Role Of TLR4 Research Articles

The Role of TLR-2 in Lethal COVID-19 Disease Involving Medullary and Resident Lung Megakaryocyte Up-Regulation in the Microthrombosis Mechanism.

The aim of this study was to study the expression of platelet glycoprotein IIIa (CD61), playing a critical role in platelet aggregation, together with TRL-2 as a marker of innate immune activation. A total of 25 patients were investigated, with the majority (24/25, 96%) having co-morbidities and dying from a fatal form of SARS-CoV-2(+) infection (COVID-19+), with 13 men and 12 females ranging in age from 45 to 80 years. When compared to a control group of SARS-CoV-2 (-) negative lungs (COVID-19-), TLR-2 expression was up-regulated in a subset of patients with deadly COVID-19 fatal lung illness. The proportion of Spike-1 (+) patients found by PCR and ISH correlates to the proportion of Spike-S1-positive cases as detected by digital pathology examination. Furthermore, CD61 expression was considerably higher in the lungs of deceased patients. In conclusion, we demonstrate that innate immune prolonged hyperactivation is related to platelet/megakaryocyte over-expression in the lung. Microthrombosis in deadly COVID-19+ lung disease is associated with an increase in the number of CD61+ platelets and megakaryocytes in the pulmonary interstitium, as well as their functional activation; this phenomenon is associated with increased expression of innate immunity TLR2+ cells, which binds the SARS-CoV-2 E protein, and significantly with the persistence of the Spike-S1 viral sequence.

New role for TLR9.

New role for TLR9.

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.

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.

The Role of TLR2 and TLR4 Immune Receptors in the Immune Response against the Giardia Lambila Parasite

The Role of TLR2 and TLR4 Immune Receptors in the Immune Response against the Giardia Lambila Parasite

Toll-like Receptor 2 Mediated Immune Regulation in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Toll-like receptors (TLRs) are crucial to the innate immune response. They regulate inflammatory reactions by initiating the production of pro-inflammatory cytokines and chemokines. TLRs also play a role in shaping the adaptive immune responses. While this protective response is important for eliminating infectious pathogens, persistent activation of TLRs may result in chronic immune activation, leading to detrimental effects. The role of TLR2 in regulating HIV-1 infection in vivo has yet to be well described. In this study, we used an SIV-infected rhesus macaque model to simulate HIV infection in humans. We evaluated the plasma of the macaques longitudinally and found a significant increase in the soluble TLR2 (sTLR2) level after SIV infection. We also observed an increase in membrane-bound TLR2 (mb-TLR2) in cytotoxic T cells, B cells, and NK cells in PBMC and NK cells in the gut after infection. Our results suggest that sTLR2 regulates the production of various cytokines and chemokines, including IL-18, IL-1RA, IL-15, IL-13, IL-9, TPO, FLT3L, and IL-17F, as well as chemokines, including IP-10, MCP-1, MCP-2, ENA-78, GRO-α, I-TAC, Fractalkine, SDF-1α, and MIP-3α. Interestingly, these cytokines and chemokines were also upregulated after the infection. The positive correlation between SIV copy number and sTLR2 in the plasma indicated the involvement of TLR2 in the regulation of viral replication. These cytokines and chemokines could directly or indirectly regulate viral replication through the TLR2 signaling pathways. When we stimulated PBMC with the TLR2 agonist in vitro, we observed a direct induction of various cytokines and chemokines. Some of these cytokines and chemokines, such as IL-1RA, IL-9, IL-15, GRO-α, and ENA-78, were positively correlated with sTLR2 in vivo, highlighting the direct involvement of TLR2 in the regulation of the production of these factors. Our findings suggest that TLR2 expression may be a target for developing new therapeutic strategies to combat HIV infection.

Let-7b-TLR7 Signaling Axis Contributes to the Anesthesia/Surgery-Induced Cognitive Impairment.

