Gene Expression Of Toll-like Receptors Research Articles

Protocatechuic acid and quercetin attenuate ETEC-caused IPEC-1 cell inflammation and injury associated with inhibition of necroptosis and pyroptosis signaling pathways

BackgroundNecroptosis and pyroptosis are newly identified forms of programmed cell death, which play a vital role in development of many gastrointestinal disorders. Although plant polyphenols have been reported to protect intestinal health, it is still unclear whether there is a beneficial role of plant polyphenols in modulating necroptosis and pyroptosis in intestinal porcine epithelial cell line (IPEC-1) infected with enterotoxigenic Escherichia coli (ETEC) K88. This research was conducted to explore whether plant polyphenols including protocatechuic acid (PCA) and quercetin (Que), attenuated inflammation and injury of IPEC-1 caused by ETEC K88 through regulating necroptosis and pyroptosis signaling pathways.MethodsIPEC-1 cells were treated with PCA (40 μmol/L) or Que (10 μmol/L) in the presence or absence of ETEC K88.ResultsPCA and Que decreased ETEC K88 adhesion and endotoxin level (P < 0.05) in cell supernatant. PCA and Que increased cell number (P < 0.001) and decreased lactate dehydrogenases (LDH) activity (P < 0.05) in cell supernatant after ETEC infection. PCA and Que improved transepithelial electrical resistance (TEER) (P < 0.001) and reduced fluorescein isothiocyanate-labeled dextran (FD4) flux (P < 0.001), and enhanced membrane protein abundance of occludin, claudin-1 and ZO-1 (P < 0.05), and rescued distribution of these tight junction proteins (P < 0.05) after ETEC infection. PCA and Que also declined cell necrosis ratio (P < 0.05). PCA and Que reduced mRNA abundance and concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8 (P < 0.001), and down-regulated gene expression of toll-like receptors 4 (TLR4) and its downstream signals (P < 0.001) after ETEC infection. PCA and Que down-regulated protein abundance of total receptor interacting protein kinase 1 (t-RIP1), phosphorylated-RIP1 (p-RIP1), p-RIP1/t-RIP1, t-RIP3, p-RIP3, mixed lineage kinase domain-like protein (MLKL), p-MLKL, dynamin- related protein 1 (DRP1), phosphoglycerate mutase 5 (PGAM5) and high mobility group box 1 (HMGB1) (P < 0.05) after ETEC infection. Moreover, PCA and Que reduced protein abundance of nod-like receptor protein 3 (NLRP3), nod-like receptors family CARD domain-containing protein 4 (NLRC4), apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D (GSDMD) and caspase-1 (P < 0.05) after ETEC infection.ConclusionsIn general, our data suggest that PCA and Que are capable of attenuating ETEC-caused intestinal inflammation and damage via inhibiting necroptosis and pyroptosis signaling pathways.

Effect of Royal Jelly on Gene Expression of Toll-like Receptors 1-9 in Patients with Hepatitis B.

By damaging the liver, hepatitis B can result in acute and chronic diseases, such as cirrhosis or hepatocellular carcinoma. Viable treatments for such diseases using natural products and determinative biomarkers have been proposed but require evaluation to improve their effects. Therefore, this study aims to examine how effectively a specific natural product (namely, royal jelly) protects the body from the copy number of the virus, as well as TLR1 to TLR9 gene expressions. The effectiveness of royal jelly was tested by giving it (orally) to 30 hepatitis B patients for one month. HBV copy number and mRNA levels of TLRs were explored using Real Time PCR technique, and liver enzymes were evaluated too. Orally treatment with royal jelly led to a significant decrease in HBV-DNA copy number, down-regulation of TLR2 and TLR8, and up-regulation of TLR3. However, mRNA levels of the TLRs were not altered in the female, while TLR1, TLR2, and TLR5 were significantly decreased in the male participants. It seems that royal jelly has anti-viral and anti-inflammatory roles in the in vivo conditions in a dependent manner in TLR3, TLR2, and TLR8. Therefore, it can be suggested as a safe complementary agent for patients with hepatitis B.

Determining Endosomal Toll-Like Receptors Gene Expression in NK Cells After Stimulation with Specific Agonists.

