Toll-like Receptor Pathway Research Articles

Guardians of Immunity: Toll-Like Receptor Signalling in the Onset and Progression of Multiple Sclerosis, Sketching an Immunopathological Scheme

Background: Multiple Sclerosis (MS) is a chronic autoimmune disorder of the central nervous system, characterized by inflammation, demyelination, and neurodegeneration. The role of toll-like receptors (TLRs) in MS pathogenesis, specifically through MYD88-dependent and -independent pathways, has become a focal point of research. Understanding these pathways offers insights into the mechanisms driving MS and potential therapeutic targets. Objective: This review aims to synthesize current knowledge on the role of TLR signaling in the onset and progression of MS, focusing on the dysregulation of MYD88-dependent and -independent pathways and their implications for disease pathogenesis and therapy. Methods: A systematic search of PubMed, Scopus, Web of Science, and Google Scholar databases was conducted to identify studies related to TLR signaling in MS. Keywords related to "toll-like receptors," "multiple sclerosis," "MYD88-dependent pathway," "MYD88-independent pathway," and "immune response" were used. The evidence synthesis was qualitative, integrating findings from both human and animal studies. Results: The review highlights the central role of NF-kB in mediating the effects of TLR signaling on the inflammatory cascade in MS. Dysregulation in TLR signaling pathways can lead to increased expression of pro-inflammatory cytokines and compromise the blood-brain barrier, exacerbating MS pathogenesis. The evidence points towards the dual role of TLR pathways in promoting inflammatory responses and potentially offering protective mechanisms against the disease. Conclusion: TLR signaling pathways play a crucial role in the immunopathology of MS, with both MYD88-dependent and -independent pathways contributing to disease progression. Targeting these pathways presents a promising therapeutic strategy, emphasizing the need for further research to develop effective treatments.

Multi-omics analysis of miRNA-mediated intestinal microflora changes in crucian carp Carassius auratus infected with Rahnella aquatilis.

Infection by an emerging bacterial pathogen Rahnella aquatilis caused enteritis and septicemia in fish. However, the molecular pathogenesis of enteritis induced by R. aquatilis infection and its interacting mechanism of the intestinal microflora associated with microRNA (miRNA) immune regulation in crucian carp Carassius auratus are still unclear. In this study, C. auratus intraperitoneally injected with R. aquatilis KCL-5 was used as an experimental animal model, and the intestinal pathological changes, microflora, and differentially expressed miRNAs (DEMs) were investigated by multi-omics analysis. The significant changes in histopathological features, apoptotic cells, and enzyme activities (e.g., lysozyme (LYS), alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate transaminase (AST), and glutathione peroxidase (GSH-Px)) in the intestine were examined after infection. Diversity and composition analysis of the intestinal microflora clearly demonstrated four dominant bacteria: Proteobacteria, Fusobacteria, Bacteroidetes, and Firmicutes. A total of 87 DEMs were significantly screened, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the potential target genes were mainly involved in the regulation of lipid, glutathione, cytosine, and purine metabolism, which participated in the local immune response through the intestinal immune network for IgA production, lysosome, and Toll-like receptor (TLR) pathways. Moreover, the expression levels of 11 target genes (e.g., TLR3, MyD88, NF-κB, TGF-β, TNF-α, MHC II, IL-22, LysC, F2, F5, and C3) related to inflammation and immunity were verified by qRT-PCR detection. The correlation analysis indicated that the abundance of intestinal Firmicutes and Proteobacteria was significantly associated with the high local expression of miR-203/NF-κB, miR-129/TNF-α, and miR-205/TGF-β. These findings will help to elucidate the molecular regulation mechanism of the intestinal microflora, inflammation, and immune response-mediated miRNA-target gene axis in cyprinid fish.

Machine-learning-based prediction of a diagnostic model using autophagy-related genes based on RNA sequencing for patients with papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer and belongs to the category of malignant tumors of the thyroid gland. Autophagy plays an important role in PTC. The purpose of this study is to develop a novel diagnostic model using autophagy-related genes (ARGs) in patients. In this study, RNA sequencing data of PTC samples and normal samples were obtained from GSE33630 and GSE29265. Then, we analyzed GSE33630 datasets and identified 127 DE-ARGs. Functional enrichment analysis suggested that 127 DE-ARGs were mainly enriched in pathways in cancer, protein processing in endoplasmic reticulum, toll-like receptor pathway, MAPK pathway, apoptosis, neurotrophin signaling pathway, and regulation of autophagy. Subsequently, CALCOCO2, DAPK1, and RAC1 among the 127 DE-ARGs were identified as diagnostic genes by support vector machine recursive feature elimination and least absolute shrinkage and selection operator algorithms. Then, we developed a novel diagnostic model using CALCOCO2, DAPK1, and RAC1 and its diagnostic value was confirmed in GSE29265 and our cohorts. Importantly, CALCOCO2 may be a critical regulator involved in immune microenvironment because its expression was related to many types of immune cells. Overall, we developed a novel diagnostic model using CALCOCO2, DAPK1, and RAC1 which can be used as diagnostic markers of PTC.

