TLR4 Gene Research Articles

Deciphering the role of TLR3 polymorphisms in oral squamous cell carcinoma pathogenesis: A case-control study.

Oral squamous cell carcinoma (OSCC) poses a significant global health burden, particularly prevalent in regions like India. Despite advancements in diagnostics, early detection of OSCC remains challenging, necessitating novel diagnostic modalities. Toll-like receptors (TLRs) and their polymorphisms have emerged as potential contributors to OSCC pathogenesis. This retrospective case-control study examined 120 individuals, including 60 OSCC cases and 60 healthy controls. Genotyping of TLR3 single-nucleotide polymorphisms (SNPs) rs3775290 and rs3775291 was conducted using TaqMan allelic discrimination real-time polymerase chain reaction. Functional consequence analysis and TLR3 expression profiling were performed to elucidate their role in OSCC pathogenesis. Significant associations were observed between TLR3 SNPs and OSCC susceptibility, particularly at loci rs3775290 and rs3775291. Functional consequence analysis revealed pathogenic mutations in TLR3 genes, potentially affecting protein structure and function. TLR3 overexpression was detected in OSCC lesions, implicating its involvement in disease progression. TLR3 polymorphisms play a pivotal role in OSCC pathogenesis, offering potential biomarkers for diagnosis and prognosis. Targeting TLR3-mediated pathways may hold promise in personalised OSCC management. Further research is warranted to elucidate the precise mechanisms underlying TLR3-mediated carcinogenesis in OSCC, facilitating the development of tailored therapeutic strategies.

Motor, Cognitive, and Behavioral Impairment in TLR3 and TLR9 Deficient Male Mice: Insights into the Non-Immunological Roles of Toll-Like Receptors

Toll-like receptors (TLRs) play a critical role in initiating the innate immune response to infection or injury. Recent studies have uncovered their intriguing functions as moonlighting proteins involved in various biological processes, including development, learning, and memory. However, the specific functions of individual TLRs are still largely unknown. We investigated the effects of TLR3 and TLR9 receptor deficiency on motor, cognitive, and behavioral functions during development using genetically modified male mice of different ages. We evaluated the motor coordination, anxiety-like behavior, spatial learning, and working memory of male mice lacking the TLR3 and TLR9 genes at different ages (two, four, six, and eight months) using the rotarod, open field, water maze, and T-maze tests. We observed that the deletion of either TLR3 or TLR9 resulted in impaired motor performance. Furthermore, young TLR3-deficient mice exhibited reduced anxiety-like behavior and spatial learning deficits; however, their working memory was unaffected. In contrast, young TLR9-knockout mice showed hyperactivity and a tendency toward decreased working memory. These findings provide valuable insights into the broader roles of the TLR system beyond the innate immune response, revealing its involvement in pathways associated with the central nervous system. Importantly, our results establish a strong association between the endosomal receptors TLR3 and TLR9 and the performance of motor, cognitive, and behavioral tasks that change over time. This study contributes to the growing body of research on the multifaceted functions of TLRs and enhances our understanding of their participation in non-immune-related processes.

Effect of rifampicin on TLR4-signaling pathways in the nucleus accumbens of the rat brain during abstinence of long-term alcohol treatment.

The treatment with the antibiotic rifampicin (Rif) led to a decrease in the frequency of neurodegenerative pathologies. There are suggestions that the mechanism of action of Rif may be mediated by its effect on toll-like receptor (TLR)4-dependent pathways. We evaluated the expression status of TLR4-dependent genes during abstinence from long-term alcohol treatments in the nucleus accumbens (NAc) of the rat brain, and also studied the effects of Rif to correct these changes. The long-term alcohol treatment was performed by intragastric delivery of ethanol solution. At the end of alcohol treatment intraperitoneal injections of Rif (100mg/kg) or saline were made. Extraction of the brain structures was performed on the 10th day of abstinence from alcohol. We used the SYBR Green qPCR method to quantitatively analyze the relative expression levels of the studied genes. The long-term alcohol treatment promotes an increase in the level of TLR4 mRNA and mRNA of its endogenous ligand high-mobility group protein B1 during abstinence drop alcohol in NAc of rats. The use of Rif in our study led to a decrease in the increased expression of high-mobility group protein B1, Tlr4, and proinflammatory cytokine genes (Il1β, Il6) in the NAc of the rat brain during abstinence of long-term alcohol treatment. In addition, Rif administration increased the decreased mRNA levels of anti-inflammatory cytokines (Il10, Il11). The data obtained indicate the ability of Rif to correct the mechanisms of the TLR4 system genes in the NAc of the rat brain during alcohol abstinence.