Poor knowledge is currently available about the biology of Toll-like receptors (TLRs) in natural killer (NK) cells. This is particularly due to the old belief that NK cells are unable to specifically eliminate microbes without presensitization. On the contrary, it has been clearly demonstrated that not only they can be activated through the engagement of Toll-like receptors (TLRs) by microbial molecules, but also that this interaction induces NK cells to release cytokines that, in turn, activate other cells of both innate and adaptive immunity. For this reason, immunotherapy based on local infusion of TLRs ligands is currently considered as a novel potential strategy to treat solid tumors. Here, we provide a protocol to efficiently stimulate NK cells via endosomal TLRs agonists and to determine endosomal TLRs gene expression level. This protocol can be used for in vitro investigation into endosomal TLRs function in NK cells under different conditions.

Physical Activity Maintain Immune Response Through TLR-2/TLR-4 Gene Expression in Type-2 Diabetes Mellitus Patient at Medan City.

The Increasing in type-2 diabetes mellitus (T2DM) needs to solve comprehensively and holistically. Patients with T2DM should have self-coping due to lifestyle modification. Abdominal fat accumulation can release pro-inflammatory cytokine that leads TLR-2 and TLR-4 to the response. These two kinds of toll-like receptors exist on the monocyte surface membrane which is an innate immunity cell. The aims of this study were to get the profile of physical activity, metabolic state, and mononuclear cell response to the expression of the TLR2 and TLR4 genes in T2DM patients. It was a descriptive-analytic study with a cross-sectional study design. Thirty-two eligible patients with inclusion criteria participated as subjects. All subjects answered questions by IPAQ, and checked metabolic state with body composition analysis. The TLR2 and TLR4 gene expression was determined with quantitative Real- Time PCR. This study result found that most T2DM patients were in a highly active category in which most of their activity was walking (light intensity). The average abdominal circumferences were 91.81 ± 15.4 cm, body fat percentage was 29.5 ± 8.8%, and fasting blood sugar was 187.07 ± 67.03 mg/dl. Mononuclear cells number were normal. The expression of the TLR2 gene was lower by 0.71 fold and TLR4 gene expression was lower by 0.9 fold compared with non-DM (p<0.05). By chi-square test, there was a positive correlation between TLR2 gene expression with fasting blood glucose (p=0.011, and a positive correlation between the abdominal circumference and TLR4 gene expression (p=0.011). Type-2 Diabetes mellitus patients in primary health care keep walking as their physical activity to maintain blood glucose. Patients need to do moderate to vigorous exercise regularly to reduce body fat percentage especially abdominal fat to reduce Toll-like receptor gene expression, so insulin resistance and blood glucose level might decline to normal.

Candida Administration in 5/6 Nephrectomized Mice Enhanced Fibrosis in Internal Organs: An Impact of Lipopolysaccharide and (1→3)-β-D-Glucan from Leaky Gut.

Uremic toxins and gut dysbiosis in advanced chronic kidney disease (CKD) can induce gut leakage, causing the translocation of gut microbial molecules into the systemic circulation. Lipopolysaccharide (LPS) and (1→3)-β-D-glucan (BG) are the major gut microbial molecules of Gram-negative bacteria and fungi, respectively, and can induce inflammation in several organs. Here, the fibrosis in the kidney, liver, and heart was investigated in oral C. albicans-administered 5/6 nephrectomized (Candida-5/6 Nx) mice. At 20 weeks post 5/6 Nx, Candida-5/6 Nx mice demonstrated increased 24 h proteinuria, liver enzymes, and serum cytokines (TNF-α, IL-6, and IL-10), but not weight loss, systolic blood pressure, hematocrit, serum creatinine, or gut-derived uremic toxins (TMAO and indoxyl sulfate), compared to in 5/6 Nx alone. The gut leakage in Candida-5/6 Nx was more severe, as indicated by FITC-dextran assay, endotoxemia, and serum BG. The areas of fibrosis from histopathology, along with the upregulated gene expression of Toll-like receptor 4 (TLR-4) and Dectin-1, the receptors for LPS and BG, respectively, were higher in the kidney, liver, and heart. In vitro, LPS combined with BG increased the supernatant IL-6 and TNF-α, upregulated the genes of pro-inflammation and pro-fibrotic processes, Dectin-1, and TLR-4 in renal tubular (HK-2) cells and hepatocytes (HepG2), when compared with LPS or BG alone. This supported the pro-inflammation-induced fibrosis and the possible LPS-BG additive effects on kidney and liver fibrosis. In conclusion, uremia-induced leaky gut causes the translocation of gut LPS and BG into circulation, which activates the pro-inflammatory and pro-fibrotic pathways, causing internal organ fibrosis. Our results support the crosstalk among several organs in CKD through a leaky gut.