Anticipation and Verification of Dendrobium-Derived Nanovesicles for Skin Wound Healing Targets, Predicated Upon Immune Infiltration and Senescence.

Dendrobium, with profound botanical importance, reveals a rich composition of bioactive compounds, including polysaccharides, flavonoids, alkaloids, and diverse amino acids, holding promise for skin regeneration. However, the precise mechanism remains elusive. Seeking a potent natural remedy for wound healing, exocyst vesicles were successfully isolated from Dendrobium. This investigation aimed to employ bioinformatics and in vivo experiments to elucidate target genes of Dendrobium-derived nanovesicles in skin wound healing, focusing on immune infiltration and senescence characteristics. C57 mice experienced facilitated wound healing through Dendrobium-derived nanovesicles (DDNVs). Bioinformatics analysis and GEO database mining identified crucial genes by intersecting immune-related, senescence-related, and PANoptosis-associated genes. The identified genes underwent in vivo validation. DDNVs remarkably accelerated skin wound healing in C57 mice. Bioinformatics analysis revealed abnormal expression patterns of immune-related, senescence-related, and pan-apoptosis-related genes, highlighting an overexpressed IL-1β and downregulated IL-18 in the model group, Exploration of signaling pathways included IL-17, NF-kappa B, NOD-like receptor, and Toll-like receptor pathways. In vivo experiments confirmed DDNVs' efficacy in suppressing IL-1β expression, enhancing wound healing. Plant-derived nanovesicles (PDNV) emerged as a natural, reliable, and productive approach to wound healing. DDNVs uptake by mouse skin tissues, labeled with a fluorescent dye, led to enhanced wound healing in C57 mice. Notably, IL-1β overexpression in immune cells and genes played a key role. DDNVs intervention effectively suppressed IL-1β expression, accelerating skin wound tissue repair.

Toll-Like Receptor 4, 2, and Interleukin 1 Receptor Associated Kinase4: Possible Diagnostic Biomarkers in Myelodysplastic Syndrome Patients.

Myelodysplastic syndrome (MDS) is a clonal hematologic disorder that requires the integration of morphologic, cytogenetic, hematologic, and clinical findings for a successful diagnosis. Trying to find ancillary tests such as biomarkers improve the diagnosis process. Several studies showed that a disordered immune system is associated with MDS. The chronic activated innate immune system, particularly the Toll-like receptors (TLRs) pathway could be involved in the induction of the inflammation. In the present study, we investigated the expression of TLR2, TLR4, and IRAK4 in bone marrow (BM) of MDS patients, the leukemia group, and the healthy group. For this purpose, we assessed the expression of TLR2, TLR4, and IRAK4 by real time-PCR. In line with new findings, we demonstrated that the expression of TLR2, TLR4, and IRAK4 significantly increased in MDS BM compared with the healthy group. Moreover, IRAK4 expression raised significantly in MDS patients compared with other studied hematologic neoplasms. Also, the expression levels of TLR2 and TLR4 significantly increased in MDS in comparison to some studied non-MDS malignancies (P ˂ 0.05). Receiver operating characteristics (ROC) analysis and area under the curve (AUC) suggested that the expression of TLR2, TLR4, and IRAK4 (AUC = 0.702, AUC = 0.75, and AUC = 0.682, respectively) had acceptable diagnostic values to identify MDS from the other understudied leukemias. Overall, the expression of TLR2, TLR4, and IRAK4 could be potential biomarkers for discriminating MDS from some hematologic disorders.

Colchicine Alleviates Rosacea by Inhibiting Neutrophil Inflammation Activated by the TLR2 Pathway.

