Toll-like Receptor Signaling Pathway Genes Research Articles

Regional modulation of toll-like receptor signaling pathway genes in acute epididymitis in mice.

Region-specific immune environments in the epididymis influence the immune responses to uropathogenic Escherichia coli (UPEC) infection, a relevant cause of epididymitis in men. Toll-like receptors (TLRs) are essential to orchestrate immune responses against bacterial infections. The epididymis displays region-specific inflammatory responses to bacterial-derived TLR agonists, such as lipopolysaccharide (LPS; TLR4 agonist) and lipoteichoic acid (LTA; TLR2/TLR6 agonist), suggesting that TLR-associated signaling pathways could influence the magnitude of inflammatory responses in epididymitis. To investigate the expression and regulation of key genes associated with TLR4 and TLR2/TLR6 signaling pathways during epididymitis induced by UPEC, LPS, and LTA in mice. Epididymitis was induced in mice using UPEC, ultrapure LPS, or LTA, injected into the interstitial space of the initial segment or the lumen of the vas deferens close to the cauda epididymidis. Samples were harvested after 1, 5, and 10 days for UPEC-treated animals and 6 and 24h for LPS-/LTA-treated animals. Ex vivo epididymitis was induced by incubating epididymal regions from naive mice with LPS or LTA. RT-qPCR and Western blot assays were conducted. UPEC infection up-regulated Tlr2, Tlr4, and Tlr6 transcripts and their associated signaling molecules Cd14, Ticam1, and Traf6 in the cauda epididymidis but not in the initial segment. In these epididymal regions, LPS and LTA differentially modulated Tlr2, Tlr4, Tlr6, Cd14, Myd88, Ticam1, Traf3, and Traf6 expression levels. NFKB and AP1 activation was required for LPS- and LTA-induced up-regulation of TLR-associated signaling transcripts in the cauda epididymidis and initial segment, respectively. The dynamic modulation of TLR4 and TLR2/TLR6 signaling pathways gene expression during epididymitis indicates bacterial-derived antigens elicit an increased tissue sensitivity to combat microbial infection in a spatial manner in the epididymis. Differential activation of TLR-associated signaling pathways may contribute to fine-tuning inflammatory responses along the epididymis.

Distinct evolution of toll-like receptor signaling pathway genes in cetaceans.

The relatively rapid spread and diversity of marine pathogens posed an initial and ongoing challenge for cetaceans (whales, dolphins, and porpoises), descendants of terrestrial mammals that transitioned from land to sea approximately 56 million years ago. Toll-like receptors (TLRs) play important roles in regulating immunity against pathogen infections by detecting specific molecular patterns and activating a wide range of downstream signaling pathways. The ever-increasing catalogue of mammalian genomes offers unprecedented opportunities to reveal genetic changes associated with evolutionary and ecological processes. This study aimed to explore the molecular evolution of TLR signaling pathway genes in cetaceans. Genes involved in the TLR signaling pathway were retrieved by BLAST searches using human coding sequences as queries. We tested each gene for positive selection along the cetacean branches using PAML and Hyphy. Physicochemical property changes of amino acids at all positively selected residues were assessed by TreeSAAP and visualized with WebLogo. Bovine and dolphin TLR4 was assessed using human embryonic kidney cell line HEK293, which lacks TLR4 and its co-receptor MD-2. We demonstrate that eight TLR signaling pathway genes are under positive selection in cetaceans. These include key genes in the response to Gram-negative bacteria: TLR4, CD14, and LY96 (MD-2). Moreover, 41 out of 65 positively selected sites were inferred to harbor substitution that dramatically changes the physicochemical properties of amino acids, with most of them situated in or adjacent to functional regions. We also found strong evidence that positive selection occurred in the lineage of the Yangtze finless porpoise, likely reflecting relatively recent adaptions to a freshwater milieu. Species-specific differences in TLR4 response were observed between cetacean and terrestrial species. Cetacean TLR4 was significantly less responsive to lipopolysaccharides from a terrestrial E. coli strain, possibly a reflection of the arms race of host-pathogen co-evolution faced by cetaceans in an aquatic environment. This study provides further impetus for studies on the evolution and function of the cetacean immune system.