Molecular Characterization and Gene Expression Of TLR4 Gene Associated with Mastitis in Goats

In this study characterization of Toll-like receptor 4 (TLR4) gene of goats; detection of polymorphisms in the nucleotides, and determination of the association of identified genotypes with the occurrence of subclinical mastitis was done using chi-square and odds ratio. Analyzing gene expression using two-sided Student's T-test was also done. Results of Study 1 revealed high similarity (99%) of TLR4 nucleotide sequence of all breeds of goats with that of C. hircus (NM_001285574.1) and sheep (Ovis aries) sequences and slightly lower similarity with cattle (Bos taurus, and Bos indicus) (96%), and water buffalo (Bubalus bubalis) (95%). In Study 2, restriction fragment length polymorphism (RFLP) analysis revealed three genotypes with nine restriction patterns using AluI enzyme. Genotype AA has odds ratio of 0.28 and 0.08 in all breeds, and in Anglo-Nubian, respectively, with significant association (P0.05) that inferred 0.28 and 0.08 times greater probability in all breeds, and in Anglo-Nubian, respectively, for subclinical mastitis to occur than those of other genotypes. Genotype AB showed odds ratio of 3.83, 13.00 and 2.40 in all breeds, in Anglo-Nubian, and in Upgraded, respectively, with significant association (P0.05) that indicated 3.83, 13.00 and 2.40 times more likely in all breeds, in Anglo-Nubian, and in Upgraded, respectively, to suffer subclinical mastitis than those of other genotypes. Â In Study 3, genetic expression analysis showed a significant upregulation of TLR4 gene up to maximum of 3.63-fold in goats without subclinical mastitis compared to subclinically mastitic animals with only 0.65-fold which suggest a prompt role of TLR4 gene in the protection of animal against possible infection.Â

Effects of fumonisin and Salmonella infection in the expression of Toll-like receptors in chicken ovary

Mycotoxins are secondary metabolites produced mainly by fungi that contaminate animal feed and basic food products throughout the world. Currently, more than 500 mycotoxins are reported and one of the most important concern to public health and agriculture is Fumonisin B1 (FB1). FB1, a mycotoxin produced by Fusarium moniliforme, is a contaminant of animal feed with various and complex cellular effects. Although FB1 has been associated with various diseases in animals, to date few studies have been performed to evaluate the endocrine disrupting effects of FB1 and more particularly the effects in the innate immune mechanisms of farm animals and more specifically in poultry species. As the family of Toll-like receptors (TLRs) is one of the key molecules of the innate immune system, the aim of the present study was to evaluate the transcriptional changes of TLRs in the chicken ovary in vivo, in chicks fed with FB1 and in response to Salmonella Enteritidis (SE) infection. RNA was extracted from the ovaries of sexually mature and aged birds, treated with FB1 and SE. Quantitative real-time PCR analysis revealed that FB1 and SE infection resulted in a significant down regulation of 5 TLR genes in the ovary of sexual mature and aged birds. These findings suggest that the mycotoxin FB1 suppresses the innate immune system of the chicken reproductive organs, through alteration in the expression of certain TLR genes.

Non-invasive sampling procedure revealing the molecular events at different abutments of bone-anchored hearing systems-A prospective clinical pilot study.

To investigate the molecular activities in different compartments around the bone-anchored hearing system (BAHS) with either electropolished or machined abutments and to correlate these activities with clinical and microbiological findings. Twelve patients received machined or electropolished abutments after implant installation of BAHS. Peri-abutment fluid and tissue were collected from baseline to 12 months. Gene expression of cytokines and factors related to tissue healing and inflammation, regeneration and remodelling, as well as bacterial recognition were determined using quantitative-polymerase chain reaction (qPCR). The clinical status was evaluated using the Holgers scoring system, and bacterial colonisation was investigated by culturing. The gene expression of inflammatory cytokines (IL-8, IL-1β, and IL-10) and bacteria-related Toll-like receptors (2 and 4) was higher in the peri-abutment fluid than at baseline and in the peri-abutment tissue at 3 and 12 months. Conversely, the expression of genes related to tissue regeneration (Coll1a1 and FOXO1) was higher in the tissue samples than in the peri-abutment fluid at 3 and 12 months. Electropolished abutments triggered higher expression of inflammatory cytokines (IL-8 and IL-1β) (in peri-abutment fluid) and regeneration factor FOXO1 (in peri-abutment tissue) than machined abutments. Several cytokine genes in the peri-abutment fluid correlated positively with the detection of aerobes, anaerobes and Staphylococcus species, as well as with high Holger scores. This study provides unprecedented molecular information on the biological processes of BAHS. Despite being apparently healed, the peri-abutment fluid harbours prolonged inflammatory activity in conjunction with the presence of different bacterial species. An electropolished abutment surface appears to be associated with stronger proinflammatory activity than that with a machined surface. The analysis of the peri-abutment fluid deserves further verification as a non-invasive sampling and diagnostic procedure of BAHS.