Rosacea is a chronic facial inflammatory skin disease that occurs with dysfunction of the immune system. Colchicine was reported to have anti-inflammatory properties. However, the impact of colchicine on rosacea remains unclear. In the present study, the phenotype of rosacea lesions was evaluated by the redness score, inflammatory biomarkers were analyzed by reverse transcription PCR (RT‒PCR), and the infiltration of inflammatory cells was assessed by IHC analysis and immunofluorescence in a rosacea-like mouse model. In vitro, RT‒PCR was used to identify the inflammatory factors that Toll-like receptor 2 (TLR2) agonist caused neutrophils to produce, and immunofluorescence and coimmunoprecipitation were used to identify putative signalling pathways. We found that skin erythema and histopathological alterations, as well as elevated proinflammatory factors (IL-1β, IL-6, TNFα, CXCL2) and CAMP, were significantly ameliorated by colchicine treatment in LL37-induced rosacea-like mice. In addition, colchicine reduced the colocalization of TLR2 and neutrophils and the formation of neutrophil extracellular trap networks (NET) in mouse lesions. In neutrophils, colchicine markedly reduced TLR2 agonist-induced inflammatory biomarker expression, NET formation, and ROS production. Moreover, we found that LL37 could bind to TLR2 upon activation of TLR2 in neutrophils. Importantly, colchicine could repress the combination of TLR2 and LL37 in vivo. Finally, bioinformatics methods further validated the key molecules of neutrophil-related inflammation in rosacea, which is consistent with our experimental findings. Collectively, colchicine ameliorated rosacea-like dermatitis by regulating the neutrophil immune response activated by the TLR2 pathway, indicating that it could be an effective therapeutic option for patients with rosacea.

Characteristics of Ovarian Cancer Immune Cell Invasion and Bioinformatics to Predict the Effect of Immunotherapy.

Recent studies have confirmed that tumor immune cell infiltration (ICI) is associated with sensitivity of ovarian cancer (OC) immunotherapy and disease progression of OC patients. However, studies related to immune infiltration in OC, has not been elucidated. Two algorithms are used to analyze the OC data in the TCGA and GEO databases. After combining the two data sets, the immune cell content of the sample was estimated by Cell-type Identification By Estimate Relative Subsets of RNA Transcripts (CIBERSORT method). An unsupervised consistent clustering algorithm was used to analyze ICI subtypes and their differentially expressed genes (DEGs). Two subgroups and three ICI gene clusters were identified by unsupervised consensus clustering algorithm. The ICI score was obtained by analyzing the gene characteristics through principal component analysis (PCA). The ICI score ranged from -15.8132 to 18.7211, which was associated with the prognosis of OC patients with immunotherapy. The Toll-like receptor pathway, B-cell receptor pathway, antigen processing and presentation pathway, NK-cell-mediated cytotoxicity pathway, and arginine-proline metabolism pathway were activated in the high ICI score group, suggesting that immune cells in the high ICI score group were activated, thus leading to a better prognosis in this group of patients. Patients with G3-G4 in the high ICI rating group were more sensitive to immunotherapy and had a better prognosis in patients with high tumor mutation burden (TMB). This study suggests that ICI scores can be used as a feasible auxiliary indicator for predicting the prognosis of patients with OC.

Physical activity levels, gut permeability, circulating inflammatory cytokines and TLR4 signalling in PBMCs of COVID-19 patients

Physical exercise could confer some protection against the inflammatory cytokine storm induced by SARS-CoV-2 infection and against severe COVID-19 symptomatology. However, the mechanisms underlying this protective effect of exercise have not been elucidated yet. Therefore, the aim of this study was to investigate the activation of the inflammatory pathway in peripheral blood mononuclear cells (PBMCs) and to test a possible association between the regulation of this pathway and the expression of plasma inflammatory cytokine markers as well as with physical activity levels. All patients (n = 31; 21 males and 10 females) had at least three symptoms associated with COVID-19 and were recruited in the emergency unit. They were immediately tested for COVID-19 by polymerase chain reaction (COVID-19 n = 18, amongst which 9 were hospitalized and 9 quarantined; Non-COVID-19 n = 13) and a blood sample was collected. Physical activity levels were determined using the international physical activity questionnaire (IPAQ). No differences in physical activity levels nor sitting time were found between the COVID-19 and the Non-COVID-19 group. However, moderate to vigorous physical activity was lower in the hospitalized compared to the quarantined group. Toll-like receptor 4 (TLR4) and MyD88 protein expression in PBMCs was lower in the COVID-19 than in the Non-COVID-19 group. Moreover, TLR4 protein expression was lower in the hospitalized than in the quarantined subgroup. While no difference was observed in plasma zonulin levels between COVID-19 and Non-COVID-19 patients, quarantined patients had lower plasma zonulin levels compared to hospitalized patients. In conclusion, patients hospitalized with severe COVID-19 were less physically active and presented greater gut permeability and a downregulation of the TLR4 pathway in PBMCs compared to quarantined and Non-COVID-19 individuals. The lower expression of TLR4 could be a mechanism to prevent, or at least to mitigate, the inflammatory cytokine storm related to the severe form of COVID-19.

