TLR4 Gene Research Articles

A comparative study of the effects of crude chicory and inulin on gut health in weaning piglets

Dysfunction of the host-microbial balance and an impaired intestinal barrier can trigger inflammation and increase the antigen penetration. Inulin, commonly extracted from chicory root, is a prebiotic beneficial to gut health. The objective of this study was to compare the effect of chicory flour to inulin on gut health, few weeks after weaning. Two dose-dependent experiments (E1 and E2) were performed sequentially, each consisting of 80 castrated male piglets, weaned at day 21 and subsequently divided in 3 groups with ad libitum feed: control (Ctrl), inulin (INU) and crude chicory flour (CHI). For INU and CHI groups, a daily supplementation with the equated ‘inulin content’ increasing weekly was done by oral force-feeding, while the Ctrl groups received an isotonic sucrose solution. For E1, these doses were 1.5 g/day, 2 g/day and 2.5 g/day in W1, W2 and W3, respectively. For E2, these doses were 3 g/day, 4 g/day and 5 g/day in W1, W2 and W3, respectively. For each experiment at W0, W1 and W3, eight piglets per group were euthanized to assess gut structural and functional parameters. In E1, the CHI had lower average daily calorie intake (kcal/day) only at W3, while in E2 it was consistently lower than Ctrl and INU. In W3 of E2, CHI showed improved villi-to-crypt ratio and lower diarrhea occurrence than INU and Ctrl. Both supplemented groups in E2 showed higher butyrate production and lower D-xylose permeability (W3), compared to Ctrl. Interestingly, in E2, CHI had a more dominant effect on increasing the abundance of health promoting genera like Catenisphaera, Butyricicoccus, etc. and decreasing harmful genera like Erysipelotrichaceae_UCG-002 and Turicibacter. In E2, on W3 several inflammatory target genes (CXCL10, IL18, TNFα) and inflammation signalling genes (MyD88, NFκB1) were downregulated in ileum of INU and CHI. In colon, both chicory and inulin, proved to be beneficial, as the inflammation signalling and inflammatory targets genes NFκB1, DEFβ4A, TLR2 and IFNα were significantly downregulated. Therefore, crude chicory flour might also be a promising cost-effective alternative supplement to improve gut health in weaned piglets.

HDL Cholesterol-Associated Shifts in the Expression of Preselected Genes Reveal both Pro-Atherogenic and Atheroprotective Effects of HDL in Coronary Artery Disease.

The associations of high-density lipoprotein (HDL) level and functionality with lipid metabolism, inflammation, and innate immunity in coronary artery disease (CAD) remain controversial. The differential expression of a set of genes related to HDL metabolism (24 genes) and atherogenesis (41 genes) in peripheral blood mononuclear cells (PBMC) from CAD and control patients with varied HDL cholesterol (HDL-C) levels was compared. 76 male patients 40-60 years old with CAD diagnosed by angiography and 63 control patients were divided into three groups with low, normal (1.0-1.4 mM), and increased HDL-C levels. Transcript levels were measured by real-time PCR. The differentially expressed genes (DEGs) and associated metabolic pathways were analyzed for three groups, with prevalent CAD as an outcome. The common feature was the increased odds ratio values for liver X receptor (LXR) gene expression for three patient groups. CAD patients with low HDL-C possessed 24 DEGs with lower expression of genes involved in cholesterol efflux, and down-regulated SREBF1 and ABCG1 are suggested as gene signatures. CAD patients with normal HDL-C possessed nine DEGs with down-regulated ITGAM and ALB as gene signatures. CAD patients with increased HDL-C possessed 19 DEGs with down-regulated APOA1 and HMGCR as gene signatures. With gene expression signatures, one standard deviation higher average gene expressions were associated with 5.1-, 48.8-, and 38.9-fold fewer CAD cases for three patient groups. As HDL-C increased in CAD patients, the expression of ABCG1, CUBN, and HDLBP genes increased, while the expression of HMGCR and NPC2 genes, involved in cholesterol synthesis and trafficking, decreased. The expression of CD14, CD36, S100A8, S100A9, S100A12, TLR5, TLR8, and VEGFA genes, involved in angiogenesis and inflammation mainly via nuclear factor-κB (NF-κB), decreased. The increased accumulation of cholesteryl ester in PBMC from patients with low HDL-C was suggested. This assumption contrasts with the suggested accumulation of free cholesterol in PBMC from patients with increased HDL-C, concomitant with suppression of cholesterol synthesis and traffic to the plasma membrane, and with an inflammatory state controlled by depressed CD36-mediated and upregulated apoE-mediated immunometabolic signaling. Gene signatures may be used for the diagnosis, prognosis, and treatment of CAD in dependence on HDL-C levels.

