SESN1 functions as a new tumor suppressor gene via Toll-like receptor signaling pathway in neuroblastoma.

AIMS: Neuroblastoma (NB) is the most common extracranial solid tumor in children, with a 5-year survival rate of <50% in high-risk patients. MYCN amplification is an important factor that influences the survival rate of high-risk patients. Our results indicated MYCN regulates the expression of SESN1. Therefore, this study aimed to investigate the role and mechanisms of SESN1 in NB. METHODS: siRNAs or overexpression plasmids were used to change MYCN, SESN1, or MyD88's expression. The role of SESN1 in NB cell proliferation, migration, and invasion was elucidated. Xenograft mice models were built to evaluate SESN1's effect in vivo. The correlation between SESN1 expression and clinicopathological data of patients with NB was analyzed. RNA-Seq was done to explore SESN1's downstream targets. RESULTS: SESN1 was regulated by MYCN in NB cells. Knockdown SESN1 promoted NB cell proliferation, cell migration, and cell invasion, and overexpressing SESN1 had opposite functions. Knockdown SESN1 promoted tumor growth and shortened tumor-bearing mice survival time. Low expression of SESN1 had a positive correlation with poor prognosis in patients with NB. RNA-Seq showed that Toll-like receptor (TLR) signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway in cancer were potential downstream targets of SESN1. Knockdown MyD88 or TLRs inhibitor HCQ reversed the effect of knockdown SESN1 in NB cells. High expression of SESN1 was significantly associated with a higher immune score and indicated an active immune microenvironment for patients with NB. CONCLUSIONS: SESN1 functions as a new tumor suppressor gene via TLR signaling pathway in NB.

Short chain fatty acids inhibit corneal inflammatory responses to TLR ligands via the ocular G-protein coupled receptor 43.

PURPOSE: Short chain fatty acids (SCFAs) produced by gut microbiota are known to play primary roles in gut homeostasis by immunomodulation partially through G-protein coupled receptors (GPR) 43. Using mouse models of TLR ligand induced keratitis, we investigated whether SCFAs and GPR43 play any regulatory roles in the pathogenesis of inflammatory responses in the eye. METHODS: Both human and mouse eyes were labeled with a specific antibody for GPR43 and imaged by a laser scanning confocal microscope. Corneal cups from naïve C57BL/6J (B6) and GPR43 knockout (KO) mice were stimulated with TLR ligands in the presence or absence of sodium butyrate overnight and then processed for RT-PCR assay for expression of GPR43 and cytokines. Keratitis was induced by Poly I:C in wild type (WT) B6, GPR43KO and chimeric mice and the disease severity was evaluated by the corneal fluorescein staining test, and infiltrating cell staining and calculating in corneal whole mount. RESULTS: GPR43 is expressed in both human and mouse eyes and the expression is bidirectionally regulated by TLR ligands and butyrate. Butyrate significantly inhibited inflammation caused by several TLR ligands such as Poly I:C, Flagellin, and CpG-ODN (TLR-3, 5 and 9 agonists, respectively) in WT, but not GPR43KO, mice. Butyrate inhibition of TLR-induced keratitis is mediated by the GPR43 expressed in tissue but not hematopoietic, cells. CONCLUSIONS: This is the first report to demonstrate of the protective effect of SCFAs on microbial keratitis, and the dynamic expression and anti-inflammatory function of GPR43 in the eye. SCFAs can modulate inflammation and immunity in the eye through GPR43.

The expression of Nrf2 and TLRs in ear effusion in children with different types of otitis media and their relationship with inflammatory factors.