GO-PEG Represses the Progression of Liver Inflammation via Regulating the M1/M2 Polarization of Kupffer Cells.

As a highly promising nanomaterial, exploring the impact of the liver, a vital organ, stands out as a crucial focus in the examination of its biological effects. Kupffer cells (KCs) are one of the first immune cells to contact with exotic-substances in liver. Therefore, this study investigates the immunomodulatory effects and mechanisms of polyethylene glycol-modified graphene oxide (GO-PEG) on KCs. Initial RNA-seq and KEGG pathway analyses reveal the inhibition of the TOLL-like receptor, TNF-α and NOD-like receptor pathways in continually stimulated KCs exposed to GO-PEG. Subsequent biological experiments validate that a 48-hour exposure to GO-PEG alleviates LPS-induced KCs immune activation, characterized by a shift in polarization from M1 to M2. The underlying mechanism involves the absorption of double-stranded RNA/single-stranded RNA, inhibiting the activation of TLR3 and TLR7 in KCs. Employing a Kupffer/AML12 cell co-culture model and animal studies, it is observed that GO-PEG indirectly inhibit oxidative stress, mitochondrial dysfunction, and apoptosis in AML12 cells, partially mitigating systemic inflammation and preserving liver tissue/function. This effect is attributed to the paracrine interaction between KCs and hepatocytes. These findings suggest a meaningful and effective strategy for treating liver inflammation, particularly when combined with anti-inflammatory drugs.

Dishevelled Segment Polarity Protein 3 (DVL3) Induced by Bacterial LPS Promotes the Proliferation and Migration of Prostate Cancer Cells through the TLR4 Pathway.

Chronic inflammation is associated with various malignant tumors. Bacterial lipopolysaccharides (LPSs) play a significant part in the event and development of prostate cancer. Dishevelled segment polarity protein 3 (DVL3) is a shared component of the Wnt/β-catenin and Notch signaling pathways, which are involved in tumor progression, chemoresistance, and maintenance of stem cell-like properties. According to reports, prostatic cancer cell invasion and proliferation are mediated by toll-like receptor 4 (TLR4). However, the role and regulation of DVL3 in prostate cancer and its relationship with TLR4 remain unclear. Survival curves were plotted to evaluate the relationship between DVL3 expression and prognosis in patients with prostate cancer. DVL3 was silenced in PC3 and DU145 cells using small interfering RNAs (siRNAs). Subsequently, cell counting kit-8 (CCK-8) assay, colony formation assay, transwell migration assay, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were performed to investigate the role of DVL3 in cell proliferation and migration in vitro. The protein markers of potential pathways were analyzed via western blotting. DVL3 expression was linked to prognosis in patients with prostate cancer; In particular, patients with high DVL3 expression had a poor prognosis. LPS stimulation increased (p < 0.01) the expression of DVL3 in PC3 cells. DVL3 regulated tumor cell proliferation and migration by mediating the increase (p < 0.01) in TLR4 expression. Knockout of TLR4 validated that TLR4 played a crucial role in LPS-induced DVL3 expression. Silencing of DVL3 decreased (p < 0.01) the LPS-induced proliferation and migration of PC3 cells. Bacterial LPS-induced DVL3 promoted the multiplication and migration of prostate cancer cells through the TLR4 pathway. This study offers a valuable reference for the development and clinical application of targeted drugs for prostate cancer.

IL1A regulates the inflammation in gout through the Toll-like receptors pathway.