The effect and mechanism of volatile oil emulsion from leaves of Clausena lansium (Lour.) Skeels on Staphylococcus aureus in vitro.

This study aimed to develop a suitable dosage form of volatile oil from wampee leaves and to explore its antibacterial mechanism in vitro. The chemical composition of the volatile oil from wampee leaves was determined by gas chromatography-mass spectrometry (GC-MS). Different microemulsion ratios were tested and their stabilities were investigated to determine the optimal ratio. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the wampee leaves volatile oil emulsion (WVOE) against Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) were determined using double-dilution and plate-counting methods, respectively. Morphological changes in these two bacteria were observed using scanning electron microscopy. Death, ultrastructural morphology, and biofilm formation were also assessed for S. aureus. Finally, we established an S. aureus-infected Lewis lung carcinoma (LLC) cell model to evaluate the protective effects of the volatile oil emulsion and the associated mechanisms. The volatile oil extracted from wampee leaves contained 37 compounds, of which 96.49% were aromatic hydrocarbons, terpenoids, and their oxygen-containing derivatives. The emulsion was most stable at 1:1 in the oil phase and 1:9 in the water phase. WVOE had poor antibacterial activity against S. typhimurium, but the MIC and MBC against S. aureus were 312.5 and 2,500 μg/mL, respectively. S. aureus survival rates were 84.6%, 14.5%, and 12.8% in the 1/2, 1, and 4 × MIC groups, respectively, compared with 97.2% in the control group. S. typhimurium survival was not affected by WVOE treatment. WVOE administration induced cavity formation and abnormal binary fission, and significantly inhibited biofilm formation in S. aureus cells. The WVOE notably reduced the number of S. aureus and inhibited TLR4, NLRP3, NF-κB, IL-6, IL-18, and TNF-α gene expression in S. aureus-infected LLC cells. The WVOE had a significant inhibitory effect on S. aureus and altered its cell membrane permeability. Moreover, it alleviated inflammation by inhibiting the NF-κB-NLRP3 pathway in S. aureus-infected LLC cells.

Doppler ultrasonographic scan, gene expression and serum profile of immune, APPs and antioxidant markers in Egyptian buffalo–cows with clinical endometritis

The objective of this study was to elaborate Doppler ultrasonographic scan, genetic resistance and serum profile of markers associated with endometritis susceptibility in Egyptian buffalo–cows. The enrolled animals were designed as; twenty five apparently healthy buffalo–cows considered as a control group and twenty five infected buffalo with endometritis. There were significant (p < 0.05) increased of cervical diameter, endometrium thickness, uterine horn diameter, TAMEAN, TAMAX and blood flow through middle uterine artery with significant decrease of PI and RI values in endometritis buffalo–cows. Gene expression levels were considerably higher in endometritis-affected buffaloes than in resistant ones for the genes A2M, ADAMTS20, KCNT2, MAP3K4, MAPK14, FKBP5, FCAMR, TLR2, IRAK3, CCl2, EPHA4, and iNOS. The RXFP1, NDUFS5, TGF-β, SOD3, CAT, and GPX genes were expressed at substantially lower levels in endometritis-affected buffaloes. The PCR-DNA sequence verdicts of healthy and affected buffaloes revealed differences in the SNPs in the amplified DNA bases related to endometritis for the investigated genes. However, MAP3K4 elicited a monomorphic pattern. There was a significant decrease of red blood cells (RBCs) count, Hb and packed cell volume (PCV) with neutrophilia, lymphocytosis and monocytosis in endometritis group compared with healthy ones. The serum levels of Hp, SAA, Cp, IL-6, IL-10, TNF-α, NO and MDA were significantly (P˂0.05) increased, along with reduction of CAT, GPx, SOD and TAC in buffalo–cows with endometritis compared to healthy ones. The variability of Doppler ultrasonographic scan and studied genes alongside alterations in the serum profile of investigated markers could be a reference guide for limiting buffalo endometritis through selective breeding of natural resistant animals.