Comparative study of immune responses elicited by outer membrane vesicles of different Pseudomonas aeruginosa strains

BackgroundPseudomonas aeruginosa (P. aeruginosa) is an opportunistic mucosal human pathogen that naturally releases outer membrane vesicles (OMVs) in different environments, as do other Gram-negative bacteria. The intestinal tract infections caused by P. aeruginosa increase the risk of respiratory infections and mortality of other diseases related to gut infections. Therefore, in this study, we attempted to investigate toll-like receptor (TLR) signaling pathways and immune response profiles of human colon adenocarcinoma (Caco2) cell line exposed to the OMVs of three different P. aeruginosa strains (i.e., antibiotic-susceptible, multi-drug resistant (MDR), and standard lab ATCC 17933). Materials and methodsReal-time quantitative reverse transcription PCR array was carried out to determine mRNA expression in 84 TLRs signaling pathway genes, and the production of specific cytokines was measured by ELISA. ResultsOMVs of different strains could induce unique changes in regulating TLRs signaling pathways, such that there were remarkable differences in pro-inflammatory effects and anti-inflammatory responses among the three strains. ConclusionThe more complete immune responses observed through the MAMPs caused by MDR strain OMVs interactions with Caco2 lead us to the conclusion that the use of MDR or cystic fibrosis strain OMVs for better known the host immune responses seems preferable.

The Combination of PARP Inhibitors and DNMT Inhibitors Modulates Immune Activity and Suggests a Role for Immune Therapy in AML

Acute myeloid leukemia (AML) is a genetically heterogeneous disease where malignant clonal proliferation of immature myeloid cells in the bone marrow leads to disruption of normal hematopoiesis and bone marrow failure. Most treatment improvements have consisted of limited chemotherapeutic interventions, and morbidity and mortality remain unacceptably high. While patients unfit for intensive chemotherapy have the options of epigenetic treatments with DNA methyltransferase inhibitors (DNMTis), and the preclinical research underpinning these treatments is robust and promising, clinical response rates have been poor. Therefore, we have derived a unique treatment strategy combining poly-ADP ribose polymerase inhibitors (PARPis) with DNMTis, and we have previously demonstrated efficacy of this combination in AML in both in vitroand in vivomodels. This combination increases the amount and retention time of PARP1 at laser-induced DNA damage sites, increases DNA DSBs and tumor cytotoxicity, blunts cell self-renewal, and leads to strong anti-tumor responses. These results form the basis for our ongoing Phase I/II clinical trial of this combination therapy in patients with relapsed/refractory AML.To identify molecular pathways underpinning this combination, genome-wide expression microarray analysis was performed on RNA extracted from multiple BRCA-intact sporadic AML cell lines treated with single agents or drug combination. Interestingly, combination drug treatment, more than either single treatment alone, up regulated genes in immune response pathways, including interferons, natural killer cell-mediated cytotoxicity and Toll-like receptor signaling pathway genes. In both AML cell lines (N=10) and primary samples (N=8), we used qPCR to validate these expression increases, with particular focus on interferon type 1-inducible genes, including IFI6, IFI27, IFI30, IRF7, ISG15, and OASL. These interferon-inducible genes were similarly increased in selected AML clinical trial samples post combination treatment. Interferon stimulation studies showed that treatment of multiple cell lines with IFN-β similarly up regulates our genes of interest, and suggests that the immune system plays a pivotal role in mediating the effects of our combination treatment.To functionally validate this immune involvement in vivo, we use an immune competent mouse model that spontaneously develops AML. The bone marrow cells collected from leukemic FLT3/ITD;NHD13 mice are transplanted into sublethally irradiated syngeneic recipients where they undergo clonal leukemic expansion and expression of specific surface markers (CD45.1 vs CD45.2) allow for the tracking of both host and transplanted blood cells. After validating that our combination treatment increases survival and reduces tumor burden in this in vivo model, we isolated mononuclear cells from spleens and livers and found that mice in the combination therapy group had increased T cell populations with increased PD1, CD28 and 4-1BB expression. Tal single agent and combination therapy treated mice also had decreased myeloid-derived suppressor cells and increased antigen presenting cells, specifically dendritic cells, monocytes and macrophages. Finally, upon ex vivo stimulation, T cells from mice receiving combination therapy produced significantly more IFN-γ and GM-CSF than mice from other treatment groups.These results indicate that our combination therapy results in immune activation, and suggest the potential to target the immune system in order to induce anti-tumor effects, enhance efficacy of our combination treatment and improve clinical outcomes in AML. DisclosuresLapidus:KinaRx, LLC: Other: Founder and Scientific Advisor.