Alterations in Circular RNAs circOprm1 and circSerpini in the Striatum are Associated with Changes in Spatial Working Memory Performance after Morphine Dependence and Withdrawal in Rats.

Modulating role of circRNAs and microRNAs in neurobiological changes induced by drug exposure remains unclear. We examined alterations in some circRNAs and microRNAs in the striatum after morphine dependence and withdrawal and their associations with the changes in spatial working memory performance. Male Wistar rats were used in which 10 days morphine exposure induced dependence. Withdrawal effects were assessed 30 days after stopping morphine exposure. Spatial working memory was assessed using a Y maze test on days 1 and 10 of the drug exposure and 30 days after withdrawal. The gene and protein expression were assessed after dependence and withdrawal. The results revealed that 10 days morphine exposure impaired working memory, which partially reinstated after withdrawal. After 10 days morphine exposure, significant increases in Oprm1 gene and OPRM1 protein levels were detected, which persisted even after withdrawal. The expression of circOprm1 and miR-339-3p decreased in the morphine-dependent group, but they returned to normal levels after withdrawal. The expression of Tlr4 gene and TLR4 protein levels decreased after dependence. While Tlr4 mRNA levels returned to normal after withdrawal, TLR4 protein levels remained lower than the control group. In the morphine-dependent group, both Serpini1 and circSerpini expression significantly increased, but they restored after withdrawal. Expression of miR-181b-3p, miR-181b-5p, miR-181c-3p, and miR-181c-5p decreased after dependence, but they reinstated after withdrawal. It can be concluded that circOprm1 and circSerpini via regulating the OPRM1 and TLR4 expression in the striatum are associated with the neuroadaptation underlying spatial working memory after both morphine dependence and withdrawal.

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.

ROLE OF TOLL-LIKE RECEPTOR POLYMORPHISMS IN THE DEVELOPMENT OF EXTREMELY SEVERE COVID-19 WITH FATAL OUTCOME

COVID-19 is a respiratory disease caused by the Sars-Cov-2 virus that can proceed to acute respiratory distress syndrome (ARDS) and trigger immunopathological mechanisms that lead to excessive inflammation. Toll-like receptors, as pattern recognition receptors, play an indispensable role in virus identification and activate the innate immune system, which can lead to the secretion of proinflammatory cytokines such as IL-1, IL-6, TNFα, and type I IFN. Antiviral toll-like receptors, including TLR3, TLR7, and TLR9, can activate the TRIF-dependent pathway, induce the production of proinflammatory cytokines, chemokines, and type I and type III interferons, participate in recognition of dsRNA and detection of unmethylated CpG DNA. In addition, an important role of TLR2 in COVID-19, which recognizes peptide glycans on the surface of gram-positive bacteria, and TLR4, which recognizes gram-negative bacteria lipopolysaccharide and plays a role in hyperinflammation in COVID-19, has been shown. It was confirmed that TLRs can be involved both in the initial failure of virus clearance and in the subsequent development of severe COVID-19 clinical manifestations mainly acute respiratory distress syndrome (ARDS) with fatal respiratory failure. The aim of this work was to study the polymorphisms of TLR3 (rs3775291), TLR9 (rs352140), TLR7 (rs3853839), TLR4 (rs4986790), TLR4 (rs4986791), TLR2 (rs574708) in patients with COVID-19 depending on disease outcome. The study included 187 patients, of whom 157 recovered and 30 deceased. Genetic analysis for polymorphisms of the TLR3 (rs3775291), TLR9 (rs352140), TLR7 (rs3853839), TLR4 (rs4986790), TLR4 (rs4986791), TLR2 (rs574708) genes was performed by real-time PCR. Statistical processing of the obtained results was performed using STATISTICA 12.0 (USA) software. The inter-group difference was considered statistically significant at p 0.05. The strength of associations was estimated and presented as odds ratio (OR) and 95% confidence interval. It was noted that the genotypes GC TLR7 (rs3853839), AG TLR2 (rs574708), GG TLR4 (rs4986790), TT TLR4 (rs4986791) can be predictors of a fatal COVID-19 outcome. On the contrary, the genotypes GG TLR7 (rs3853839), AA TLR2 (rs574708), AA TLR4 (rs4986790), CC TLR4 (rs4986791) can be protective and contribute to the recovery of patients.