OBJECTIVE: This study aimed to analyze the differences in the expression of Toll-like receptors (TLRs) and nuclear factor erythroid 2-related factor 2 (Nrf2) in ear effusion in children with different types of otitis media (OM), to elaborate the relationship between the expression of TLRs and Nrf2 in ear effusion and the pathogenesis of OM, and to explore the relationship between the two indicators and pro-inflammatory cytokines in children with OM, thereby laying a scientific foundation for revealing the underlying molecular mechanisms of the progression of different types of OM. METHODS: A total of 73 children with OM who were treated in our hospital from March 2019 to July 2021 were selected as the study subjects. By using the cross-sectional investigation method, participants were divided into three groups according to the different pathological types, including the secretory OM group (30 cases), the chronic suppurative OM group (27 cases), and the cystic lesional OM group (16 cases). The levels of Nrf2, TLR2, TLR4 and proinflammatory cytokines [interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), transforming growth factor-ß 1(TGF-ß1), procalcitonin (PCT) and interleukin-1ß (IL-1ß)] were detected in ear effusion of children with different types of OM. Linear regression was used to analyze the correlation between the Nrf2, TLR2 and TLR4 expression levels and pro-inflammatory cytokines in ear effusion. RESULTS: The expression levels of TNF-α and PCT in the ear effusion of the children under 3 years old were significantly higher than that of the children between 3 and 5 years old and that of the children between 6 and 8 years old (all P < 0.001). The mRNA levels of Nrf2, TLR2 and TLR4 in the ear effusion of the children from the chronic suppurative OM group were higher than these from the secretory OM group (P < 0.001, P = 0.008 and P = 0.021). The mRNA levels of Nrf2, TLR2, and TLR4 in the ear effusion of the children from the cystic lesional OM group were higher than those from the chronic suppurative OM group (P < 0.001, P = 0.029 and P = 0.018). A prominent increase in the concentrations of IFN-γ, TNF-α, TGF-ß1, PCT and IL-1ß was found in the ear effusion of children from the chronic suppurative OM group compared to these from the secretory OM group (P = 0.021, P = 0.044, P = 0.048, P = 0.004 and P = 0.001). The concentrations of IFN-γ, TNF-α, TGF-ß1, PCT and IL-1ß in the ear effusion of the children from the cystic lesional OM group were markedly increased as compared with these from the chronic suppurative OM group (P < 0.001, P = 0.004, P = 0.003, P < 0.001 and P < 0.001). Nrf2, TLR2 and TLR4 were taken as independent variables, and inflammatory indexes, including IFN-γ, TNF-α, TGF-ß1, PCT and IL-1ß were used as dependent variables for the linear regression analysis. The results showed that Nrf2, TLR2 and TLR4 were positively correlated with the secretion levels of pro-inflammatory cytokines after adjusting for age, sex, course and the OM classification (all P < 0.05). CONCLUSION: The expressions of Nrf2, TLR2 and TLR4 in the ear effusion of children with different types of OM gradually increased with the severity of the disease, these were significantly positively correlated with the pro-inflammatory cytokines of the children. Nrf2/TLR signaling pathway maintained chronic inflammation in OM, induced damage of middle ear tissue, and promoted the transition from acute OM to chronic OM.

The inflammatory response to birth requires MyD88 and is driven by both mother and offspring.

Birth is an inflammatory event for the newborn, characterized by elevations in interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α peripherally and/or centrally, as well as changes in brain microglia. However, the mechanism(s) underlying these responses is unknown. Toll-like receptors (TLRs) play crucial roles in innate immunity and initiate inflammatory cascades upon recognition of endogenous or exogenous antigens. Most TLR signaling depends on the adaptor molecule myeloid differentiation primary response 88 (MyD88). We independently varied MyD88 gene status in mouse dams and their offspring to determine whether the inflammatory response to birth depends on MyD88 signaling and, if so, whether that signaling occurs in the offspring, the mother, or both. We find that the perinatal surges in plasma IL-6 and brain expression of TNF-α depend solely on MyD88 gene status of the offspring, whereas postnatal increases in plasma IL-10 and TNF-α depend on MyD88 in both the pup and dam. Interestingly, MyD88 genotype of the dam primarily drives differences in offspring brain microglial density and has robust effects on developmental neuronal cell death. Milk cytokines were evaluated as a possible source of postnatal maternal influence; although we found high levels of CXCL1/GROα and several other cytokines in ingested post-partum milk, their presence did not require MyD88. Thus, the inflammatory response previously described in the late-term fetus and newborn depends on MyD88 (and, by extension, TLRs), with signaling in both the dam and offspring contributing. Unexpectedly, naturally-occuring neuronal cell death in the newborn is modulated primarily by maternal MyD88 gene status.