Perioperative neurocognitive disorders (PNDs) are severe and common neurological complications among elderly patients following anesthesia and surgery. As the first line of defense of the innate immune system, Toll-like receptors (TLRs) have been found to be involved in the occurrence of neurodegenerative diseases in recent years. However, the role of TLR7 in the pathology and development of PNDs remains largely unclear. In our current study, we hypothesized that increased microRNA let-7b (let-7b) during anesthesia and surgical operation would activate TLR7 signaling pathways and mediate PNDs. Using a mouse model of PNDs, 18-20months wild-type (WT) mice were undergoing unilateral nephrectomy, and increased TLR7 and let-7b expression levels were found in the surgery group compared with the Sham group. Of note, increased TLR7 was found to be co-localized with let-7b in the hippocampal area CA1 in the PNDs model. In addition, TLR7 and let-7b inhibition could improve hippocampus-dependent memory and attenuate the production of inflammatory cytokines. Together, our results indicated that TLR7 activation and up-regulation might be triggered by increased let-7b under stressful conditions and initiated the downstream inflammatory signaling, playing a substantial role in the development of PNDs.

Toll-like receptor 2 deficiency alleviates acute pancreatitis by inactivating the NF-κB/NLRP3 pathway.

The early aseptic immune response is the key factor leading to the aggravation of acute pancreatitis (AP). Toll-like receptor (TLR) 2 is an important member of the TLR family, but the role of TLR2 in AP remains to be investigated. In the present study, we found that TLR2 expression was significantly increased in AP patients. In a mouse model of cerulein-induced AP, TLR2 deficiency resulted in reduced inflammation, reduced infiltration of pancreatic neutrophils and macrophages, and decreased expression of proinflammatory cytokines such as interleukin (IL)-1β, IL-6, IL-17 and IL-18. In addition, transcriptomic analysis revealed that nod-like receptor family pyrin domain-containing 3 (NLRP3) expression was increased in AP, and there was a significant correlation between NLRP3 and TLR2. This study found that TLR2 deficiency can lead to a decrease in the activation of the NF-κB/NLRP3 signalling pathway, and the NLRP3 inhibitor MCC950 can alleviate AP in mice. Therefore, this study confirmed that TLR2 participates in the development of AP by activating the NF-κB/NLRP3 pathway. This study suggested that TLR2 might be a novel therapeutic target for AP.

The role of TLR2 in exercise-induced immunomodulation in normal weight individuals

Toll-like receptors (TLRs) have been targeted for therapeutic drug development for several disorders, including cardiovascular diseases (CVD), and diabetes mellitus. Daily levels physical activity (PA) has been purported to influence the systemic circulation of cytokines, affecting the overall activation of TLRs and influencing the inflammatory milieu. Objective and self-reported daily PA was tracked in 69 normal-weight adults. Freedson's cut-offs categorized daily PA intensity into the 25th lowest, medium, and top percentiles. Monocytic TLR2 expression was quantified by flow cytometry in fresh whole blood. Cross-sectional associations between flow cytometry measured TLR2+ subsets and clinical biomarkers were evaluated. PA increased circulation of TLR2+ monocytes. TLR2 expression was adversely corelated with reduced diastolic blood pressure (DBP), triglyceride (TG), and matrix metallopeptidase 9 (MMP9) levels. However, regression analysis indicated that only TG levels were independently linked with TLR2+ subsets in circulation in active participants. Higher daily levels of physical activity are associated with improved cardiovascular blood markers and elevated circulatory monocytic TLR2+ subsets. These findings suggest that TLR2 may play a role in modulating CVD risk factors in individuals leading physically active lifestyles.

Platelet TLR7 is essential for the formation of platelet-neutrophil complexes and low-density neutrophils in lupus nephritis.