Can the Analysis of Toll-like Receptors (TLR) on NK and NKT-like Cells Improve Gastric Cancer Diagnostics and Treatment?

Background/Objectives: The aim of this study was to determine the assessment of the percentage of NK and NKT-like cells expressing Toll-like receptors (TLR-2, TLR-3, TLR-4, and TLR-9) in patients with gastric cancer (GC) compared with healthy volunteers (HV) and to investigate differences according to cancer subtype. We also assessed TLR gene expression by RT-qPCR to assess whether TLRs could be diagnostic and prognostic biomarkers. Methods: The study included 86 patients with histologically confirmed gastric cancer and 30 healthy volunteers. Peripheral blood samples were collected from the participants, and TLR expression on NK and NKT-like cells was assessed by flow cytometry and RT-qPCR. The expression of TLR2, TLR3, TLR4, and TLR9 genes was assessed using genetic material derived from NK and NKT-like cells sourced from PBMC. The obtained results were statistically analyzed using Mann-Whitney U and Kruskal-Wallis tests, and the predictive ability of variables was assessed using ROC curve analysis. Results: A significantly higher expression of TLR receptors (TLR-2, TLR-3, TLR-4, and TLR-9) was found in patients with gastric cancer compared to healthy volunteers (p < 0.05). TLR expression also differed depending on the cancer subtype, and higher expression was observed in more advanced GC subtypes. RT-qPCR analysis showed significantly increased expression of TLR genes in the group of GC patients. ROC curves indicate a high ability of TLRs to differentiate between GC patients and healthy individuals. Conclusions: The expression of TLRs on NK and NKT-like cells is clearly increased in patients with gastric cancer, especially in more advanced subtypes of the tumor. The results suggest that TLRs could potentially be used as diagnostic and prognostic biomarkers and represent potential targets for immune therapies in GC. However, further studies are needed to determine the functional role of TLRs in disease progression and the possibility of their use in personalized treatment.

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.

Association of TLR-2 gene polymorphisms with susceptibility to Pulmonary Tuberculosis at Northwest Ethiopian population, a case control study.

Background: Tuberculosis is one of the leading causes of mortality and morbidity worldwide. Polymorphisms in the TLR-2 gene have a diverse influence on the innate immune response and may result in the susceptibility of active pulmonary tuberculosis diseases among particular families, ethnicities, and races. However, the genetic variation in TLR2 in the susceptibility to PTB in our population has not been previously investigated. Thus, this study assesses the association of TLR-2 gene polymorphisms with susceptibility to PTB in Northwest Ethiopian population.

Liposome encapsulating pine bark extract in Nile tilapia: Targeting interrelated immune and antioxidant defense to combat coinfection with Aeromonas hydrophila and Enterococcus faecalis