Objective: Gout is a dangerous metabolic condition related to monosodium urate (MSU). Our aim is to study the molecular mechanisms underlying gout and to identify potential clinical biomarkers by bioinformatics analysis and experimental validation. Methods: In this study, we retrieved the overlapping genes between GSE199950-Differential Expressed Genes (DEGs) dataset and key module in Weighted Gene Co-Expression Network Analysis (WGCNA) on GSE199950. These genes were then analyzed by protein-protein interaction (PPI) network, expression and Gene Set Enrichment Analysis to identify the hub gene related to gout. Then, the gene was investigated by peripheral blood mononuclear cells (PBMCs), immunoassay and cell experiments like western blotting to uncover its underlying mechanism in gout cells. Results: From the turquoise module and 83 DEGs, we identified 62 overlapping genes, only 11 genes had mutual interactions in PPI network and these genes were highly expressed in MSU-treated samples. Then, it was found that the IL1A (interleukin 1 alpha) was the only one gene related to Toll-like receptor signaling pathway that was associated with the occurrence of gout. Thus, IL1A was determined as the hub gene in this study. In immunoassay, IL1A was significantly positively correlated with B cells and negatively correlated with macrophages. Moreover, IL1A is highly expressed in gout patients,it has a good clinical diagnostic value. Finally, the results of in vitro experiments showed that after knocking down IL1A, the expressions of pro-inflammatory cytokines and Toll-like receptor signaling pathway-related proteins (TLR2, TLR4, MyD88) were all reduced. Conclusion: It is confirmed that IL1A is a promoting gene in gout with a good diagnostic value, and specifically it affects the inflammation in gout through Toll-like receptor pathway. Our research offers fresh perspectives on the pathophysiology of gout and valuable directions for future diagnosis and treatment.

Circ_0087199 depletion attenuates lipopolysaccharides-induced human periodontal ligament cell injury through the miR-527/TLR4 axis.

Circular RNAs participate in the development of periodontitis. The present work aims to reveal the role and mechanism of circ_0087199 in human periodontal ligament cell (PDLC) injury during periodontitis. PDLCs were treated with lipopolysaccharides (LPS) to establish a periodontitis cell model. Quantitative real-time polymerase chain reaction was used to detect the expression of circ_0087199, miR-527, toll-like receptor 4 (TLR4). Western blot analysis assay was performed to assess protein expression. Cell viability, proliferation, apoptosis and inflammation were investigated by cell counting kit-8, EdU assay, flow cytometry and enzyme-linked immunosorbent assay, respectively. Oxidative stress was evaluated by malondialdehyde assay kit and superoxide dismutase activity assay kit. The interaction between miR-527 and circ_0087199 or TLR4 was confirmed by a dual-luciferase reporter assay. Circ_0087199 and TLR4 expression levels were significantly increased, while miR-527 was decreased in the periodontal ligament tissues of periodontitis patients and LPS-stimulated PDLCs when compared with controls. LPS treatment inhibited cell viability and proliferation but induced cell apoptosis, inflammation and oxidative stress, whereas these effects were attenuated after circ_0087199 knockdown. Circ_0087199 bound to miR-527 and regulated LPS-caused PDLC damage by targeting miR-527. Additionally, the overexpression of TLR4, a target gene of miR-527, rescued miR-527 mimic-mediated effects on LPS-treated PDLCs. Further, the regulation of circ_0087199 toward TLR4 involved miR-527. Circ_0087199 knockdown attenuated LPS-induced apoptosis, inflammation and oxidative stress of PDLCs by regulating the miR-527/TLR4 pathway.

Molecular mechanism of Xuebijing in treating pyogenic liver abscess complicated with sepsis.

Xuebijing (XBJ) can alleviate the inflammatory response, improve organ function, and shorten the intensive care unit (ICU) stay in patients with pyogenic liver abscess (PLA) complicated with sepsis, but the molecular mechanisms have not been elucidated. This study aimed to explore the molecular mechanism of XBJ in treating PLA complicated with sepsis using a network pharmacology approach. The active ingredients and targets of XBJ were retrieved from the ETCM database. Potential targets related to PLA and sepsis were retrieved from the GeneCards, PharmGKB, DisGeNet, Online Mendelian Inheritance in Man (OMIM), Therapeutic Targets Database (TTD), and DrugBank databases. The targets of PLA complicated with sepsis were mapped to the targets of XBJ to identify potential treatment targets. Protein-protein interaction networks were analyzed using the STRING database. Potential treatment targets were imported into the Metascape platform for Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Molecular docking was performed to validate the interactions between active ingredients and core targets. XBJ was found to have 54 potential treatment targets for PLA complicated with sepsis. Interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF) were identified as core targets. KEGG enrichment analysis revealed important pathways, including the interleukin-17 (IL-17) signaling pathway, the TNF signaling pathway, the nuclear factor-kappa B (NF-κB) signaling pathway, and the Toll-like receptor (TLR) signaling pathway. Molecular docking experiments indicated stable binding between XBJ active ingredients and core targets. XBJ may exert therapeutic effects on PLA complicated with sepsis by modulating signaling pathways, such as the IL-17, TNF, NF-κB, and TLR pathways, and targeting IL-1β, IL-6, and TNF.