The targeted anti-Salmonella bacteriophage attenuated the inflammatory response of laying hens challenged with Salmonella Gallinarum

Fowl typhoid is a severe disease caused by Salmonella Gallinarum with considerable mortality and morbidity in laying hen farms. The current study has focused on controlling the infection in laying hens using anti-Salmonella spp. bacteriophage. The treatments included, PC, without challenge; NC, S. Gallinarum challenged (SGC); B5, 5 mg bacteriophage/kg+SGC; B10, 10 mg bacteriophage/kg+SGC. The Salmonella shedding, inflammatory responses, and gene expression of pro-inflammatory cytokines, toll-like receptor (TLR), and heat shock protein (HSP) in the jejunum, liver, and thigh muscle were tested in laying hens. Supplementation of bacteriophage reduced the abundance of S. Gallinarum in the excreta at d 3, 7, and 14. The abundance of S. Gallinarum was lower in the B10 than the B5 at d 7. Supplementation of bacteriophage decreased the abundance of S. Gallinarum in the oviduct, spleen, and cecum at d 14. The laying hens in the NC group showed an increased relative spleen weight compared with the PC and B10 treatments. Among the SGC treatments, the NC treatment showed higher gene expressions of IL-4 compared with the B5, higher gene expressions of interferon (IFNγ), TLR4, and tumor necrosis factor-α (TNF-α) compared with the B5 and B10, and higher gene expressions of HSP27 compared with the B10 in the jejunum. Dietary supplementation of B10 decreased the mRNA expressions of TLR4 and TNF-α compared with the B5 treatment in the jejunum. The NC treatment showed the highest gene expressions of HSP27, TLR4, and TNF-α in the liver. Dietary supplementation of B10 showed lower mRNA expressions of HSP27 compared with the B5 treatment in the liver. Moreover, the IFNγ and HSP27 were upregulated in the NC treatment compared with the B5 and B10 in the muscle. In conclusion, it can be suggested that bacteriophage is an effective supplement to control S. Gallinarum infection in laying hens and possibly lower horizontal contaminations in laying hen flocks.

Genome-Wide Identification and Characterization of Toll-like Receptors (TLRs) in Diaphorina citri and Their Expression Patterns Induced by the Endophyte Beauveria bassiana.

Toll-like receptors (TLRs) are pathogen recognition receptors (PRRs), which play key roles in helping the host immune system fight pathogen invasions. Systematic information on TLRs at the genome-wide level and expression profiling in response to endophytic colonization is very important to understand their functions but is currently lacking in this field. Here, a total of two TLR genes were identified and characterized in Diaphorina citri. The TLR genes of D. citri were clustered into five families according to the phylogenetic analysis of different species’ TLRs. The domain organization analyses suggested that the TLRs were constituted of three important parts: a leucine-rich repeat (LRR) domain, a transmembrane region (TR) and a Toll/interleukin-1 receptor (TIR) domain. The mRNA expression levels of the two TLR genes (DcTOLL and DcTLR7) were highly regulated in both nymphs and adults of D. citri. These results elucidated the potentiated TLR gene expression in response to endophytically colonized plants. Furthermore, the 3D structures of the TIR domain were highly conserved during evolution. Collectively, these findings elucidate the crucial roles of TLRs in the immune response of D. citri to entomopathogens systematically established as endophytes, and provide fundamental knowledge for further understanding of the innate immunity of D. citri.

Potentiality and Inflammatory Marker Expression Are Maintained in Dental Pulp Cell Cultures from Carious Teeth.