Platelets and low-density neutrophils (LDNs) are major players in the immunopathogenesis of SLE. Despite evidence showing the importance of platelet-neutrophil complexes (PNCs) in inflammation, little is known about the relationship between LDNs and platelets in SLE. We sought to characterize the role of LDNs and Toll-like receptor 7 (TLR7) in clinical disease. Flow cytometry was used to immunophenotype LDNs from SLE patients and controls. The association of LDNs with organ damage was investigated in a cohort of 290 SLE patients. TLR7 mRNA expression was assessed in LDNs and high-density neutrophils (HDNs) using publicly available mRNA sequencing datasets and our own cohort using RT-PCR. The role of TLR7 in platelet binding was evaluated in platelet-HDN mixing studies using TLR7-deficient mice and Klinefelter syndrome patients. SLE patients with active disease have more LDNs, which are heterogeneous and more immature in patients with evidence of kidney dysfunction. LDNs are platelet bound, in contrast to HDNs. LDNs settle in the peripheral blood mononuclear cell (PBMC) layer due to the increased buoyancy and neutrophil degranulation from platelet binding. Mixing studies demonstrated that this PNC formation was dependent on platelet-TLR7 and that the association results in increased NETosis. The neutrophil:platelet ratio is a useful clinical correlate for LDNs, and a higher NPR is associated with past and current flares of LN. LDNs sediment in the upper PBMC fraction due to PNC formation, which is dependent on the expression of TLR7 in platelets. Collectively, our results reveal a novel TLR7-dependent crosstalk between platelets and neutrophils that may be an important therapeutic opportunity for LN.

Role of TLR In The Cell Fate Decision of Fibroblastic Reticular Cells

Abstract During sepsis, massive PAMPs and DAMPs are released and initiate the TLR signaling to mediate immune response and cell fate decision in cells. We have shown that Fibroblastic Reticular Cells (FRC)-based therapy improves outcome in a mouse polymicrobial sepsis model. Modulating TLR4 and TLR9 signaling in FRC regulates the efficacy of FRC-based therapy in sepsis. However, whether TLR signaling regulates FRC cell fate decision and thus affects the efficacy of FRC therapy is unknown. Here we aim to understand the role of TLR9 and TLR4 in FRC cell fate decision. FRCs were treated with or without LPS/ODN1585 for 6, 12, and 24h. To test the role of TLR4/9 in FRC proliferation, Ki67 expression was assessed using quantitative PCR and flow cytometry. To test the role of TLR4/9 in FRC cell death, the expression levels of protein related to apoptosis and necroptosis were assessed using flow cytometry and western blot. Treatment of ODN1585 decreased the expression levels of Ki67, indicating that TLR9 signaling suppressed FRC proliferation. While the expression levels of Ki67 remain at similar levels in the LPS-treated FRC as the control. Furthermore, treatment of ODN1585 drove FRC towards apoptosis cell death, evidenced by increased levels of activated caspase-3 and caspase-8. Interestingly, treatment of LPS increased the expression levels of p-RIPK1 and MLKL, suggesting that TLR4 signaling drives FRC towards necroptotic cell death. Our result indicated that TLR4 and TLR9 signaling are important for the cell fate decision of FRC. Understanding the mechanism of TLR4/9 in controlling FRC cell fate decision may help with designing new therapeutic strategies to improve the efficacy of FRC-based therapy for sepsis. R01 AI152044

Regulation of megakaryo/thrombopoiesis by endosomal toll-like receptor 7 and 8 activation of CD34+ cells in a viral infection model