Genetic variant of IRAK2 in the toll-like receptor signaling pathway and survival of non-small cell lung cancer.

The toll-like receptor (TLR) signaling pathway plays an important role in the innate immune responses and antigen-specific acquired immunity. Aberrant activation of the TLR pathway has a significant impact on carcinogenesis or tumor progression. Therefore, we hypothesize that genetic variants in the TLR signaling pathway genes are associated with overall survival (OS) of patients with non-small cell lung cancer (NSCLC). To test this hypothesis, we first performed Cox proportional hazards regression analysis to evaluate associations between genetic variants of 165 TLR signaling pathway genes and NSCLC OS using the genome-wide association study (GWAS) dataset from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). The results were further validated by the Harvard Lung Cancer Susceptibility GWAS dataset. Specifically, we identified IRAK2 rs779901 C > T as a predictor of NSCLC OS, with a variant-allele (T) attributed hazards ratio (HR) of 0.78 [95% confidence interval (CI) = 0.67-0.91, P = 0.001] in the PLCO dataset, 0.84 (0.72-0.98, 0.031) in the Harvard dataset, and 0.81 (0.73-0.90, 1.08x10-4 ) in the meta-analysis of these two GWAS datasets. In addition, the T allele was significantly associated with an increased mRNA expression level of IRAK2. Our findings suggest that IRAK2 rs779901 C > T may be a promising prognostic biomarker for NSCLC OS.

Genomic signature of highland adaptation in fish: a case study in Tibetan Schizothoracinae species

BackgroundGenome-wide studies on highland adaptation mechanism in terrestrial animal have been widely reported with few available for aquatic animals. Tibetan Schizothoracinae species are ideal model systems to study speciation and adaptation of fish. The Schizothoracine fish, Gymnocypris przewalskii ganzihonensis had underwent the ecological niche shift from salt water to freshwater, and also experienced a recent split from Gymnocypris przewalskii przewalskii. In addition, G. p. ganzihonensis inhabited harsh aquatic environment including low temperature and hypoxia as well as other Schizothoracinae species, its genetic mechanism of highland adaptation have yet to be determined.ResultsOur study used comparative genomic analysis based on the transcriptomic data of G. p. ganzihonensis and other four fish genome datasets to investigate the genetic basis of highland adaptation in Schizothoracine fish. We found that Schizothoracine fish lineage on the terminal branch had an elevated dN/dS ratio than its ancestral branch. A total of 202 gene ontology (GO) categories involved into transport, energy metabolism and immune response had accelerated evolutionary rates than zebrafish. Interestingly, we also identified 162 genes showing signature of positive selection (PSG) involved into energy metabolism, transport and immune response in G. p. ganzihonesis. While, we failed to find any PSG related to hypoxia response as previous studies.ConclusionsComparative genomic analysis based on G. p. ganzihonensis transcriptome data revealed significant genomic signature of accelerated evolution ongoing within Tibetan Schizothoracinae species lineage. Molecular evolution analysis suggested that genes involved in energy metabolism, transport and immune response functions in Schizothoracine fish underwent positive selection, especially in innate immunity including toll-like receptor signaling pathway genes. Taken together, our result as a case study in Schizothoracinae species provides novel insights in understanding the aquatic animal adaptation to extreme environment on the Tibetan Plateau, and also provides valuable genomic resource for further functional verification studies.