Objectives: This investigation aimed to isolate and culture human dental pulp cells from carious teeth (cHDPCs) and compare their growth characteristics, colony-forming efficiency, mineralization potential and gene expression of Toll-like receptors (TLR)-2, TLR-4, TLR-9, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-17A, 1L-17R, IL-23A, nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK1), dentin matrix protein (DMP)-1, dentin sialophospho protein (DSPP), sex determining region Y-box 2 (SOX2) and marker of proliferation Ki-67 (MKi67) with cells isolated from healthy or non-carious teeth (ncHDPCs). Methods: Pulp tissues were obtained from both healthy and carious teeth (n = 5, each) to generate primary cell lines using the explant culture technique. Cell cultures studies were undertaken by generating growth curves, a colony forming unit and a mineralization assay analysis. The expression of vimentin was assessed using immunocytochemistry (ICC), and the gene expression of above-mentioned genes was determined using quantitative real-time reverse-transcription polymerase chain reaction. Results: ncHDPCs and cHDPCs were successfully isolated and cultured from healthy and inflamed human dental pulp tissue. At passage 4, both HDPC types demonstrated a typical spindle morphology with positive vimentin expression. No statistical difference was observed between ncHDPCs and cHDPCs in their growth characteristics or ability to differentiate into a mineralizing phenotype. ncHDPCs showed a statistically significant higher colony forming efficiency than cHDPCs. The gene expression levels of TLR-2, TLR-4, TLR-9, TNF-α, IL-6, IL-8, IL-17R, IL-23A, NF-κB, MAPK1, DMP1, DSPP and SOX2 were significantly higher in cHDPCs compared with ncHDPC cultures. Conclusion: cHDPCs retain their differentiation potential and inflammatory phenotype in vitro. The inflamed tooth pulp contains viable stem/progenitor cell populations which have the potential for expansion, proliferation and differentiation into a mineralizing lineage, similar to cells obtained from healthy pulp tissue. These findings have positive implications for regenerative endodontic procedures.

Glycoside oleandrin downregulates toll-like receptor pathway genes and associated miRNAs in human melanoma cells

Melanoma accounts for the majority of skin cancer-related deaths. Nerium oleander is a plant known to be toxic and consumed due to the cardiac glycosides it contains. Oleandrin is a cardiac glycoside obtained from of N. oleander. Beside capable of inhibiting proliferation and metastasis of cancer cells, cardiac glycoside derivative compounds cause cardiovascular side effects. Because of cardiovascular toxicity of clinically used cardiac glycosides, it is necessary to investigate cardiac glycoside derivative compounds capable of inhibiting proliferation and metastasis of cancer cells.It is known that oleandrin has anticarcinogenic effects in other cancers. Previous studies have shown that toll-like receptors (TLRs) and their related microRNAs (miRNAs) are associated with cancer. Therefore, aim was to investigate the effect of oleandrin on genes and miRNAs associated with TLRs in A375 melanoma cells in this study. The effects of oleandrin on cell viability, cytokines, apoptosis were evaluated using XTT, ELISA and TUNEL analyses, respectively. The effect of oleandrin on expression of TLR genes and 5 associated miRNAs in A375 cells has been determined by qRT-PCR. In addition, the levels of MyD88, TLR2 and TLR4 proteins were analyzed by western blot method.ELISA indicated that oleandrin treatment (47 nM at 48 h) reduced the level of proinflammatory cytokine IFNG. TUNEL analysis showed that apoptosis rate was significantly increased in the oleandrin dose group. According to qRT-PCR results, there was a significant decrease in IRAK1, IRAK4, MyD88, TLR2-TLR7 and TRAF3 expressions in the oleandrin treated group compared to the control (untreated cell). Also, a significant decrease in TLR4 protein expression has been observed. In addition, oleandrin significantly downregulated the levels of hsa-miRNA-146a-5p and hsa-miRNA-21-5p.In conclusion, it has been observed that oleandrin has an effect on TLR pathway-related genes and miRNAs in melanoma cells. We show that TLRs pathways and hsa-miR-146a-5p and hsa-miR-21-5p can participate in the oleandrin molecular mechanism of action.

The influence of parental high-fat high-sugar diet on the gut-brain axis in male offspring

PurposeThe gut-brain axis (GBA) is implicated in the development of obesity, and its role in developmental programming needs to be explored. This study uncovers the effects of a parental high-fat, high-sugar diet (HFS) on the gut (colon) and brain (hypothalamus) GBA of male Wistar rat offspring at weaning until adulthood. MethodsFor ten weeks before mating, male progenitors were fed a control diet (CD) or HFS, whereas dams were fed CD or HFS during pregnancy and lactation. Male offspring aged 21-and 90-day old were assessed for: Gene expression of toll-like receptor 4 (TLR4) pathway and zonula occludens 1 (ZO1) in the colon and hypothalamus; hypothalamic gene expression of orexigenic neuropeptides and Leptin receptor; serum levels of lipopolysaccharide (LPS), glucagon like peptide 1 (GLP-1), Ghrelin and neuropeptide Y (NPY); colonic cytokine levels; FaecalBifidobacterium spp.andLactobacillus spp. DNA. ResultsPaternal HFS showed increased endotoxaemia, reduced colonic gene expression of ZO1 and reduced colonic TNF-α at weaning. In the adult offspring, paternal HFS showed increased NPY, reduced serum Ghrelin, colonic pro-inflammatory cytokines, and lower faecalBifidobacteriumspp. DNA. Maternal diet showed increased hypothalamic gene expression of myeloid differentiation primary response 88 (MYD88) at weaning. The maternal HFS diet showed increased NPY and reduced faecalBifidobacteriumspp. andLactobacillusspp. DNA in the adult offspring. The combined effect of parental diet showed increased NPY at weaning, and lowerBifidobacteriumspp. andLactobacillus spp.in the adult offspring. ConclusionMaternal and paternal HFS diet seem to influence the programming of the gut-brain axis, leading to increased visceral adiposity and weight of male offspring at weaning, the effect that lasted until adulthood.