BackgroundCD34+ cells, megakaryocytes (MKs), and platelets express toll-like receptors (TLRs) that enable these cells to amplify the host innate immune response. However, the role of TLR7/TLR8 activation in megakaryopoiesis has not yet been investigated. ObjectivesWe evaluated the effect of coxsackievirus B3 (CVB3) and synthetic TLR7/TLR8 agonists on the development of human MKs and production of platelets. MethodsCD34+ cells from human umbilical cord were inoculated with CVB3 or stimulated with synthetic TLR7/TLR8 agonists and then cultured in the presence of thrombopoietin. ResultsCD34+ cells, MK progenitor cells, and mature MKs expressed TLR7 and TLR8, and exposure to CVB3 resulted in productive infection, as determined by the presence of viral infectious particles in culture supernatants. Cell expansion, differentiation into MKs, MK maturation, and platelet biogenesis were significantly reduced in CD34+-infected cultures. The reduction in MK growth was not due to an alteration in cellular proliferation but was accompanied by an increase in cellular apoptosis and pyroptosis. Impairment of MK generation and maturation of viable cells were also associated with decreased expression of transcription factors involved in these processes. These effects were completely abrogated by TLR7 but not TLR8 antagonists and mimicked by TLR7 but not TLR8 agonists. CVB3 infection of CD34+ cells increased the immunophenotype of MKs characterized as CD148+/CD48+ or CD41+/CD53+ cells. ConclusionThese data suggest a novel role of TLR7 in megakaryo/thrombopoiesis that may contribute to a better understanding of the molecular basis underlying thrombocytopenia and the immunologic role of MKs in viral infection processes.

Upregulation of TLR5 indicates a favorable prognosis in prostate cancer.

Toll-like receptors (TLRs) are the key sensors of innate immunity for triggering immune responses against infections. TLRs are well known to be expressed and activated in innate immune cells, such as macrophage and dendritic cells, but we and others have found that some TLRs are also functional in epithelial cells. However, the role of an epithelial TLR in prostate cancer remains elusive. TLR5 expression in messenger RNA and protein level in prostate cancer was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). The activation of TLR5 signaling in epithelial cells was detected upon nuclear factor-κB activation by luciferase assay and western blot analysis, and proinflammatory cytokine activation by RT-qPCR. Distinguishing between the TLR5 and NLRC4 pathways, both recognizing flagellin, is determined by small interfering RNA and proinflammatory cytokine activation. The role of TLR5 in prostate cancer was analyzed by IHC and bioinformatics using a general and single-cell database. In the present study, we show that TLR5, among other TLRs, is exceedingly expressed in human prostate cancer cells. This cancer epithelial cell TLR5 functions to activate the TLR5 signaling pathway in human prostate cancer cells, as it does with innate immune cell TLR5. The bacterial protein flagellin induces a robust immune response in prostate cancer cells in a TLR5-dependent but NLRC4-independent manner. TLR5 is highly expressed in prostate cancer patient specimens, and high TLR5 expression in prostate cancer patients indicates a favorable prognosis. TLR5, as an innate immunity receptor, is a functional TLR in human prostate cancer epithelial cells. TLR5 plays an important role in prostate cancer development and is a new potential prognosis biomarker. TLR5 may represent a novel immunotherapy target against prostate cancer.

Characterization analysis of TLR5a and TLR5b immune response after different bacterial infection in grass carp (Ctenopharyngodon idella)