Intracellular Toll-Like Receptors Modulate Adaptive Immune Responses in Head and Neck Cancer.

The percentage of head and neck cancer (HNC) positive for human papillomavirus (HPV) is unknown in most parts of India. How toll-like receptors (TLRs) affect the adaptive immune response in HNC is also mainly unknown. We here assessed the expressions of HPV DNA, p16, inflammation, and TLRs in oral squamous cell carcinoma (OC) and oropharyngeal squamous cell carcinoma (OPC). Patients with OC (n = 31) and OPC (n = 41), diagnosed during 2017-2018 at the Malabar Cancer Centre (tertiary cancer center), Kerala, India, were included in the study. Immunohistochemistry was performed on tumor specimens against p16, TLR3, TLR7, TLR8, TLR9, CD4, and CD8. Quantitate polymerase chain reaction for 14 high-risk HPVs (HPV16/18/31/33/35/39/45/51/52/56/58/59/66/68) was performed. Seven out of 31 OC (22.6%) were p16+ but only 3.2% (1/31) of OC were positive for HPV DNA. While 24.4% (10/41) of OPC were p16+, HPV DNA was found in only one P16+ OPC and in no P16- OPC. TLR3, TLR7, TLR8, and TLR9 were expressed both in OC and in OPC. The expression of TLR7 was significantly higher in OPC compared with OC. TLR8 expression was correlated with and TLR7 tended to be correlated with the inflammatory score in OPC (r = 0.56, p < 0.05 and r = 0.52, p = 0.08, respectively). In conclusion, the role of HPV in OC and OPC is minor, and p16 constitutes a poor biomarker for HPV positivity in Kerala, India. Intracellular TLRs are correlated with the degree of inflammation in OPC but not in OC and may potentially constitute a medical target in the therapy of HNC in the future.

[Effect of Pp2cm Gene Silencing on Mouse Macrophage Resistance Against Staphylococcus aureus Infection via TLR Pathway].

Objective: To investigate the effect of silencing protein phosphatase 2cm ( Pp2cm) gene on the expression of inflammatory factors in macrophages infected with Staphylococcus aureus ( S. aureus) and the mechanisms involved. Methods: The effects of Pp2cm knockdown on inflammatory factors, proliferation, apoptosis, and Toll-like receptor (TLR) signaling were analyzed in RAW 264.7 cells, a murine macrophage cell line, transfected with adenovirus (Ad). The cells were divided into four groups, including Ad-Ctrl group, Ad- Pp2cm group, Ad-Ctrl+ S. aureus group and Ad- Pp2cm+ S. aureus group. Cell transfection was achieved by separately introducing control adenovirus (Ad-Ctrl) or adenovirus targeting the Pp2cm gene (Ad- Pp2cm) and inflammation or the absence of inflammation was induced by applying or not applying S. aureus. The expression of tumor necrosis factor-alpha ( TNF-α), interleukin-1ß ( IL-1 ß), TLR2, TLR4, Toll-like receptor adaptor protein ( Tirap) and myeloid differentiation factor 88 ( Myd88) was determined by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). PP2Cm protein expression was determined by Western blot. Cell proliferation was determined by cell counting kit-8 (CCK-8) assay and cell apoptosis was measured by flow cytometry. Results: The expression of Pp2cmgene and PP2Cm protein was downregulated in the Ad- Pp2cm group when compared to the Ad-Ctrl group, with the diference showing statistical significance ( P<0.05). When compared to those of the Ad-Ctrl+ S. aureus group, macrophages in the Ad- Pp2cm+ S. aureus group showed significantly increase in the TNF- α and IL-1 ß gene levels ( P<0.01). Furthermore, the Ad- Pp2cm group demonstrated elevated gene expression levels of TLR2, TLR4, Tirap and Myd88 in macrophages when compared to the Ad-Ctrl group, with the difference showing statistical significance ( P<0.05). There were no statistically significant differences in cell apoptosis and proliferation between the Ad-Ctrl and Ad- Pp2cm groups. Conclusions: Silencing Pp2cm gene promotes the inflammatory response of macrophages to S. aureus infection. Moreover, the TLR pathway plays an important role in the inflammatory activation of macrophages.