TLR2 on CD4+ and CD8+ T cells promotes control of Mycobacterium tuberculosis infection.

Although a role for TLR2 on T cells has been indicated in prior studies, in vivo stimulation of TLR2 on T cells by Mtb and its impact on Mtb infection has not been tested. Furthermore, it is not known if the enhanced susceptibility to Mtb of Tlr2 gene knockout mice is due to its role in macrophages, T cells, or both. To address TLR2 on T cells, we generated Tlr2fl/flxCd4cre/cre mice, which lack expression of TLR2 on both CD4 and CD8 T cells, to study the in vivo role of TLR2 on T cells after aerosol infection with virulent Mtb. Deletion of TLR2 in CD4+ and CD8+ T cells reduces their ability to be co-stimulated by TLR2 ligands for cytokine production. These include both pro- (IFN-γ, TNF-α) and anti-inflammatory cytokines (IL-10). Deletion of TLR2 in T cells affected control of Mtb in the lungs and spleens of infected mice. This suggests that T-cell co-stimulation by mycobacterial TLR2 ligands in vivo contributes to the control of Mtb infection in the lung and spleen.

Hyperglycaemia amplifies TLR-mediated inflammatory response of M(IL4) macrophages to dyslipidemic ligands.

Hyperglycaemia is critical for initiation of diabetic vascular complications. We systemically addressed the role of hyperglycaemia in the regulation of TLRs in primary human macrophages. Expression of TLRs (1-9) was examined in monocyte-derived M(NC), M(IFNγ) and M(IL4) differentiated in normoglycemic and hyperglycaemic conditions. Hyperglycaemia increased expression of TLR1 and TLR8 in M(NC), TLR 2 and 6 in M(IFNγ), and TLR4 and TLR5 in M(IL4). The strongest effect of hyperglycaemia in M(IL4) was the upregulation of TLR4 gene and protein expression. Hyperglycaemia amplified TLR4-mediated response of M(IL4) to LPS by significantly enhancing IL1beta and modestly supressing IL10 production. In M(IL4), hyperglycaemia in combination with synthetic triacylated lipopeptide (TLR1/TLR2 ligand), amplified expression of TLR4, and production of IL1beta. In summary, hyperglycaemia enhanced inflammatory potential of homeostatic, inflammatory and healing macrophages by increasing specific profiles of TLRs. In combination with dyslipidemic ligands, hyperglycaemia can stimulate low-grade inflammatory program in healing macrophages supporting vascular diabetic complications.

Toll-like receptor 2 deficiency promotes the generation of alloreactive γδT17 cells after cardiac transplantation in mice

Homograft rejection is the main cause of heart transplantation failure. The role of TLR2, a major member of the toll-like receptor (TLR) family, in transplantation rejection is has yet to be elucidated. In this study, we used a mouse model of acute cardiac transplantation rejection to investigate whether the TLR2 signalling pathway can regulate cardiac transplantation rejection by regulating alloreactive IL-17+γδT (γδT17) cells. We found that the expression of TLR2 on the surface of dendritic cells (DCs) and macrophages increased during acute transplantation rejection. In addition, our investigation revealed that γδT17 cells exert a significant influence on acute cardiac transplantation rejection. TLR2 gene knockout resulted in an increase in alloreactive γδT17 cells in the spleen and heart grafts of recipient mice compared with wild-type recipient mice and an increase in the mRNA expression of IL-17, IL-1β, CCR6, and CCL20 in the heart grafts. In an in vitro experiment, a mixed lymphocyte reaction was conducted to assess the impact of TLR2 deficiency on the generation of γδT17 cells, which further substantiated a significant increase compared to that in wild-type controls. Furthermore, the mixed lymphocyte reaction showed that TLR2 regulated the production of γδT17 cells by regulating the ability of DCs to secrete IL-1β. These results suggest that TLR2 signalling is important for regulating the generation of γδT17 cells after cardiac allograft transplantation.

Candida Variety in the Oral Cavity of Mexican Subjects with Type 2 Diabetes Mellitus and TLR2 Gene Expression.