Profile of differentially expressed Toll-like receptor signaling genes in the natural killer cells of patients with Sézary syndrome.

Sézary syndrome (SS), an aggressive and leukemic form of cutaneous T-cell lymphoma, usually results in shortened survival. Improving innate immunity in SS by targeting natural killer (NK) cells with Toll-like receptor (TLR) agonists could be an interesting modulatory strategy. We evaluated the NK cell populations in SS patients assessing activating and inhibitory receptors expression and profiled the differential expression of TLR signaling pathway genes in unstimulated NK cells and after TLR7/8 stimulation. We observed preserved CD56bright NK cells and a low percentage of CD56dim NK cells in the peripheral blood of SS patients compared to those in the healthy control group. Both NK cell populations showed down-modulation of NKG2C and NKG2D expression, which was associated with high serum levels of the soluble form of NKG2D ligands. In contrast, an expansion of “memory” CD57+ NKG2C+ NK cells and high cytomegalovirus antibody titers were detected in SS patients. Profiling of the TLR signaling genes in NK cells from SS patients showed an abundance of differentially expressed genes (DEGs) in NK cells in the unstimulated condition, with mostly up-regulation of NFκB/JNK p38 pathway genes, but there was down-regulation of type I (IFN-α/β) and II (IFN-γ) interferon and IL-12A. After activation of NK cells with TLR7/8 agonist, the down-regulated genes correlated with the IFN response, and IL-12 became up-regulated, together with other antitumor factors. NK cell activation with a dual agonist for TLR7 and TLR8 is able to induce the expression of IFN-γ and type I IFN, which can improve immunity in SS patients.

Alternative pre-mRNA splicing of Toll-like receptor signaling components in peripheral blood mononuclear cells from patients with ARDS.

A key physiological feature of acute respiratory distress syndrome (ARDS) is inflammation. Toll-like receptor (TLR) signaling is required to combat the infection that underlies many ARDS cases but also contributes to pathological inflammation. Several TLR signaling pathway genes encoding positive effectors of inflammation also produce alternatively spliced mRNAs encoding negative regulators of inflammation. An imbalance between these isoforms could contribute to pathological inflammation and disease severity. To determine whether splicing in TLR pathways is altered in patients with ARDS, we monitored alternative splicing of MyD88 and IRAK1, two genes that function in multiple TLR pathways. The MyD88 and IRAK1 genes produce long proinflammatory mRNAs (MyD88L and IRAK1) and shorter anti-inflammatory mRNAs (MyD88S and IRAK1c). We quantified mRNA encoding inflammatory cytokines and MyD88 and IRAK1 isoforms in peripheral blood mononuclear cells (PBMCs) from 104 patients with ARDS and 30 healthy control subjects. We found that MyD88 pre-mRNA splicing is altered in patients with ARDS in a proinflammatory direction. We also observed altered MyD88 isoform levels in a second critically ill patient cohort, suggesting that these changes may not be unique to ARDS. Early in ARDS, PBMC IRAK1c levels were associated with patient survival. Despite the similarities in MyD88 and IRAK1 alternative splicing observed in previous in vitro studies, there were differences in how MyD88 and IRAK1 alternative splicing was altered in patients with ARDS. We conclude that pre-mRNA splicing of TLR signaling genes is altered in patients with ARDS, and further investigation of altered splicing may lead to novel prognostic and therapeutic approaches.