The role of CD14 and CSF1R in osteoarthritis and gastritis.

Osteoarthritis (OA) is a non-inflammatory degenerative joint disease that mainly involves articular cartilage damage and involves the whole joint tissue. Gastritis is a common stomach disorder, typically referring to inflammation or lesions of the gastric mucosa. However, the relationship between CD14 and colony stimulating factor-1 receptor (CSF1R) and these 2 diseases is not yet clear. OA datasets GSE46750, GSE82107 and gastritis datasets GSE54043 profiles were downloaded from gene expression omnibus databases generated by GPL10558 and GPL570.The R package limma was used to screen differentially expressed genes (DEGs). Weighted gene co-expression network analysis was performed. The construction and analysis of protein-protein interaction network, functional enrichment analysis, gene set enrichment analysis and comparative toxicogenomics database analysis were performed. TargetScan was used to screen miRNAs regulating central DEGs. A total of 568 DEGs were identified. According to the gene ontology (GO) and biological processes analysis, they were mainly enriched in ATP metabolism negative regulation, toll-like receptor TLR1:TLR2 signaling pathway, and intracellular transport. The enrichment terms for OA and gastritis were similar to the GO and Kyoto encyclopedia of gene and genome enrichment terms of DEGs, mainly enriched in ATP metabolism negative regulation, secretion granules, transmembrane receptor protein kinase activity, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, MAPK signaling pathway, and TGF-ß signaling pathway. In the Metascape enrichment projects, GO enrichment projects showed functions related to cell-cell receptor interaction, cell secretion, and growth. Two core genes were identified through the construction and analysis of the protein-protein interaction network. The core genes (CD14 and CSF1R) exhibited high expression in OA and gastritis samples and low expression in normal samples. Comparative toxicogenomics database analysis revealed associations between core genes (CD14 and CSF1R) and diseases such as OA, osteoporosis, gastritis, juvenile arthritis, diarrhea, and inflammation. CD14 and CSF1R are highly expressed in OA and gastritis, making them potential therapeutic targets for both diseases.

Toll-like receptor 9 signaling in chronic lymphocytic leukemia cell lines.

Chronic lymphocytic leukemia (CLL) is a prototypic neoplasia in which malignant cells strongly depend on microenvironmental stimulations in the lymphoid tissues where they accumulate; leukemic cells are exposed to interaction with bystander and accessory cells, as well as inflammatory soluble mediators. Cell lines are frequently used to model the pathobiology of this disease; however, they do not always recapitulate leukemic cell growth and response to stimulation, and no data are available on Toll-like receptors (TLR) signaling in CLL cell lines. To address this gap, we analyzed HG3, MEC2, and PCL12 cell lines, before and after CpG stimulation, by RNA-sequencing followed by bioinformatic analyses and validation experiments. We identified NFKBIZ mRNA and the corresponding IkBz protein as robust markers of TLR9 activation in both MEC2 and PCL12, but not in HG3 cells. Next, we compared our current results with previous results obtained with primary CLL patient samples and were able to conclude that MEC2 is most similar to the patients' cells in terms of global responsiveness to TLR stimulation; in particular, MEC2 better resembles the samples of patients, as it is characterized by high expression levels of IkBz, but with a lower number of genes regulated.

Multiple toll-like receptors (TLRs) display differential bacterial and ligand specificity in the earthworm, Eisenia andrei.