The aim was to diagnose Candida in the oral cavity of subjects with type 2 diabetes mellitus (T2DM) using a genotyping technique and compare the results with those from conventional diagnosis by Papanicolaou (Pap) staining. Palatal mucosa smears were performed on 18 dental care patients diagnosed with T2DM and grade I, II, and III prosthetic stomatitis who met the inclusion criteria; 18 healthy control subjects were also included in the study. Hemoglobin A1c (HbA1c) levels were determined from total blood. Using exfoliative cytology, the Pap staining technique was used to diagnose candidiasis. Exfoliative cytology was also used for molecular diagnosis; DNA was obtained for Candida genotyping, and RNA was used for gene expression studies. Clinical patterns indicated that all subjects were positive for Candida; however, Pap analysis revealed only three positive subjects, whereas end-point polymerase chain reaction (PCR) analysis revealed 15 subjects with some type of Candida. The most common Candida species found were Candida guilliermondii (38.8%), Candida krusei (33.3%), Candida tropicalis, and Candida lusitaniae (22.2%). Interestingly, the coexpression of different species of Candida was found in various patients. In all patients, HbA1c levels were increased. Gene expression analysis showed a significant decrease (p ≤ 0.05) in TLR2 expression in positive subjects, whereas TLR4 expression did not differ significantly among patients. The end-point PCR technique showed better sensitivity for the diagnosis of Candida when compared with the diagnosis by Pap staining. T2DM subjects showed an increased presence of C. guilliermondii that was correlated with decreased TLR2 expression.

Polymorphisms in Innate and Adaptive Immune Genes in Subjects with Allergic Bronchopulmonary Aspergillosis Complicating Asthma.

Innate and adaptive immunity play a crucial role in allergic bronchopulmonary aspergillosis (ABPA) pathogenesis. We performed next-generation sequencing using the Illumina TruSight One panel (4,811 human disease-associated genes, at least 20 × coverage) and selected 22 known immune genes (toll-like receptors (TLRs), C-type lectin, interleukin-4 receptor, and others). We included ABPA (n = 18), asthma without ABPA (n = 12), and healthy controls (n = 8). We analyzed 3011 SNPs from 22 genes and identified 145 SNPs (13 genes) that were present only in the disease groups and absent in controls. The SNP frequency overall was significantly higher in ABPA than in asthmatics (89/145 [61.4%] vs. 56/145 [38.6%], p = 0.0001). The SNP frequency in the TLR10 gene was also significantly higher in ABPA than in asthma (p = 0.017). Association analysis further revealed three genes having significant associations. Of these, NOS3 and HLA-DQB1 are associated with antimicrobial activity and adaptive immunity. More extensive studies are required to confirm our findings.

Effect of estradiol after bacterial infection on the Wnt/β-catenin pathway in bovine endometrium epithelial cells and organoids

Endometritis is a disease caused by a postpartum bacterial infection with a poor prognosis that primarily affects dairy cows. Three-dimensional organoids have been used as a model for endometritis, because they exhibit a structure comparable to that of the endometrium, demonstrating both expansibility and hormone responsiveness. These characteristics render them an ideal platform for in vitro investigations of endometrial diseases. Estradiol (E2) is an endogenous steroid hormone with demonstrated anti-inflammatory properties, and the objective of this study was to determine the mechanism by which E2 modulates the inflammatory response and the Wnt signal transduction pathway in bovine endometrial epithelial cells and organoids following E. coli infection. We present the techniques for isolating and culturing primary bovine endometrial epithelial cells (BEECs), and producing endometrial organoids. For the experiments, the endometrial epithelial cells and organoids were infected with E. coli for 1 h, followed by incubation with E2 for 12 h. The mRNA and protein expressions of the inflammation-related genes, IL-1β, IL-6, TLR4, and NF-κB, as well as the Wnt pathway-related genes, Wnt4, β-catenin, c-Myc, and CyclinD1, were assessed using real-time quantitative-PCR and western blotting, respectively. The CCK8 viable cell counting assay was utilized to determine the optimal concentration of the Wnt inhibitor, IWR-1. The mRNA and protein expression of Wnt pathway-related genes was assessed following IWR-1 treatment, while the expression levels of proliferation-associated genes (Ki67, PCNA) and barrier repair genes (occludin, claudin, and Zo-1) in BEECs and organoids were evaluated after E2 treatment. The results of this study show that mRNA expression of the inflammatory genes, IL-1β, TLR4, and NF-κB (P < 0.05) decreased in BEECs following E2 treatment compared to the E. coli group. The protein expression of the IL-1β, IL-6, TLR4 and NF-κB genes was also inhibited (P < 0.05). Similar results were observed in tests on the organoids. Our findings demonstrate that E2 significantly upregulates the expression of Wnt-related genes, including β-catenin and c-Myc, while concurrently downregulating the expression of GSK3β (P < 0.05). Next, we treated E. coli-infected BEECs and organoids with the Wnt inhibitor, IWR-1. Compared with E. coli and E. coli + E2, the expression of mRNA and protein from Wnt 4, β-catenin, and CyclinD1 in E. coli + E2 and E. coli + IWR-1 was down-regulated (P < 0.05). The expression of the proliferation genes, Ki67, PCNA, and the tight junction genes, occludin, claudin1, and Zo-1, in organoids was significantly higher than that in BEECs (P < 0.05). In summary, we found strong potential for E2 mitigation of the E. coli-induced inflammatory response in BEECs and organoids, through activation of the Wnt pathway. In addition, the proliferation and repair capacity of organoids was much higher than that of BEECs.