Application of smart delivery systems for encapsulation of natural ingredients provides novel avenues and is being frequently developed. Thus, we aimed to highlight the effects of cyclosome liposomal pine bark extract (CL-PBE) on Nile tilapia growth, immunomodulation, antioxidant capacity and resistance against coinfection with Aeromonas hydrophila and Enterococcus faecalis and their associated virulence genes. The experiment was conducted on four fish groups receiving a control diet (control) along with three baseline meals supplemented with 200, 400 and 600 mg/kg diet of CL-PBE (CL-PBE 200, 400 and 600, respectively). At the end of the 12-weeks feeding trial, the tilapias were intraperitoneally challenged with virulent A. hydrophila strain and five days later, E. faecalis challenge was carried out. The results revealed that tilapias fed diets fortified with CL-PBE displayed significantly enhanced growth rate and feed conversion ratio in a dose-dependent manner. Moreover, we demonstrated that CL-PBE had potent antioxidant property presented by modulation of several markers of oxidative stress; substantial reductions in reactive oxygen species, hydrogen peroxide and malondialdehyde levels, an elevation in total antioxidant capacity and boosting glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) activities in fish serum and muscle tissues. This was also correlated with augmenting the expression of CAT, SOD, GSH-Px, Nrf2 and caspase-1 genes alongside reducing those of COX-2, HSP70 and iNOS genes in response to CL-PBE. Our data demonstrated that CL-PBE fortification counteracted the overly pronounced inflammatory response-mediated induction of IL-1β, TNF-α, MHCII and TLR2 genes at the transcriptional levels post coinfection together with promotion in MUC2 and IL-10 genes expression. Notably, our findings displayed optimal well-functioning fish immune system post dietary supplementation of CL-PBE for the protection against coinfection with A. hydrophila and E. faecalis. This was evident from the decline of their counts and hence encompassing the capacity to reduce cumulative mortality percentage in conjunction with interference with their virulence via the significant downregulatory effects of CL-PBE on E. faecalis esp and gelE and A. hydrophila act and fla virulence genes. Taken together, our study strongly suggested dietary inclusion of CL-PBE for Nile tilapias with superior growth performance and significant economic benefits coupled with potent stimulatory effects on antioxidant capacity and immune response expediting our detailed understanding of how coinfection with A. hydrophila and E. faecalis was controlled.

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.

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.

Morphea, also known as localized scleroderma, is a rare fibrosing inflammatory disease of unknown pathogenesis. Although the genetic basis for morphea is important, reports on 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-β1, FGF, and VEGF levels in patients with morphea and compare these results with healthy controls. The expression levels of TLRs in the lesional and non-lesional adjacent skin of patients with morphea and in normal skin of healthy controls were evaluated by RT-PCR, whereas serum levels of IL-6, IL-17A, TGF-β1, FGF, and VEGF were evaluated by ELISA. 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. TLRs are important parts of the pathogenesis of morphea, and a better understanding of them will lead to more directed, 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.

Variants rs3804099 and rs3804100 in the TLR2 Gene Induce Different Profiles of TLR-2 Expression and Cytokines in Response to Spike of SARS-CoV-2.

The present study aimed to identify in patients with severe COVID-19 and acute respiratory distress syndrome (ARDS) the association between rs3804099 and rs3804100 (TLR2) and evaluate the expression of TLR-2 on the cell surface of innate and adaptive cells of patients' carriers of C allele in at least one genetic variant. We genotyped 1018 patients with COVID-19 and ARDS. According to genotype, a subgroup of 12 patients was selected to stimulate peripheral blood mononuclear cells (PBMCs) with spike and LPS + spike. We evaluated soluble molecules in cell culture supernatants. The C allele in TLR2 (rs3804099, rs3804100) is not associated with a risk of severe COVID-19; however, the presence of the C allele (rs3804099 or rs3804100) affects the TLR-2 ability to respond to a spike of SARS-CoV-2 correctly. The reference group (genotype TT) downregulated the frequency of non-switched TLR-2+ B cells in response to spike stimulus; however, the allele's C carriers group is unable to induce this regulation, but they produce high levels of IL-10, IL-6, and TNF-α by an independent pathway of TLR-2. Findings showed that TT genotypes (rs3804099 and rs3804100) affect the non-switched TLR-2+ B cell distribution. Genotype TT (rs3804099 and rs3804100) affects the TLR-2's ability to respond to a spike of SARS-CoV-2. However, the C allele had increased IL-10, IL-6, and TNF-α by stimulation with spike and LPS.

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.