Toll-like receptor (TLR) is an important pattern recognition receptor, which specifically recognizes microbial components, and TLR5 recognizes bacterial flagellin in vertebrates and invertebrates. In this study, two forms of TLR5 (TLR5a and TLR5b) were identified in grass carp (Ctenopharyngodon idella). Aeromonas hydrophila and Staphylococcus aureus were used to investigate the role of grass carp TLR5a and TLR5b against bacteria (flagellate and non-flagellate) in innate immunity, and the expression of TLR5a and TLR5b genes and proteins were detected in immune-related tissues. Quantitative real-time polymerase chain reaction results showed that TLR5a and TLR5b genes of grass carp were highly expressed in the liver, spleen, and head kidney, and their expression patterns were similar in tissues. Meanwhile, the TLR5b gene expression was higher than TLR5a in most tissues. Following exposure to A. hydrophila and S. aureus, the expression levels of TLR5a and TLR5b genes in the liver, spleen, and head kidney were up-regulated significantly. Moreover, the downstream gene, NF-κB, was up-regulated significantly. After A. hydrophila infection, the expression of TLR5a gene was up-regulated in the liver and spleen at 24 h, while TLR5b was up-regulated at 6 h. In the head kidney, TLR5a was up-regulated at 6 h, while TLR5b was up-regulated at 6 h and 12 h. After S. aureus infection, TLR5a and TLR5b were up-regulated at 6 h in the liver and 12 h in the spleen. However, in the head kidney, TLR5a was down-regulated, while TLR5b was up-regulated. Compared with TLR5a, TLR5b had a higher expression level and stronger response to pathogen stimulation. The immunofluorescence results showed that TLR5a and TLR5b proteins in the liver of grass carp infected with A. hydrophila and S. aureus were similar but different in the spleen and head kidney. The results indicated that TLR5a and TLR5b play a critical role in resisting bacterial infection, and TLR5a and TLR5b had obvious tissue and pathogen specificity. TLR5b may play a major role in immune tissues, while TLR5a may play an auxiliary regulatory role in early infection. In addition, TLR5a and TLR5b have an irreplaceable regulatory role in response to flagellate and non-flagellate bacteria. This lays a foundation to explore further the role of TLR5 in resisting flagellate and non-flagellate infections in fish and provides a reference for the innate immunity research of grass carp.

Intraperitoneal injection of class A TLR9 agonist enhances anti-PD-1 immunotherapy in colorectal peritoneal metastases.

Peritoneal metastases are associated with a low response rate to immune checkpoint blockade (ICB) therapy. The numbers of peritoneal resident macrophages (PRMs) are reversely correlated with the response rate to ICB therapy. We have previously shown that TLR9 in fibroblastic reticular cells (FRCs) plays a critical role in regulating peritoneal immune cell recruitment. However, the role of TLR9 in FRCs in regulating PRMs is unclear. Here, we demonstrated that the class A TLR9 agonist, ODN1585, markedly enhanced the efficacy of anti-PD-1 therapy in mouse models of colorectal peritoneal metastases. ODN1585 injected i.p. reduced the numbers of Tim4+ PRMs and enhanced CD8+ T cell antitumor immunity. Mechanistically, treatment of ODN1585 suppressed the expression of genes required for retinoid metabolism in FRCs, and this was associated with reduced expression of the PRM lineage-defining transcription factor GATA6. Selective deletion of TLR9 in FRCs diminished the benefit of ODN1585 in anti-PD-1 therapy in reducing peritoneal metastases. The crosstalk between PRMs and FRCs may be utilized to develop new strategies to improve the efficacy of ICB therapy for peritoneal metastases.

TLR9 Negatively Regulates Intracellular Bacterial Killing by Pyroptosis in Burkholderia pseudomallei-Infected Mouse Macrophage Cell Line (Raw264.7).

ABSTRACTMelioidosis is a serious infectious disease caused by Burkholderia pseudomallei. This bacterium is able to survive and multiply inside the immune cells such as macrophages. It is well established that Toll-like receptors (TLRs), particularly surface TLRs such as TLR2, TLR4, and TLR5, play an essential role in defending against this bacterial infection. However, the involvement of endosomal TLRs in the infection has not been elucidated. In this study, we demonstrated that the number of intracellular bacteria is reduced in TLR9-depleted RAW264.7 cells infected with B. pseudomallei, suggesting that TLR9 is involved in intracellular bacterial killing in macrophages. As several reports have previously demonstrated that pyroptosis is essential for restricting intracellular bacterial killing, particularly in B. pseudomallei infection, we also observed an increased release of cytosolic enzyme lactate dehydrogenase (LDH) in TLR9-depleted cells infected with B. pseudomallei, suggesting TLR9 involvement in pyroptosis in this context. Consistently, the increases in caspase-11 and gasdermind D (GSDMD) activations, which are responsible for the LDH release, were also detected. Moreover, we demonstrated that the increases in pyroptosis and bacterial killing in B. pseudomallei-infected TLR9-depleted cells were due to the augmentation of the IFN-β, one of the key cytokines known to regulate caspase-11. Altogether, this finding showed that TLR9 suppresses macrophage killing of B. pseudomallei by regulating pyroptosis. This information provides a novel mechanism of TLR9 in the regulation of intracellular bacterial killing by macrophages, which could potentially be leveraged for therapeutic intervention.IMPORTANCE Surface TLRs have been well established to play an essential role in Burkholderia pseudomallei infection. However, the role of endosomal TLRs has not been elucidated. In the present study, we demonstrated that TLR9 plays a crucial role by negatively regulating cytokine production, particularly IFN-β, a vital cytokine to control pyroptosis via caspase-11 activation. By depletion of TLR9, the percentage of pyroptosis was significantly increased, leading to suppression of intracellular survival in B. pseudomallei-infected macrophages. These findings provide a new role of TLR9 in macrophages.