Anti-sepsis effect of Xiaochaihu decoction based on the TLR4/MyD88/NF-κB signalling pathway

The aims of this study were to explore the protective effect of Xiaochaihu decoction in mice with sepsis induced by intraperitoneal injection; to explore its anti-inflammatory effect on the TLR4-MyD88-NF-κB signalling pathway; and to explore the main material basis of the anti-inflammatory effect of Xiaochaihu decoction, with the aim of supplementing and expanding the associated research and providing a scientific foundation for the clinical use of the decoction. The effects of Xiaochaihu decoction on septic mice were analysed by measurements of white blood cells (WBC) and Platelets (PLT); Nitric Oxide (NO) level in serum; IL-6, IL-1β and TNF-α levels in serum; RT-PCR; Haematoxylin-Eosin (HE) immunohistochemistry; western blotting (WB). The results showed the excellent in vivo anti-inflammatory effects of Xiaochaihu decoction in LPS-induced septic mice, through down regulation of the gene and protein expression of TLR4, MYD88, TRAF6, IKK, IKBα and p65 and the subsequent reduction in the release of inflammatory mediators IL-6, IL-1β, TNF-α and NO. Moreover, significant anti-septic effect was observed from high and medium doses of Xiaochaihu decoction, but not from the low dose.

Vitamin D Status, VDR, and TLR Polymorphisms and Pulmonary Tuberculosis Epidemiology in Kazakhstan.

Tuberculosis (TB) and vitamin D deficiency remain major public health problems in Kazakhstan. Due to the high incidence of pulmonary tuberculosis in the country and based on the importance of vitamin D in the modulation of the immune response and the association of its deficiency with many health conditions, the aim of our research was to study the vitamin D status, VDR and TLR gene polymorphisms, and pulmonary tuberculosis epidemiology in Kazakhstan. A case-control study included 411 individuals diagnosed with pulmonary TB and 686 controls with no family history of pulmonary tuberculosis. Concentrations of serum vitamin D (25-(OH)D) levels were measured by electrochemiluminescence immunoassay. The gene polymorphisms were determined by real-time polymerase chain reaction (PCR) allelic discrimination assay using TaqMan probes. The association between the risk of pulmonary TB and polymorphisms was evaluated using multimodal logistic regression and assessed with the ORs, corresponding to 95% Cis, and the significance level was determined as p < 0.05. 1097 individuals were recruited from 3 different regions of Kazakhstan. Biochemical data showed vitamin D deficiency (25-(OH)D < 20 ng/mL) was present in both groups, with the case group accounting for almost 95% and 43.7% in controls. Epidemiological data revealed that socioeconomic factors such as BMI < 25 kg/m2 (p < 0.001), employment (p < 0.001), diabetes (p < 0.001), and vitamin D deficiency (p < 0.001) were statistically different between case and control groups. Logistic regression analysis, adjusted by sex, age, BMI, residence, employment, smoking, alcohol consumption, and diabetes, showed that T/T polymorphism of the VDR gene (rs1544410, OR = 1.97, 95% CI: 1.04-3.72, p = 0.03) and A/A polymorphism of the TLR8 gene (rs3764880, OR = 2.44, 95% CI: 1.20-4.98, p = 0.01) were associated with a high risk of developing pulmonary tuberculosis. Vitamin D deficiency remains prevalent in our study cohort and is associated with TB progression. Socioeconomic determinants such as unemployment, BMI under 25 kg/m2, and diabetes are the main risk factors for the development of pulmonary TB in our study. A/A polymorphism of TLR8 (rs3764880) and T/T polymorphism (BsmI, rs1544410) of VDR genes may act as biomarkers for pulmonary tuberculosis in the Kazakh population.