Toll-like receptors (TLRs), an ancient and well-conserved group of pattern recognition receptors (PRRs), recognize conserved pathogen-associated molecular patterns. TLRs consist of three domains: the extracellular N-terminal domain, containing one or more leucine-rich repeats (LRRs), responsible for the recognizing and binding of antigens; the type-I transmembrane domain; and the intracellular domain known as the Toll/Interleukin-1 receptor (TIR) domain required for the downstream signaling pathway. We identified six new full-length complementary DNA (cDNA) sequences, Ean-TLR1/2/3/4/5/6. The deduced amino acid sequences indicate that Ean-TLRs consist of one signal peptide, one LRR N-terminal domain (Ean-TLR4/5), varying numbers of LRRs, one (Ean-TLR1/2/3/4/5) or two (Ean-TLR6) LRR C-terminal domains, one type-I transmembrane domain, and a TIR domain. In addition, a TIR domain alignment revealed that three conserved motifs, designated as Box 1, Box 2, and Box 3, contain essential amino acid residues for downstream signaling activity. Phylogenetic analysis of earthworm TLRs generated two separate evolutionary branches representing single (sccTLR) and multiple (mccTLR) cysteine cluster TLRs. Ean-TLR1/2/3/4 (sccTLR type) and Ean-TLR6 (mccTLR type) were clustered with corresponding types of previously reported earthworm TLRs as well as TLRs from Clitellata and Polychaete. As PRRs, earthworm TLRs should be capable of sensing a diverse range of pathogens. Except for Ean-TLR3, which was not responsive to any bacteria, earthworm TLR expression was significantly induced by Gram-positive but not Gram-negative bacteria. Moreover, it is likely that earthworms can differentiate between different species of Gram-positive bacteria via their TLR responses. The ligand specificity of earthworm TLRs suggests that their pathogenic ligand recognition is likely to be as specific and diverse as the mammalian TLR pathogen-sensing system.

Methods to Study TLRs in Transplantation.

Toll-like receptors (TLRs) are key regulators of immune responses, including alloimmune responses. In this chapter, we present protocols to study whether and/or how TLRs can contribute to solid-organ transplant rejection. We describe methods to reduce heterogeneity in microbiome variations between animals before beginning experiments to limit confounding factors, protocols using TLR agonists to prevent anti-CD154/donor splenocyte transfer-mediated tolerance, and recipes to heat-kill microbes or use hosts genetically deficient in TLR-dependent pathways to distinguish between TLR-dependent and live bacteria-dependent effects.

Delineating the Role of Toll-Like Receptors in Inflammatory Bowel Disease.

Toll-like receptors (TLRs) recognize altered gut microbiota triggering an immune response. These responses play a critical role in the pathogenesis and treatment of inflammatory bowel disease (IBD). IBD is characterized by inflammation of the intestinal tracts as in Crohn's disease and ulcerative colitis. However, one challenge in determining the role of a specific TLR in IBD and its underlying mechanism is disparity. Variance in age, gender, race, and ethnicity shows a dramatic difference in the disease incidence, severity, and response to treatment. Delineating the role of TLRs in IBD relies on both a knockout mouse and a disease model. Here, we describe a detailed protocol on how to use nearly identical genetic backgrounds of TLR wild-type and knockout littermate mice in a dextran sodium sulfate (DSS)-induced colitis model.

Ancient fish lineages illuminate toll-like receptor diversification in early vertebrate evolution.

Since its initial discovery over 50 years ago, understanding the evolution of the vertebrate RAG- mediated adaptive immune response has been a major area of research focus for comparative geneticists. However, how the evolutionary novelty of an adaptive immune response impacted the diversity of receptors associated with the innate immune response has received considerably less attention until recently. Here, we investigate the diversification of vertebrate toll-like receptors (TLRs), one of the most ancient and well conserved innate immune receptor families found across the Tree of Life, integrating genomic data that represent all major vertebrate lineages with new transcriptomic data from Polypteriformes, the earliest diverging ray-finned fish lineage. Our analyses reveal TLR sequences that reflect the 6 major TLR subfamilies, TLR1, TLR3, TLR4, TLR5, TLR7, and TLR11, and also currently unnamed, yet phylogenetically distinct TLR clades. We additionally recover evidence for a pulse of gene gain coincident with the rise of the RAG-mediated adaptive immune response in jawed vertebrates, followed by a period of rapid gene loss during the Cretaceous. These gene losses are primarily concentrated in marine teleost fish and synchronous with the mid Cretaceous anoxic event, a period of rapid extinction for marine species. Finally, we reveal a mismatch between phylogenetic placement and gene nomenclature for up to 50% of TLRs found in clades such as ray-finned fishes, cyclostomes, amphibians, and elasmobranchs. Collectively, these results provide an unparalleled perspective of TLR diversity and offer a ready framework for testing gene annotations in non-model species.