The expression profile of toll-like receptor signaling molecules in CD19+ B cells from patients with primary immune thrombocytopenia

BackgroundB cells play a critical role in the pathogenesis of immune thrombocytopenia (ITP), and toll-like receptor (TLR) signaling is essential for the activation of autoreactive B cells. The objective of this study was to investigate the expression profile of TLR signaling molecules in circulating and splenic CD19+ B cells isolated from ITP patients. MethodsCD19+ B cells were magnetically isolated from peripheral blood and splenocytes. Human Toll-Like Receptor Signaling Pathway RT2 Profiler™ PCR Array was used to determine the differences in mRNA expression of 84 TLR signaling pathway genes between ITP patients and controls. Flow cytometry was used to investigate intracellular expression of cytokines (IL-1β and IL-10). ResultsA total of 31 genes involving TLR signaling pathways were differentially transcribed in circulating CD19+ B cells, among which 27 were up-regulated in ITP. By comparison, differentially transcribed genes amounted to 39 in splenic B cells in ITP, among which only two were down-regulated. Up to 18 TLR signaling molecules exhibited up-regulated transcriptional levels both in splenic B cells and in circulating B cells in ITP. However, Only IL-10 and IL-1β were significantly upregulated in both the circulating and splenic B cells of patients with ITP. Intracellular staining of IL-10 and IL-1β confirmed the results of PCR Array. ConclusionsThe expression of TLRs and downstream cytokines, including IL-10 and IL-1β, is up-regulated in circulating and splenic B cells in ITP patients, suggesting that activated TLR signaling pathways in B cells may play dual roles in the pathophysiology of primary ITP.

Evolutionary patterns of Toll-like receptor signaling pathway genes in the Suidae

BackgroundThe Toll-like receptor (TLR) signaling pathway constitutes an essential component of the innate immune system. Highly conserved proteins, indicative of their critical roles in host survival, characterize this pathway. Selective constraints could vary depending on the gene’s position within the pathway as TLR signaling is a sequential process and that genes downstream of the TLRs may be more selectively constrained to ensure efficient immune responses given the important role of downstream genes in the signaling process. Thus, we investigated whether gene position influenced protein evolution in the TLR signaling pathway of the Suidae. The members of the Suidae examined included the European Sus scrofa (wild boar), Asian Sus scrofa (wild boar), Sus verrucosus, Sus celebensis, Sus scebifrons, Sus barbatus, Babyrousa babyrussa, Potamochoerus larvatus, Potamochoerus porcus and Phacochoerus africanus.ResultsA total of 33 TLR signaling pathway genes in the Suidae were retrieved from resequencing data. The evolutionary parameter ω (dn/ds) had an overall mean of 0.1668 across genes, indicating high functional conservation within the TLR signaling pathway. A significant relationship was inferred for the network parameters gene position, number of protein-protein interactions, protein length and the evolutionary parameter dn (nonsynonymous substitutions) such that downstream genes had lower nonsynonymous substitution rates, more interactors and shorter protein length than upstream genes. Gene position was significantly correlated with the number of protein-protein interactions and protein length. Thus, the polarity in the selective constraint along the TLR signaling pathway was due to the number of molecules a protein interacted with and the protein’s length.ConclusionResults indicate that the level of selective constraints on genes within the TLR signaling pathway of the Suidae is dependent on the gene’s position and network parameters. In particular, downstream genes evolve more slowly as a result of being highly connected and having shorter protein lengths. These findings highlight the critical role of gene network parameters in gene evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0602-7) contains supplementary material, which is available to authorized users.

Mapping toll-like receptor signaling pathway genes of Zhikong scallop (Chlamys farreri) with FISH

Toll-like receptor (TLR) signaling pathway plays a pivotal role in the innate immune system. Studies on TLR signaling pathway genes in Zhikong scallop (Chlamys farreri) have mainly focused on sequence analysis and expression profiling, no research has been carried out on their localization. The chromosomal position of TLR signaling pathway genes can be valuable for assemblying scallop genome and analysizing gene regulatory networks. In the present study, five key TLR signaling pathway genes (CfTLR, CfMyd88, CfTRAF6, CfNFκB, and CfIκB) containing bacterial artificial chromosomes (BACs) were isolated and physically mapped through fluorescence in situ hybridization on five non-homologous chromosome pairs, showing a similar distribution to another five model species. The isolation and mapping of these key immune genes of C. farreri will aid to the research on innate immunity, assignment of interested genes to chromosomes, and integration of physical, linkage and cytogenetic maps of this species.