The single nucleotide polymorphism rs4986790 (c.896A>G) in the gene TLR4 as a protective factor in corona virus disease 2019 (COVID-19).

Several factors, such as hypertension and diabetes mellitus, are known to influence the course of coronavirus disease 2019 (COVID-19). However, there is currently little information on genetic markers that influence the severity of COVID-19. In this study, we specifically investigated the single nucleotide polymorphism (SNP) rs4986790 in the TLR4 gene to identify a universal marker for preclinical prediction of COVID-19 disease progression. We analyzed the influence of demographics, pre-existing conditions, inflammatory parameters at the time of hospitalization, and TLR4 rs4986790 genotype on the outcome of COVID-19 in a comprehensive cohort (N = 1570). We performed multivariable analysis to investigate the impact of each factor. We confirmed that younger patient age and absence of pre-existing conditions were protective factors against disease progression. Furthermore, when comparing patients with mild SARS-CoV-2 infection with patients who required hospitalization or intensive care or even died due to COVID-19, the AG/GG genotype of TLR4 rs4986790 was found to be a protective factor against COVID-19 disease progression (OR: 0.51, 95% CI: 0.34 - 0.77, p = 0.001). In addition, we demonstrated that low levels of interleukin-6 (IL-6) and procalcitonin (PCT) had a favorable effect on COVID-19 disease severity. In the subsequent multivariable analysis, we confirmed the absence of cardiovascular disease, low levels of IL-6 and PCT, and TLR4 rs4986790 AG/GG genotypes as independent predictors of potential hospitalization and reduction of severe or fatal disease course. In this study, we identified an additional genetic factor that may serve as an invariant predictor of COVID-19 outcome. The TLR4 rs4986790 AG/GG genotype reduced by half the risk of COVID-19 patients requiring hospitalization, intensive care or to have a fatal outcome. In addition, we were able to confirm the influence of previously known factors such as pre-existing conditions and inflammatory markers upon the onset of disease on the course of COVID-19. Based on these observations, we hereby provide another prognostic biomarker that could be used in routine diagnostics as a predictive factor for the severity of COVID-19 prior to SARS-CoV-2 infection.

Conventional dendritic cell 2 links the genetic causal association from allergic asthma to COVID-19: a Mendelian randomization and transcriptomic study

Recent evidence suggests that allergic asthma (AA) decreases the risk of Coronavirus Disease 2019 (COVID-19). However, the reasons remain unclear. Here, we systematically explored data from GWAS (18 cohorts with 11,071,744 samples), bulk transcriptomes (3 cohorts with 601 samples), and single-cell transcriptomes (2 cohorts with 29 samples) to reveal the immune mechanisms that connect AA and COVID-19. Two-sample Mendelian randomization (MR) analysis identified a negative causal correlation from AA to COVID-19 hospitalization (OR = 0.968, 95% CI 0.940–0.997, P = 0.031). This correlation was bridged through white cell count. Furthermore, machine learning identified dendritic cells (DCs) as the most discriminative immunocytes in AA and COVID-19. Among five DC subtypes, only conventional dendritic cell 2 (cDC2) exhibited differential expression between AA/COVID-19 and controls (P < 0.05). Subsequently, energy metabolism, intercellular communication, cellular stemness and differentiation, and molecular docking analyses were performed. cDC2s exhibited more differentiation, increased numbers, and enhanced activation in AA exacerbation, while they showed less differentiation, reduced number, and enhanced activation in severe COVID-19. The capacity of cDC2 for differentiation and SARS-CoV-2 antigen presentation may be enhanced through ZBTB46, EXOC4, TLR1, and TNFSF4 gene mutations in AA. Taken together, cDC2 links the genetic causality from AA to COVID-19. Future strategies for COVID-19 prevention, intervention, and treatment could be stratified according to AA and guided with DC-based therapies.Graphical

Expression of TLR2, IL-1β, and IL-10 Genes as a Possible Factor of Successful or Pathological Aging in Nonagenarians.