Evaluation of the Impact of Some Single-Nucleotide Gene Polymorphisms on the Development of Adenomatous Polyps of the Colon in Patients with Irritable Bowel Syndrome.

BACKGROUND: The number of cases of irritable bowel syndrome is growing worldwide, in which adenomatous polyps can develop as a result of microinflammation of the colonic epithelium. Our study was aimed at the identification of the possible effect of single-nucleotide polymorphisms on the risk of the development of irritable bowel syndrome-related colonic adenomatous polyps. MATERIALS AND METHODS: The study involved 187 irritable bowel syndrome patients. The single-nucleotide polymorphisms were investigated by the polymerase chain reaction method and DNA was extracted with the phenol-chloroform: interleukin-1ß gene-31C/T (rs1143627), -511C/T (rs16944); interleukin-6 gene-174G/C (rs1800795); interleukin-10 gene-592C/A (rs1800872), -819T/C (rs1800871), -1082A/G (rs1800896); Toll-like receptor-2 gene Arg753Gln (rs5743708); Toll-like receptor-4 gene Thr399ile (rs4986791), Asp299Gly (rs4986790); and metalloproteinase-9 gene-8202A/G (rs11697325). The study of polymorphic loci was checked for compliance with the Hardy- Weinberg equilibrium using Fisher's exact test along with the analyses of the frequency of alleles and the genotypes. RESULTS: The association of diseases with G allele Toll-like receptor-2 gene Arg753Gln (rs5743708) was revealed in irritable bowel syndrome patients with adenomatous polyps of the colon (P < .0006) and AG single-nucleotide polymorphisms s of Toll-like receptor-2 gene (χ2 = 12.78, P < .002); A allele had a protective effect. The AG genotype metalloproteinase-9 gene-8202A/G (rs11697325) polymorphism in irritable bowel syndrome patients with adenomatous polyps of the colon had a protective effect (P < .05). AA genotype interleukin-10 gene-1082A/G (rs1800896) polymorphism in the irritable bowel syndrome patient (χ2 = 33.97, 4.0E-8) can be considered as the risk for adenomatous polyps of the colon in irritable bowel syndrome. CONCLUSION: G allele Toll-like receptor-2 gene Arg753Gln (rs5743708) and AA genotype interleukin-10 gene-1082A/G (rs1800896) polymorphisms can be the marker of the emergence of adenomatous polyps of the colon concomitant with irritable bowel syndrome.

Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies.

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage.

Gene expression of Toll-like receptors, cytokines and a nuclear factor and cytokine secretion in DH82 canine macrophage cells infected with Brucella canis.

Canine brucellosis caused by Brucella canis infection occurs mainly in dogs, and is a zoonotic disease that also has the possibility of infection in humans. Many studies have been conducted to understand the immunopathological mechanism of B. canis infection. However, the precise immune mechanism remains to be elucidated because compared to other Brucella spp., B. canis has different immune evasion mechanisms. In this study, gene expression levels of Toll-like receptors (TLRs) and TLR-associated molecules and cytokine production were analyzed to figure out the roles of immune-related host factors in B. canis infection. Time-dependent gene expression of TLRs (1-10) and TLR-related molecules (TNF-α, IL-5, IL-23, CCL4, CD40 and NFκ-B) and release of Th1, Th2 and Th17-related cytokines (IFN-γ, IL-1ß, IL-4, IL-6, IL-10 and IL-17A) were investigated in DH82 canine macrophages infected with B. canis. Time-dependent induction of TLRs 3, 7 and 8 was observed, and TLR 7 had the highest expression level (p <0.05). The expression levels of all TLR-related genes were significantly increased after infection. In particular, the expression of the CCL4 and IL-23 genes was highly induced. The amounts of IL-1ß, IL-6 and IL-10 were significantly increased by B. canis infection, but the amounts of IL-4 and IL-17A were not. The production of IL-1ß and IL-6 was the highest at 24 hr after B. canis infection (p <0.05). This study demonstrates that TLRs 3, 7 and 8 are prominent sites of to immune response induction with the production of related cytokines and a nuclear factor in DH82 cells infected with B. canis. These results suggest a sequential immune mechanism of B. canis infection, involving TLRs, cytokines and their associated factors.