The role of TLR-3 in the course and outcomes of rotavirus infection in infants

It was found that the clinical picture of rotavirus infection in infants with an initially low number of immunocompetent cells expressing TLR-3 does not differ significantly from that of patients with an initially large number of cells expressing TLR-3. When analyzing the treatment, it turned out that antibacterial therapy due to the activation of bacterial microflora was used only in children with an initially low number of immunocompetent cells expressing TLR-3. In the same group of patients, the development of atopic dermatitis and food allergies was noted during catamnestic observation for 6—12 months after rotavirus infection.

Abstract 1296: MEK inhibition overcomes chemoimmunotherapy resistance by inducing CXCL10 in cancer cells

Abstract Chemotherapy with anti PD-1/PD-L1 mAb has become the standard of care for patients with metastatic non-small cell lung cancer (NSCLC). Using lung tumor models, where pemetrexed-platinum chemotherapy (PEM/CDDP) remains unable to synergize with immune checkpoint inhibitors (ICI), we linked failure of this treatment with its inability to induce CXCL10 expression and CD8+ T cell recruitment. Using drug screening, we showed that combining a MEK inhibitor (MEKi) with PEM/CDDP triggers CXCL10 secretion by cancer cells and CD8+ T cell recruitment, and restores ICI efficacy. PEM/CDDP plus MEKi promotes optineurin (OPTN)-dependent mitophagy, resulting in CXCL10 production in a mitochondrial DNA and TLR9-dependent manner. TLR9 or autophagy/mitophagy processes genetic inactivation of abort the antitumor efficacy of PEM/CDDP plus MEKi/anti PD-L1 therapy. In human NSCLC, high OPTN, TLR9 and CXCL10 expression is associated with better response to ICI. Our results underline the role of TLR9 and OPTN-dependent mitophagy in enhancing chemoimmunotherapy efficacy. Citation Format: Emeric Limagne, Lisa Nuttin, Marion Thibaudin, Elise Jacquin, Romain Aucagne, Marjorie Bon, Elise Ballot, Solène Revy, Robby Barnestein, Caroline Truntzer, Valentin Derangère, Jean-David Fumet, Rébé Cédric, Pierre-Simon Bellaye, Coureche-Guillaume Kaderbhai, Aodrenn Spill, Bertrand Collin, Mary Callanan, Aurélie Lagrange, Laure Favier, Bruno Coudert, Laurent Arnould, Sylvain Ladoire, Bertrand Routy, Philippe Joubert, François Ghiringhelli. MEK inhibition overcomes chemoimmunotherapy resistance by inducing CXCL10 in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1296.

