 21). Carriage of the minor frequency A alleles at -238 of the promoter region of TNF was greater in patients than in controls (11.4% vs 2.6 %, p < 0.001). In a stepwise regression model including host variables and TNF-238 G A-1 genotype, only the last one was associated with brucellar spondylodiscitis [OR 2.91 (CI95 % 1.02–8.31), p = 0.047]. ConclusionsIn our cohort, the association of one TNF SNP of patients with brucellosis, in particular spondylodiscitis, might be prognostic whereas further investigation of the exact role in the host immune response is required.

The pro-inflammatory responses of innate immune cells to Leishmania RNA virus 2-infected L. major support the survival and proliferation of the parasites

Infection of Leishmania by Leishmania RNA virus (LRV) has been proposed as a pathogenic factor that induces pro-inflammatory responses through the TLR3/TLR4 signaling pathway. We investigated the effect of L. major infection by LRV2 on innate immune cell responses (human neutrophil (HL-60) and macrophage (THP-1) cell lines). The expression levels of pro- and anti-inflammatory cytokine and chemokine genes as well as genes involved in the amino acid metabolism of arginine were then investigated by RT-qPCR. Moreover, the expression of TLR genes and their downstream signaling pathways were compared in THP-1 cells infected with the two isolates. Apoptosis was also evaluated in infected THP-1 and HL-60 cells using the PI/Annexin V flow cytometry assay. In both cell lines, the expression of pro-inflammatory cytokines increased in response to LRV2+ L. major (Lm+), and the expression of chemokines shifted toward macrophage recruitment. In contrast to LRV2- L. major (Lm-), Lm+ infected THP-1 cells acquired the M2-like phenotype. The presence of LRV2 increased the gene expression of TLRs and their signaling pathways, especially TLR3 and TLR4, which was proportional to the increase in pro-inflammatory cytokines. In addition, Lm+ increased the expression of IL-10 and IFN-β, which contribute to the survival and growth of the parasite in the phagolysosome. Altogether, our results showed that Lm+ could stimulate pro-inflammatory responses that promote parasite replication and stabilization in the host.

Evaluation of Gene Expression Levels of TLRs 2, 4, and 5 Genes in Patients with Chronic Suppurative Otitis Media (CSOM) and Relationship with Bacterial Infection

Toll-like receptors (TLRs), the building blocks of the innate immune response, are largely responsible for the development of otitis media. The illness may worsen due to mistakes or faults in TLR expression in immune cells found in the blood or middle ear tissue. People with various bacterial infections and chronic otitis media were evaluated for their TLR (TLR2, TLR4, and TLR5) gene expression. The results showed that higher expression levels of TLR2 were associated with Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa in mucosal cells, whereas lower expression levels were associated with Moraxella catarrhalis and Haemophilus influenzae. The study found that TLR4 gene expression was higher in the mucosal cells of patients infected with Moraxella catarrhalis, Pseudomonas aeruginosa, Haemophilus influenzae and Streptococcus pneumoniae, compared to healthy controls, while Staphylococcus aureus was associated with lower expression. The study found that patients with infections caused by Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus had no significant expression levels of the TLR5 gene in their mucosa cells, while patients with infections caused by Pseudomonas aeruginosa and Streptococcus pneumoniae had higher expression levels. TLRs2 mRNA expression levels increased in patients with chronic otitis media infected with Gram-positive bacteria (Staphylococcus aureus and Streptococcus pneumoniae), while TLR-4 and TLR-5 levels increased in patients infected with Gram-negative bacteria (Pseudomonas aeruginosa and Streptococcus pneumoniae).

Role of rs5743708 SNP in the risk of Bacterial Respiratory Infections in Children of Ramadi city/Iraq

Background and Aim: Respiratory Infections are a major global health and financial burden, so among cause the five most common and deadly for respiratory illnesses is acute lower respiratory tract infections (RTIs), these infections claim over 4 million lives annually. Toll-like receptors (TLRs) are key components of the innate immune system that recognize pathogen which associated a molecular patterns) PAMPs) and initiate an immune response. Understanding the role of TLR polymorphisms in susceptibility to respiratory infections in children is important for developing strategies to prevent and treat these infections. So, the current study aims to "Investigate the potential association between TLR2 gene polymorphisms and susceptibility to respiratory infections in children". Methodology: The study included collect of 300 samples with respiratory infection from both sex with range ages(1-12) years, and other 50 healthy control samples. The samples were collected from general and private hospitals in Ramadi city, Iraq. PCR was carried out using primer for gene target and amplification of target gene. Sanger Sequencing conducted with both direction for all samples. Results and Conclusion: The result of sputum culture indicate that 100 of suspected samples were positive culturing while other demonstrate a negative growth. The results analysis of sequencing according to Query and subjected samples indicated that 4 of patient have SNP of rs5743708 G&gt;A, while there is not found in control samples, and the results of genotype frequencies analysis it was found that TLR2 polymorphism was associated with increased risk for bacterial respiratory infections in children according to under study samples, but no statistical significant differences correlation of rs5743708 G&gt;A and infection, with adjusted odds ratio [OD:5.3; 95%Cl: 0.3-101; P: 0.2].