Nutrigenomic profiling of reduced specification diets and phytogenic inclusion effects on critical toll-like receptor signaling, mitogen-activated protein kinase-apoptosis, and PI3K-Akt-mTOR gene components along the broiler gut.

The effects of concurrent reduction of dietary metabolizable energy (ME) and crude protein (CP) levels combined or not with the dietary inclusion of a phytogenic feed additive (PFA) were studied using a nutrigenomics approach. In particular, the expression of 26 critical genes relevant for inflammation control (TLR pathway), cellular apoptosis (MAPK pathway) cell growth and nutrient metabolism (PI3K-Akt-mTOR pathway) was profiled along the broiler intestine. Two dietary types (L and H) differing in metabolizable energy and crude protein levels (L: 95% and H: 100% of optimal Cobb 500 recommendations for ME and CP requirements) supplemented or not with PFA (- or +) and their interactions (L-, L+, H-, H+) were evaluated. There were only 3 total interactions (mTOR, IL8, and HRAS P < 0.05) between diet type and PFA inclusion indicating limited concurrent effects. Diet type, L upregulated genes related with inflammation mainly in the jejunum, ileum, and cecum (P < 0.05) and MAPK pathway in the ileum and cecum (P < 0.05). Moreover, diet type L negatively affected the expression of genes related to PI3K-Akt-mTOR pathway mainly in duodenum and cecum (P < 0.05). On the other hand, PFA inclusion downregulated (P < 0.05) genes related with TLR signaling pathway (TLR2B, MyD88, TLR3, IL8, LITAF) along the intestine and MAPK pathway genes (APO1, FOS) in jejunum (P < 0.05). Finally, PFA supplementation regulated nutrient sensing and metabolism in the cecum in a manner perceived as beneficial for growth. In conclusion, the study results highlight that the reduced ME and CP specifications, especially in the absence of PFA, regulate inflammation, apoptosis and nutrient metabolism processes at homeostatic control levels that hinder maximizing the availability of dietary energy and nutrients for growth purposes. Inclusion of PFA helped to adjust the respective homeostatic responses and control to levels supporting broiler performance, especially at reduced specification diets.

Upregulation of TLR5 indicates a favorable prognosis in prostate cancer.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Influence of genetic variability in toll-like receptors (TLR 2, TLR 4, and TLR 9) on human immunodeficiency virus-1 disease progression.

Background: It has been demonstrated that toll-like receptors (TLR2), TLR4, and TLR9 which were initially known for recognizing bacterial products are involved in the detection of viral components. It was planned to undertake a prospective longitudinal study among ethnically homogeneous antiretroviral treatment and antitubercular treatment naïve human immunodeficiency virus (HIV)-positive patients representing the north Indian population. The aim of the study was to investigate the influence of TLR2, TLR4, and TLR9 polymorphism in HIV disease progression. Methods: The present study was designed to investigate genetic polymorphism in TLRs (TLR2, TLR4, and TLR9) among HIV-infected patients with and without TB coinfection. The study population consisted of two groups: (i) HIV-positive patients without TB infection and disease (n = 223, HIV-positive patients); (ii) HIV-positive patients with latent tuberculosis infection (LTBI) (n = 150, HIV-positive LTBI patients). These participants were of either gender between 18 and 60 years of age and treatment naïve for both TB and HIV. HIV-positive and HIV-positive LTBI patients were longitudinally followed up for t2 years to study HIV disease progression. Results: On comparing TLR2 and TLR4 allelic and genotypic frequencies between 306 HIV-positive patients (no TB/AIDS) and 47 HIV-positive patients progressed to active TB/AIDS, no significant difference was observed between the two groups. The frequency of "A" allele in TLR9 was found to be significantly increased in 47 HIV-positive patients who progressed to active TB/AIDS (61.7%) as compared to 42.16% in 306 HIV-positive patients (no TB/AIDS), (P < 0.001). Furthermore, a significantly increased frequency of "AA" genotype in TLR9 was observed in 47 HIV-positive patients progressed to active TB/AIDS (55.32%) as compared to 20.26% in HIV-positive patients (no TB/AIDS). Conclusion: Findings of the present study revealed that genetic variability in TLR9 may influence HIV disease progression. The AA genotype in TLR9 may be associated with progression to TB/AIDS for 2 years in HIV-positive patients.