The dualistic role of Lyn tyrosine kinase in immune cell signaling: implications for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE, lupus) is a debilitating, multisystem autoimmune disease that can affect any organ in the body. The disease is characterized by circulating autoantibodies that accumulate in organs and tissues, which triggers an inflammatory response that can cause permanent damage leading to significant morbidity and mortality. Lyn, a member of the Src family of non-receptor protein tyrosine kinases, is highly implicated in SLE as remarkably both mice lacking Lyn or expressing a gain-of-function mutation in Lyn develop spontaneous lupus-like disease due to altered signaling in B lymphocytes and myeloid cells, suggesting its expression or activation state plays a critical role in maintaining tolerance. The past 30 years of research has begun to elucidate the role of Lyn in a duplicitous signaling network of activating and inhibitory immunoreceptors and related targets, including interactions with the interferon regulatory factor family in the toll-like receptor pathway. Gain-of-function mutations in Lyn have now been identified in human cases and like mouse models, cause severe systemic autoinflammation. Studies of Lyn in SLE patients have presented mixed findings, which may reflect the heterogeneity of disease processes in SLE, with impairment or enhancement in Lyn function affecting subsets of SLE patients that may be a means of stratification. In this review, we present an overview of the phosphorylation and protein-binding targets of Lyn in B lymphocytes and myeloid cells, highlighting the structural domains of the protein that are involved in its function, and provide an update on studies of Lyn in SLE patients.

Dysregulation of Toll-Like Receptor Signaling-Associated Gene Expression in X-Linked Agammaglobulinemia: Implications for Correlations Genotype-Phenotype and Disease Expression.

In X-linked agammaglobulinemia (XLA), the diversity of BTK variants complicates the study of genotype-phenotype correlations. Since BTK negatively regulates toll-like receptors (TLRs), we investigated if distinct BTK mutation types selectively modulate TLR pathways, affecting disease expression. Using reverse transcription-quantitative polymerase chain reaction, we quantified ten TLR signaling-related genes in XLA patients with missense (n = 3) and nonsense (n = 5) BTK mutations and healthy controls (n = 17). BTK, IRAK2, PIK3R4, REL, TFRC, and UBE2N were predominantly downregulated, while RIPK2, TLR3, TLR10, and TLR6 showed variable regulation. The missense XLA group exhibited significant downregulation of IRAK2, PIK3R4, REL, and TFRC and upregulation of TLR3 and/or TLR6. Hypo-expression of TLR3, TLR6, and TLR10 may increase susceptibility to infections, while hyper-expression might contribute to chronic inflammatory conditions like arthritis or inflammatory bowel disease. Our findings shed light on the important inflammatory component characteristic of some XLA patients, even under optimal therapeutic conditions.

Expression profile of Toll-like Receptors Pathway Genes in Chicken Erythrocytes Infected with Mycoplasma synoviae

Expression profile of Toll-like Receptors Pathway Genes in Chicken Erythrocytes Infected with Mycoplasma synoviae

Neurosteroid [3α,5α]-3-hydroxy-pregnan-20-one enhances IL-10 production via endosomal TRIF-dependent TLR4 signaling pathway.