The expression of the gene of pattern recognition receptor TLR2, proinflammatory cytokine IL-1β, and anti-inflammatory cytokine IL-10 was analyzed in the peripheral blood of nonagenarians (n=219; mean age 92.1 years, 77 men and 142 women) in comparison with healthy young donors (n=24; mean age 22.5 years, 16 women and 8 men). Nonagenarians were interviewed, medical records were analyzed, and a comprehensive geriatric assessment was performed according to the Clinical Guidelines on Frailty. The level of gene expression was determined by real-time PCR. The participation of inflammatory mechanisms in the immunosenescence was revealed. It was shown that increased expression of IL1B and TLR2 genes is associated with the development of frailty in nonagenarians and can be a factor of pathological aging. Increased expression of IL10 gene can be considered as a factor of successful aging in nonagenarians.

TLR4, IgA and EpCAM Expression in Colorectal Cancer and Their Possible Association with Microbiota as a Pathogenic Factor; An Immunohistochemical and Genetic Study.

The pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC) is thought to be related to immune response against gut microbiota. TLR4, IgA, and EpCAM have a role in intestinal local immune response and their altered expression related to both IBD and CRC. Lipopolysaccharide (LPS) is the main activator of TLR4. The objective of this study is to evaluate the possible role of intestinal microbiota in the pathogenesis of IBD and CRC through expression of TLR4, IgA and EpCAM. One hundred five cases were divided into (Group 1/ Control: 10 sections of normal colonic mucosa, Group 2/CRC: 51 cases, Group 3/IBD: 44 cases). Immunohistochemistry for TLR4, IgA, and EpCAM was done. LPS was assessed in all groups. TLR4 gene and protein expression were assessed in colorectal cancer cell line by RT-PCR and immunocytochemistry. There was a significant correlation between TLR4 and tumor grade (P value 0.003 and 0.01 respectively). A significant correlation was found between IgA expression and T stage (P value 0.02) and between EpCAM expression and histologic type (P value 0.02). In comparison of CRC patients to controls; there was a statistically significant different expression of TLR4 positivity, IgA positivity and EpCAM (P value <0.001, 0.004, <0.001 respectively). Patients with CRC were compared to colitis patients and there was a statistically significant different expression of IgA positivity and EpCAM expression (P value <0.001). There was significant higher expression of TLR4 in CRC cell line than the fibroblast by both PCR and immunocytochemistry (P-value: 0.003 and 0.024 respectively). LPS level in CRC patients was significantly higher than the control and IBD groups (P values <0.001 and <0.001 respectively). TLR4, IgA, EpCAM expression in both CRC and IBD might be related to the pathogenic role of microbiota and could represent potential prevention modalities and therapeutic targets.

Interface Gain-of-Function Mutations in TLR7 Cause Systemic and Neuro-inflammatory Disease

TLR7 recognizes pathogen-derived single-stranded RNA (ssRNA), a function integral to the innate immune response to viral infection. Notably, TLR7 can also recognize self-derived ssRNA, with gain-of-function mutations in human TLR7 recently identified to cause both early-onset systemic lupus erythematosus (SLE) and neuromyelitis optica. Here, we describe two novel mutations in TLR7, F507S and L528I. While the L528I substitution arose de novo, the F507S mutation was present in three individuals from the same family, including a severely affected male, notably given that the TLR7 gene is situated on the X chromosome and that all other cases so far described have been female. The observation of mutations at residues 507 and 528 of TLR7 indicates the importance of the TLR7 dimerization interface in maintaining immune homeostasis, where we predict that altered homo-dimerization enhances TLR7 signaling. Finally, while mutations in TLR7 can result in SLE-like disease, our data suggest a broader phenotypic spectrum associated with TLR7 gain-of-function, including significant neurological involvement.

Effect of human umbilical cord blood-mesenchymal stem cells on cisplatin-induced nephrotoxicity in rats.