The role of TLR-2 and TLR-4 gene polymorphisms in the development of sepsis in children with severe burns

Recently, many studies are based on the study of innate immunity genes, namely the role of TLRs in the development of various diseases. Severe burn injury is characterized by the development of hyperimmune reactions, which further leads to the development of multiple organ complications and sepsis. The aim of our study was to identify the frequency and prognostic value of polymorphism of TLR-2 Arg753Gln and TLR-4 Thr399 Ile genes and their role in the development of sepsis and MOD in children with severe burns. Materials and methods: genetic analysis of TLR-2 Arg 753 Gln and TLR-4 Thr399 Ile gene polymorphisms was performed in children with severe and extremely severe burns (n ​​= 22) who were treated in the anesthesiology department with intensive care beds. Results: as a result of the obtained data, the heterozygous genotype TLR-2 Arg 753 Gln was detected in 69.5% (n = 16), in 31.8% (n = 7) patients with burn injury polymorphism was not detected. In 13.6% (n = 3) cases, polymorphism was associated with sepsis and MOD. Sepsis and normal homozygous genotype were diagnosed in 4.5% (n = 1). Heterozygous genotype TLR-4 Thr399 Ile was observed in 18.2% (n = 4) patients, of which only 4.5% (n = 1) patients had polymorphism associated with sepsis and MOD. Analysis of the association of TLR 2 genotypes and markers of acute inflammation revealed statistically significant differences between the heterozygous TLR 2 Arg 753 Gln genotype and C-reactive protein (CRP) levels in the blood of patients on day 3 of burn disease. Conclusions: The study of the role of innate immune system genes is promising in predicting the development of sepsis and complications in severe burns and requires further careful study.

A40 IDENTIFYING NOVEL ROLES FOR TLR2 SIGNALING IN THE INTESTINAL EPITHELIUM USING ORGANOIDS

Abstract Background Inflammatory bowel disease (IBD) is thought to result from an imbalance between protective and damaging immune responses to the resident microbiota and other luminal antigens. Increasing evidence suggests that toll-like-receptor (TLR)-mediated innate immune dysfunction contributes to the pathogenesis of IBD. Among the receptors in the TLR family, TLR2 and its signalling pathway appears to play an important protective role in the gastrointestinal (GI) tract. Notably, our group previously discovered that Tlr2 expression by nonhematopoietic cells played an important protective role in the Citrobacter rodentium model of infectious colitis. Aims To determine if intestinal epithelial cells (IECs) control Tlr2 dependent tissue protective responses, we sought to characterize the role of Tlr2 signalling in intestinal organoids. In addition, to better define the role of Tlr2 during C. rodentium infection, we developed a novel in vitro model of C. rodentium infection using organoid-derived monolayers. Methods Organoids were derived from the colonic tissue of wild type (C57BL/6J) and Tlr2 deficient (-/-) mice and then stimulated with Tlr2 agonists, and their responses were evaluated by qPCR, ELISA, Western blot and immunostaining. In addition, 2D monolayers were grown from organoids, and infected with C. rodentium to explore whether Tlr2 signaling regulates other protective IEC functions such as barrier proteins and cell death in response to noxious stimuli. Results Stimulation of WT, but not Tlr2-/- mouse orgnaoids led to increased transcription of chemokine and cytokine genes Ccl20, Mcp-1, Cxcl1 and Tnf-α. This was accompanied by increased protein secretion through a NF-κB and p38 MAP kinase dependent mechanism. Interestingly, organoids derived from the distal colon displayed stronger Tlr2 responses than organoids from the proximal colon. During C. rodentium infection of monolayers, Tlr2 signaling had no effect on the distribution of tight junction proteins such as ZO-1 or Claudin3, however, Tlr2 did regulate levels of IEC apoptosis upon C. rodentium infection. Conclusions Our study demonstrates that colonic organoids express functional Tlr2, which upon stimulation, leads to pro-inflammatory responses as well as control over cell death. Our novel C. rodentium infection model enables us to further study the role of IEC in promoting host defense during bacterial infection. Futher work will examine how interactions with Tlr2 dependent cytokines (ie. IL-6, IL-22) impact IEC responses to C. rodentium. Funding Agencies CCC, CIHRCH.I.L.D