Previous studies demonstrated the inhibitory effect of allopregnanolone (3α,5α-THP) on the activation of inflammatory toll-like receptor 4 (TLR4) signals in RAW264.7 macrophages and the brains of selectively bred alcohol-preferring (P) rats. In the current study, we investigated the impact of 3α,5α-THP on the levels of IL-10 and activation of the TRIF-dependent endosomal TLR4 pathway. The amygdala and nucleus accumbens (NAc) of P rats, which exhibit innately activated TLR4 pathways as well as RAW264.7 cells, were used. Enzyme-linked immunosorbent assays (ELISA) and immunoblotting assays were used to ascertain the effects of 3α,5α-THP on the TRIF-dependent endosomal TLR4 pathway and endosomes were isolated to examine translocation of TLR4 and TRIF. Additionally, we investigated the effects of 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) on the levels of IL-10 in RAW264.7 macrophages. Finally, we examined whether inhibiting TRIF (using TRIF siRNA) in RAW264.7 cells altered the levels of IL-10. 3α,5α-THP administration facilitated activation of the endosomal TRIF-dependent TLR4 pathway in males, but not female P rats. 3α,5α-THP increased IL-10 levels (+13.2 ± 6.5%) and BDNF levels (+21.1 ± 11.5%) in the male amygdala. These effects were associated with increases in pTRAM (+86.4 ± 28.4%), SP1 (+122.2 ± 74.9%), and PI(3)K-p110δ (+61.6 ± 21.6%), and a reduction of TIRAP (-13.7 ± 6.0%), indicating the activation of the endosomal TRIF-dependent TLR4 signaling pathway. Comparable effects were observed in NAc of these animals. Furthermore, 3α,5α-THP enhanced the accumulation of TLR4 (+43.9 ± 11.3%) and TRIF (+64.8 ± 32.8%) in endosomes, with no significant effect on TLR3 accumulation. Additionally, 3α,5α-THP facilitated the transition from early endosomes to late endosomes (increasing Rab7 levels: +35.8 ± 18.4%). In RAW264.7 cells, imiquimod (30 µg/mL) reduced IL-10 while 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) restored IL-10 levels. To determine the role of the TRIF-dependent TLR4 signaling pathway in IL-10 production, the downregulation of TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%). TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%) and 3α,5α-THP (1.0 µM) no longer restored the reduced IL-10 levels. The results demonstrate 3α,5α-THP enhancement of the endosomal TLR4-TRIF anti-inflammatory signals and elevations of IL-10 in male P rat brain that were not detected in female P rat brain. These effects hold significant implications for controlling inflammatory responses in both the brain and peripheral immune cells.

Comparative Analysis of Nucleic Acid-Binding Polymers as Potential Anti-Inflammatory Nanocarriers.

Conventionally, nanocarriers are used to regulate the controlled release of therapeutic payloads. Increasingly, they can also be designed to have an intrinsic therapeutic effect. For example, a positively charged nanocarrier can bind damage-associated molecular patterns, inhibiting toll-like receptor (TLR) pathway activation and thus modulating inflammation. These nucleic acid-binding nanomaterials (NABNs), which scavenge pro-inflammatory stimuli, exist in diverse forms, ranging from soluble polymers to nanoparticles and 2D nanosheets. Unlike conventional drugs that primarily address inflammation symptoms, these NABPs target the upstream inflammation initiation pathway by removing the agonists responsible for inflammation. Many NABNs have demonstrated effectiveness in murine models of inflammatory diseases. However, these scavengers have not been systematically studied and compared within a single setting. Herein, we screen a subset of the most potent NABNs to define their relative efficiency in scavenging cell-free nucleic acids and inhibiting various TLR pathways. This study helps interpret existing in vivo results and provides insights into the future design of anti-inflammatory nanocarriers.

Abstract A011: Targeting the FES tyrosine kinase in antigen presenting cells to enhance anti-tumor cytotoxic lymphocyte responses