Bacillus licheniformis PF9 improves barrier function and alleviates inflammatory responses against enterotoxigenic Escherichia coli F4 infection in the porcine intestinal epithelial cells

BackgroundEnterotoxigenic Escherichia coli (ETEC) F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses. Although Bacillus licheniformis (B. licheniformis) has been reported to enhance intestinal health, it remains to be seen whether there is a functional role of B. licheniformis in intestinal inflammatory response in intestinal porcine epithelial cell line (IPEC-J2) when stimulated with ETEC F4.MethodsIn the present study, the effects of B. licheniformis PF9 on the release of pro-inflammation cytokines, cell integrity and nuclear factor-κB (NF-κB) activation were evaluated in ETEC F4-induced IPEC-J2 cells.ResultsB. licheniformis PF9 treatment was capable of remarkably attenuating the expression levels of inflammation cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-8, and IL-6 during ETEC F4 infection. Furthermore, the gene expression of Toll-like receptor 4 (TLR4)-mediated upstream related genes of NF-κB signaling pathway has been significantly inhibited. These changes were accompanied by significantly decreased phosphorylation of p65 NF-κB during ETEC F4 infection with B. licheniformis PF9 treatment. The immunofluorescence and western blotting analysis revealed that B. licheniformis PF9 increased the expression levels of zona occludens 1 (ZO-1) and occludin (OCLN) in ETEC F4-infected IPEC-J2 cells. Meanwhile, the B. licheniformis PF9 could alleviate the injury of epithelial barrier function assessed by the trans-epithelial electrical resistance (TEER) and cell permeability assay. Interestingly, B. licheniformis PF9 protect IPEC-J2 cells against ETEC F4 infection by decreasing the gene expressions of virulence-related factors (including luxS, estA, estB, and elt) in ETEC F4.ConclusionsCollectively, our results suggest that B. licheniformis PF9 might reduce inflammation-related cytokines through blocking the NF-κB signaling pathways. Besides, B. licheniformis PF9 displayed a significant role in the enhancement of IPEC-J2 cell integrity.

Artemisia annua L. Aqueous Extract Promotes Intestine Immunity and Antioxidant Function in Broilers.

This study was conducted to investigate the effects of Artemisia annua L. aqueous extract (AAE) on intestinal immune and antioxidative function of broilers. A total of 200 one-day-old Arbor Acre broilers were randomly allotted into five dietary treatment groups, with five replicates per treatment and eight broilers per replicate. The five treatment diets were formulated by adding, respectively, 0 (control group), 0.5, 1.0, 1.5, and 2.0 g/kg AAE in the basal diet. The results showed that dietary inclusion of AAE quadratically decreased interleukin (IL)-1β content, linearly decreased IL-6 content in the small intestine through regulating the nuclear factor-kappa B signal pathway, and quadratically increased immunoglobulin (Ig)M and sIgA content in ileum and jejunum. Besides, there was a quadratic decrease in the gene expression of IL-1β, IL-6, and toll like receptor 4 (TLR4) in ileum on day 21, and the gene expression of IL-6 and TLR4 in duodenum on day 42, thereby improving small intestinal immune function in broilers. Additionally, dietary inclusion of AAE improves antioxidative function through the nuclear factor-erythroid 2-related factor 2 (Nrf2) signal pathway in the small intestinal mucosa of broilers, especially, quadratically increased catalase (CAT) and superoxidase dismutase activity in ileum, and total antioxidant capacity and glutathione peroxidase activity in duodenum, and quadratically decreased malondialdehyde concentration in ileum, besides, linearly increased heme oxygenase-1 and Nrf2 gene expression in jejunum and ileum on day 42, quadratically increased CAT gene expression in the small intestine. Furthermore, regression analyses of the above parameters showed that the optimal dose range of AAE in the diet of broilers was 1.12–1.38 g/kg.