Screening and Bioinformatics Analyses of Key miRNAs Associated with Toll-like Receptor Activation in Gastric Cancer Cells.

Background and Objectives: To screen key miRNAs and their target genes related to Toll-like receptor (TLR) activation in gastric cancer (GC) cells and analyze them bioinformatically. Materials and Methods: Venn diagrams were obtained to screen miRNAs that were upregulated/downregulated in both GSE54129 and GSE164174. The miRTarBase database was used to predict the target genes of upregulated miRNAs. The differentially expressed genes in the regulatory network were analyzed. miR-16-5p expression in different tissue samples and the variations in the methylation states of four hub genes were measured. Results: We found that GSE54129 included 21 normal gastric tissues and 111 gastric cancer tissues, GSE164174 included 1417 normal gastric tissues and 1423 gastric cancer tissues. Venn diagram analysis results showed that compared with the control group, a total of 68 DEmiRNAs were upregulated in the GSE54129 and GSE164174 datasets, and no common downregulated DEmiRNAs were found. On further analysis of the GSE108345 dataset, we obtained the competing endogenous RNA (ceRNA) network associated with the activation of TLRs, and listed the top 10 lncRNA-miRNA-mRNA networks, including 10 miRNAs, 86 mRNA and 134 lncRNAs. Cytological HuBBA scores yielded a total of 1 miRNA, 16 mRNAs and 45 lncRNAs, of which miR-16-5p scored the highest as it was considered a key miRNA for TLR activation in GC cells, which are important in response against microorganisms. The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that endocytosis, microRNAs in cancer and the PI3K-Akt signaling pathway are related to TLR signaling. The results of in vivo experiments indicated that miR-16-5p was highly expressed in gastric cancer cells and tissues. Conclusions: Hsa-miR-16-5p's target genes mainly play a role by regulating the expression of four genes-MCL1, AP2B1, LAMB1, and RAB11FIP2. The findings provide a scientific basis for the development of immunotherapy for GC.

Toxoplasma gondii microneme protein MIC3 induces macrophage TNF-α production and Ly6C expression via TLR11/MyD88 pathway.

Toxoplasma gondii is the most successful parasite worldwide. It is of great interest to understand how T. gondii induce different immune responses in different hosts. In this study, we found that a peptide of T. gondii microneme protein MIC3 induced TNF-α production, NF-κB phosphorylation, iNOS transcription and Ly6C expression in mouse macrophage RAW264.7 cells. MyD88 inhibition, small interfering RNA against Tlr11 and CRISPR/Cas9-mediated knock-out of Tlr11 all reduced MIC3-induced TNF-α production, NF-κB phosphorylation, iNOS transcription and Ly6C expression. Additionally, we determined the location of MIC3 peptide in mouse macrophages using immunofluorescence. MIC3 could both adhere to the cell membrane of mouse macrophages and enter the cells. These results suggest that MIC3 triggered the immune responses in mouse macrophages via TLR11/MyD88/NF-κB pathway. It is known that human macrophages lacking TLR11. We predicted that the immune responses induced by MIC3 in human macrophages were significantly different from those in mouse macrophages. As expected, MIC3 peptide failed to induce TNF-α expression, iNOS expression and NF-κB phosphorylation in human THP-1 derived macrophages. MIC3 induced macrophage immune responses via TLR11. Intriguingly, the amino acid sequence of MIC3 is completely different from the well-known TLR11 ligand profilin, which generates a potent IL-12p40, TNF-α and IL-6 response. In marked contrast to profilin, MIC3 could not induce IL-12p40 expression in both mouse RAW264.7 cells and human THP-1 derived macrophages. Furthermore, the simulated tertiary structure of MIC3 peptide shows poor similarity with the crystal structure of profilin, suggesting that MIC3 might be a different ligand from profilin. These findings about MIC3 and TLR11 will provide us with important insights into the pathogenesis of toxoplasmosis and coevolution during host-parasite interaction.