Abstract For cytotoxic lymphocytes (CTLs) to effectively kill cancer cells, inflammatory signals from antigen presenting cells (APCs) are required. Targeting the signaling pathways that regulate production of these inflammatory cytokines represents an exciting strategy to bolster CTL activation in the context of cancer immunotherapy. Achieving optimal levels of CTL activation is critical to the success of immunotherapy treatments. Compelling advancements in CTL therapies including CAR-T cells and immune checkpoint inhibitors are showing great promise for improving patient outcomes. However, enhancing the innate immune system’s ability to strengthen anti-tumor CTL immune responses has been explored to a lesser extent. We intend to fill this gap by studying the APC-intrinsic role of the non-receptor tyrosine kinase FES in regulating the production of inflammatory cytokines. The first evidence of FES’s potential immune regulating role came from observations of increased lipopolysaccharide (LPS) sensitivity in fes-null mice. A transgenic mouse model of breast cancer expressing activated HER2/Neu in the mammary glands showed delayed tumor onset in mice targeted with a fes mutation that catalytically inactivated FES; and this delay correlated with increased immune infiltration and inflammation in pre-malignant mammary tissue. Previous studies have implicated FES in the activation of the SHP-2 phosphatase in macrophages, which can suppress toll-like receptor (TLR) pathways. TLR pathways govern strong innate inflammatory responses, including the expression of the so-called signal 3 cytokines required for full CTL activation (e.g. IL-12, IFNα/β). We therefore hypothesized that disrupting FES in APCs will relieve suppression of inflammatory cytokine production pathways, leading to increased CTL activation and cancer cell cytotoxicity. To investigate the impact of FES disruption on inflammatory signaling cascades, immunoblotting analysis of proteins in these signaling pathways was conducted on LPS-stimulated fes−/− and WT mouse bone marrow derived macrophages or dendritic cells. qRT-PCR was used to measure inflammatory cytokine transcript levels, including IFNα/β, IL-12, IL-1β and TNFα. To compare the ability of WT and fes−/− macrophages to present antigen and activate CTLs, OT-1 CTLs were co-cultured with OVA-presenting WT or fes−/− macrophages; and then evaluated by flow cytometry for IFNγ production. Evidence of increased activation of downstream mediators of TLR signaling including NFκB and TBK1 were seen in fes−/− macrophages and dendritic cells post LPS stimulation. fes−/− macrophages displayed increased inflammatory cytokine mRNA production in response to LPS stimulation. Finally, CTLs showed increased IFNγ expression when co-cultured with LPS-stimulated fes−/− macrophages compared to WT macrophages, indicating a higher degree of activation by fes−/− macrophages. The immunosuppressive function of FES makes it an actionable target for improving the efficacy of cancer immunotherapies and anti-cancer adaptive immune responses. Citation Format: Natasha Dmytryk, Brian Laight, Peter Greer. Targeting the FES tyrosine kinase in antigen presenting cells to enhance anti-tumor cytotoxic lymphocyte responses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A011.

Abstract C053: Multi-omics guided design of lymphoid organoids to study cooperative cell signaling in lymphomas

Abstract Our collaborative research (Cell1) used 1001 patients of diffuse large B-cell lymphoma (DLBCL) to show that genetic alterations interact with clinical factors to impact overall survival (OS) in DLBCL. B cell receptor (BCR) signaling-driven activated B cell-like (ABC) DLBCL subtypes is the most chemo-resistant subtype to the frontline therapy R-CHOP, with 40% of patients experiencing no response or relapse. We have systematically examined disparities in lymphoma outcomes and identified African-American (AA) patients as a poor-risk population in DLBCL, with a diagnosis age 10 years younger than other racial groups. We found that AA-DLBCL patients experienced inferior 5-year OS compared to non-Hispanic whites (38% vs 46%)2,3. We performed the first-ever characterization of genetic alterations among AA-DLBCL patients and demonstrated distinct mutation patterns across DLBCL arising in AA ancestry groups4. While some of the most frequent genetic mutations in AA-DLBCL are related to chromatin and epigenetics, few studies have investigated racial disparities in patients with aggressive ABC DLBCLs. The mechanisms that determine this racial inequality are unknown but may lie in the particular spectrum of mutations that act in concert with intricate survival signals imparted by the lymph node tumor microenvironment (Ly-TME). The pathogenesis of ABC DLBCLs has been linked to constitutive activation of and cooperation between the BCR and Toll-like receptor (TLR) pathways. These pathways are emerging as novel therapeutic targets, with a particular interest in MALT1 (mucosa-associated lymphoid tissue translocation protein 1). However, the genetic heterogeneity of ABC DLBCLs and the cooperative signaling mediated by the Ly-TME have led to discrepancies in drug responses. As a first step towards understanding Ly-TME in DLBCLs (Nature Materials5), we used DLBCL patient samples from multiple cohorts, covering &amp;gt;1100 samples and imaging analysis to determine immune and ECM cues in the lymphoid TME. These samples were not classified into racial categories. We designed a biomaterials-based modular lymphoma organoid that presents specific integrin ligands at controlled densities to DLBCLs. Human ABC-DLBCLs responded to MALT1 inhibition in an integrin-ligand-dependent manner. TLR activation with TLR agonists rescued cells from MALT1 inhibitors, possibly due to increased MALT1 expression. The inclusion of T-cell signals increased MALT1 expression and rescued cells from MALT1 inhibitor-mediated killing in cell lines and human PDX organoids. Changes in RNA-seq and phosphorylation in the TLR &amp; BCR pathways were used to inform effective combination therapy approaches with MALT1 inhibitors in vitro and in vivo5. Ongoing work is determining the racial disparities in AA lymphomas through complex interactions between Ly-TME and DLBCLs.