Peptidomimetic Lipid-Nanoparticle-Mediated Knockdown of TLR4 in CNS Protects against Cerebral Ischemia/Reperfusion Injury in Mice

Ischemic stroke activates toll-like receptor 4 (TLR4) signaling, resulting in proinflammatory polarization of microglia and secondary neuronal damage. Herein, we report a novel lipid-nanoparticle (LNP)-mediated knockdown of TLR4 in microglia and amelioration of neuroinflammation in a mouse model of transient middle cerebral artery occlusion (tMCAO). siRNA against TLR4 (siTLR4) complexed to the novel LNP (siTLR4/DoGo310), which was based on a dioleoyl-conjugated short peptidomimetic (denote DoGo310), was readily internalized by the oxygen–glucose-deprived (OGD) mouse primary microglia, knocked-down TLR4, and polarized the cell to the anti-inflammatory phenotype in vitro. Systemic administration of siTLR4/DoGo310 LNPs in the tMCAO mice model resulted in the accumulation of siRNA mainly in the Iba1 positive cells in the peri-infarct. Analysis of the peri-infarct brain tissue revealed that a single injection of siTLR4/DoGo310 LNPs led to significant knockdown of TLR4 gene expression, reversing the pattern of cytokines expression, and improving the neurological functions in tMCAO model mice. Our data demonstrate that DoGo310 LNPs could be a promising nanocarrier for CNS-targeted siRNA delivery for the treatment of CNS disorders.

Role of Toll-like receptors in local effects in a model of experimental envenoming induced by Bothrops jararacussu snake venom and by two phospholipases A2

Literature shows that phospholipases A2 isolated from snake venoms of the genus Bothrops are involved in the local inflammatory response. However, the mechanisms by which these enzymes trigger this process have not yet been clarified. Toll-Like receptors (TLRs) are transmembrane proteins that recognize pathogens associated molecular patterns (PAMPs), or even damage associated molecular patterns (DAMPs). After this recognition, an innate immune response is activated resulting in cytokines liberation contributing to inflammation. Thus, the purpose of this work was to study the participation of different TLRs during the local inflammatory process induced by B. jararacussu snake venom and by two isolated phospholipases A2, BthTX-I or BthTX-II, from this venom in a model of experimental envenoming. For this, sub-lethal doses of B. jararacussu venom (BjussuV), BthTX-I or BthTX-II were injected in the gastrocnemius muscle. Myotoxic activity was evaluated by histological analysis and by quantification of plasma levels of total-creatine kinase (CK). The pro-inflammatory cytokines TNF-α and IL-1β was measured in both muscle tissue homogenate and plasma. A quantification of the gene expression of TLRs 2, 4, 5 and 9 in muscle tissue homogenate was performed by the real-time polymerase chain reaction (RTq-PCR). According to the results, it can be observed that, when compared to the control, there was a significant increase of CK and TNF-α in the bloodstream of the animals injected with both BjussuV, BthTX-I and BthTX-II. In muscle tissue homogenate, it was observed a significant increase in both cytokines, TNF-α and IL-1β, levels compared to the control animals. The results point to an important increase in the gene expression of TLR2 and TLR4, suggesting that these TLRs can be important targets for the development of future therapies for local treatment for victims of snakebites.

Promising cardioprotective effect of baicalin in doxorubicin-induced cardiotoxicity through targeting toll-like receptor 4/nuclear factor-κB and Wnt/β-catenin pathways

Doxorubicin (Dox) is an indispensable chemotherapeutic agent associated with damaging cardiotoxicity. Baicalin (BA) is a flavonoid, extracted from the medicinal plant Scutellariae baicalensis Georgi. BA is well known for its anti-inflammatory and antioxidant effects. Our study investigated the potential effect of BA in attenuating Dox-induced cardiotoxicity. To this end, male Swiss albino mice were given BA (100 mg/kg/d, orally) for 4 wk and were challenged with Dox (six intraperitoneal doses, each 2.5 mg/kg, every other day with a final cumulative dose of 15 mg/kg). Serum activities of cardiac biomarkers (cardiac troponin-I, creatine kinase–membrane bound, lactate dehydrogenase, and aspartate aminotransferase) were assessed along with the histopathological examination of the heart tissues. Gene expression of Toll-like receptor 4 (TLR4) was analyzed by quantitative reverse transcription real-time polymerase chain reaction. Analysis of the protein levels of β-catenin and nuclear factor-κB (NF-κB) was done immunohistochemically. Cardiac Dickkopf-1 (DKK1) and interleukin-1beta (IL-1β) were quantified by enzyme-linked immuno-sorbent assay. Cardiac levels of reduced glutathione (GSH) and malondialdehyde (MDA) were detected spectrophotometrically. Pretreatment with BA significantly prevented Dox-induced elevation of serum activities of cardiac biomarkers and alterations to the heart. Moreover, BA suppressed the gene overexpression of cardiac TLR4 and subsequently prevented Dox-induced elevation of both cardiac NF-κB and IL-1β. BA also significantly reduced the cardiac levels of DKK1 and elevated the level of β-catenin. Dox-induced elevation of MDA and reduction of GSH were reversed by BA. BA exhibited a novel cardioprotective effect against Dox-induced cardiotoxicity. The cardioprotective effect was indicated through the inhibition of the inflammatory TLR4/NF-κB pathway and the activation of the protective Wnt/β-catenin pathway by the suppression of DKK1.