In vitro screening of LPS-induced miRNAs in leukocytes derived from cord blood and their possible roles in regulating TLR signals

microRNAs (miRNAs) are involved in a wide variety of biological processes and play roles in the regulation of Toll-like receptor (TLR) signals. We hypothesized that lipopolysaccharide (LPS)-induced miRNAs in the leukocytes from cord blood (CB) play an important role in newborn innate immunity, specifically in the regulation of the TLR signaling pathway. The expression profiles of LPS-induced miRNAs and TLR-related genes were studied by microarray and PCR arrays, respectively. A bioinformatics analysis was used to identify the potential biological processes and targets involved in the TLR signals affected by these miRNAs. A total of 85 miRNAs and 41 TLR-related genes were differentially expressed. The bioinformatics analysis showed that the potential target genes of these miRNAs were involved in regulation of cellular biosynthetic process, regulation of gene expression, regulation of macromolecule biosynthetic process, etc. Thirteen potential miRNA-mRNA interaction sites were found within the cDNA sequences of 11 differentially expressed TLR signaling pathway genes. We identified a global miRNA expression signature occurring during LPS-induced acute inflammation in leukocytes derived from CB. The target genes were mainly involved in several biological processes, and these miRNAs may play important roles in the regulation of TLR signals. However, the precise mechanisms require further validation.

Variants in the Toll-interacting protein gene are associated with susceptibility to sepsis in the Chinese Han population

IntroductionDeregulated or excessive host immune responses contribute to the pathogenesis of sepsis. Toll-like receptor (TLR) signaling pathways and their negative regulators play a pivotal role in the modulation of host immune responses and the development of sepsis. The objective of this study was to investigate the association of variants in the TLR signaling pathway genes and their negative regulator genes with susceptibility to sepsis in the Chinese Han population.MethodsPatients with severe sepsis (n = 378) and healthy control subjects (n = 390) were enrolled. Five genes, namely TLR2, TLR4, TLR9, MyD88 and TOLLIP, were investigated for their association with sepsis susceptibility by a tag single nucleotide polymorphism (SNP) strategy. Twelve tag SNPs were selected based on the data of Chinese Han in Beijing from the HapMap project and genotyped by direct sequencing. The mRNA expression levels of TOLLIP were determined using real-time quantitative Polymerase Chain Reaction (PCR) assays, and concentrations of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA).ResultsOur results showed that the minor C-allele of rs5743867 in TOLLIP was significantly associated with the decreased risk of sepsis (Padj = 0.00062, odds ratio (OR)adj = 0.71, 95% confidence interval (CI) 0.59 to 0.86) after adjustment for covariates in multiple logistic regression analysis. A 3-SNP haplotype block harboring the associated SNP rs5743867 also displayed strong association with omnibus test P value of 0.00049. Haplotype GTC showed a protective role against sepsis (Padj = 0.0012), while haplotype GCT showed an increased risk for sepsis (Padj = 0.00092). After exposure to lipopolysaccharide (LPS), TOLLIP mRNA expression levels in peripheral blood mononuclear cells (PBMCs) from homozygotes for the rs5743867C allele were significantly higher than in heterozygotes and homozygotes for the rs5743867T allele (P = 0.013 and P = 0.01, respectively). Moreover, the concentrations of TNF-α and IL-6 in culture supernatants were significantly lower in the subjects of rs5743867CC genotype than in CT and TT genotype subjects (P = 0.016 and P = 0.003 for TNF-α; P = 0.01 and P = 0.002 for IL-6, respectively).ConclusionsOur findings indicated that the variants in TOLLIP were significantly associated with sepsis susceptibility in the Chinese Han population.