Cisplatin-containing regimen is an effective treatment for several malignancies. However, cisplatin is an important cause of nephrotoxicity. So, many trials were performed to transplant stem cells systemically or locally to control cisplatin-induced nephrotoxicity. Stem cell therapeutic effect may be dependent on the regulation of inflammation and oxidant stress. To investigate the effect of human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) on the histological structure, the oxidant stress, and the inflammatory gene expression in an experimental model of cisplatin-induced nephrotoxicity in rats. The rats were divided into 6 equal groups (each of 10 rats): Group I included normal rats that received no treatment. Group II included healthy rats that received IV hUCB-MSCs. Group III included untreated cisplatin-induced nephrotoxic rats. Group IV included cisplatin-induced nephrotoxic rats that received magnesium (Mg) injections after injury. Group V was injected with hUCB-MSCs after injury. Group VI received both Mg and hUCB-MSCs after injury. In tissue homogenates, reduced glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) activities were measured. Quantitative real-time-polymerase chain reaction (qRT-PCR) was performed to assess iNOS, TLR4, and NF-kB gene expression. Hematoxylin and eosin (H&E) staining was performed to study the histological structure of the kidney. Immunohistochemical staining of iNOS and NF-κB was performed, as well. Disturbed kidney functions, oxidative status, and histological structure were seen in the rats that received cisplatin. Treated groups showed improvements in kidney functions, oxidative status, and histological structure, particularly in the combined treatment group. In the cisplatin-induced nephrotoxicity model, hUCB-MSCs could improve the functional and morphological kidney structure by modulation of oxidative and inflammatory status.

P099 Neutrophil extracellular traps enhance profibrotic activity of intestinal fibroblasts in Crohn's disease through TLR2/NF-kB pathway

Abstract Background Neutrophil extracellular traps (NETs) consist of DNA filaments and cytoplasmic protein granules, extruded by neutrophils after PAD4-dependent activation. NETs release occurs early during inflammation in various immune-related diseases, further aggravating tissue injury and contributing to fibrosis. Our aim was to investigate the potential profibrotic effect of NETs in Crohn’s disease (CD). Methods NETs and activated fibroblasts were labelled by multiplex immunofluorescence staining on unaffected, inflamed and fibrotic ileum derived from patients with stricturing CD undergoing ileocaecal resection. Intestinal fibroblasts isolated from unaffected CD ileum were co-cultured with NETs for 24 hours to analyse their transcriptome and quantify collagen released with SIRCOL®. Fibroblast migratory activity was investigated with a scratch test. Immunofluorescence intensity of collagen and fibroblast activation protein (FAP) was quantified using the Operetta® Analysis System. Transfection of fibroblasts with NF-kB-luciferase reporter plasmid was performed to evaluate the TLR2/NF-kB pathway, and a specific TLR2 inhibitor (TL2-C29) tested. Finally, mice with selective deletion of PAD4 in neutrophils (PAD4fl/flMRP8Cre+) were subjected to chronic Dextran Sulphate Sodium (DSS) colitis to assess the role of PAD4-mediated NETosis in intestinal fibrosis development. Results We found spatial tissue distribution of clusters of NETs adjacent to FAP+ fibroblasts in ileal ulcerations in patients with active CD. Transcriptomics demonstrated upregulation of various profibrotic processes in fibroblasts stimulated with NETs, including TGFβ-receptor signalling pathway, positive regulation of collagen metabolic process, and TLR-signalling pathways. This correlated with increased proliferation rate (p&amp;lt;0.0001), slower wound healing capability (p&amp;lt;0.0001) and higher collagen release in the medium (p&amp;lt;0.01) in the NET-treated group, as well as higher expression of collagens and FAP. Among TLR genes, TLR2 was found upregulated after stimulation with NETs (p=0.02). Transfection data showed significant upregulation (p&amp;lt;0.05) of NF-kB in the NETs-treated group, whereas its expression and soluble collagen release decreased with the addition of the TLR2 antagonist TL2-C29. In line, a significantly lower amount of collagen deposition was observed in the colon of PAD4fl/flMRP8Cre+ mice subjected to chronic DSS colitis, as well as modulation of MMP2/TIMP2 balance and reduced fibroblast activation. Conclusion NETs may represent an early trigger of intestinal fibroblast activation via the TLR2/NF-kB axis. As NETs are early players during inflammation, blocking PAD4 may reduce inflammation and thus fibrogenesis in CD.