Extracellular vesicles from ALI models induce endothelial dysfunction

One distinguishing characteristic of acute respiratory distress syndrome (ARDS) or its lesser form, acute lung injury (ALI), is dysfunction of the lung endothelium. This dysfunction leads to increased permeability and eventual pulmonary edema. Despite the globally climbing rates of ARDS, the mechanisms of endothelial dysfunction are poorly understood. Recently, extracellular vesicles (EVs) have been shown to induce various microvascular disease states, as they contain various endothelial‐targeting cargo in the form of mRNA, proteins, lipids, and miRNAs. Therefore, we hypothesized that EVs play a role in the endothelial dysfunction observed in ARDS/ALI. EVs were isolated from the plasma of male Sprague Dawley rats which were given saline or P. aeruginosa (PA103) via intratracheal (IT) injection for 48h. ALI was confirmed by determining the wet to dry weights of the rodent lungs. We then tested the effect of the EVs isolated from saline‐ and ALI treated animals (sEVs and ALI‐EVs, respectively) on endothelial barrier function and modifications in gene expression. We found that treatment with ALI‐EVs, but not sEVs, increased rat lung microvascular endothelial cell (RLMVEC) permeability in the presence of lipopolysaccharide (LPS). Interestingly, ALI‐EVs did not induce endothelial dysfunction on their own, rather exacerbated the damage induced by LPS. We further noted ALI‐EVs, but not sEVs, increased gene expression of toll‐like receptor 4 (TLR4), which is the main pattern recognition receptor (PRR) for LPS and its downstream signaling molecule, MyD88 in the presence of LPS in RLMVEC. Further, we found that ALI‐EVs, together with LPS, increased gene expression of intracellular adhesion molecule 1 (ICAM‐1), vascular adhesion molecule 1 (VCAM‐1), and interleukin 6 (IL‐6), which are all involved in inflammatory signaling. Thus, it is likely that through the modulation of inflammatory signaling mediators, ALI‐EVs contain select cargo that worsen the endothelial barrier in settings where LPS is present, such as bacterial‐ARDS.

Distinctive Toll-like Receptors Gene Expression and Glial Response in Different Brain Regions of Natural Scrapie.

Prion diseases are chronic and fatal neurodegenerative diseases characterized by the accumulation of disease-specific prion protein (PrPSc), spongiform changes, neuronal loss, and gliosis. Growing evidence shows that the neuroinflammatory response is a key component of prion diseases and contributes to neurodegeneration. Toll-like receptors (TLRs) have been proposed as important mediators of innate immune responses triggered in the central nervous system in other human neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. However, little is known about the role of TLRs in prion diseases, and their involvement in the neuropathology of natural scrapie has not been studied. We assessed the gene expression of ovine TLRs in four anatomically distinct brain regions in natural scrapie-infected sheep and evaluated the possible correlations between gene expression and the pathological hallmarks of prion disease. We observed significant changes in TLR expression in scrapie-infected sheep that correlate with the degree of spongiosis, PrPSc deposition, and gliosis in each of the regions studied. Remarkably, TLR4 was the only gene upregulated in all regions, regardless of the severity of neuropathology. In the hippocampus, we observed milder neuropathology associated with a distinct TLR gene expression profile and the presence of a peculiar microglial morphology, called rod microglia, described here for the first time in the brain of scrapie-infected sheep. The concurrence of these features suggests partial neuroprotection of the hippocampus. Finally, a comparison of the findings in naturallyinfected sheep versus an ovinized mouse model (tg338 mice) revealed distinct patterns of TLRgene expression.