TLR4 mRNA Expression Research Articles

H3K4me3 Histone ChIP-Seq Analysis Reveals Molecular Mechanisms Responsible for Neutrophil Dysfunction in HIV-Infected Individuals.

Peripheral neutrophils in HIV-infected individuals are characterized by impairment of chemotaxis, phagocytosis, bactericidal activity, and oxidative burst ability regardless of whether patients are receiving antiretroviral therapy or not. Neutrophil dysfunction leads not only to increased susceptibility to opportunistic infections but also to tissue damage through the release of reactive oxygen species (ROS), proteases, and other potentially harmful effector molecules contributing to AIDS progression. In this study, we demonstrated high levels of histone H3 lysine K4 trimethylated (H3K4me3) and dysregulation of DNA transcription in circulating neutrophils of HIV-infected subjects. This dysregulation was accompanied by a deficient response of neutrophils to LPS, impaired cytokine/chemokine/growth factor synthesis, and increased apoptosis. Chromatin immunoprecipitation sequencing (ChIPseq) H3K4me3 histone analysis revealed that the most spectacular abnormalities were observed in the exons, introns, and promoter-TSS regions. Bioinformatic analysis of Gene Ontology, including biological processes, molecular function, and cellular components, demonstrated that the main changes were related to the genes responsible for cell activation, cytokine production, adhesive molecule expression, histone remodeling via upregulation of methyltransferase process, and downregulation of NF-κB transcription factor in canonical pathways. Abnormalities within H3K4me3 implicated LPS-mediated NF-κB canonical activation pathway that was a result of low amounts of κB DNA sites within histone H3K4me3, low NF-κB (p65 RelA) and TLR4 mRNA expression, and reduced free NF-κB (p65 RelA) accumulation in the nucleus. Genome-wide survey of H3K4me3 provided evidence that chromatin modifications lead to an impairment within the canonical NF-κB cell activation pathway causing the neutrophil dysfunction observed in HIV-infected individuals.

The Expression of Hemagglutinin by a Recombinant Newcastle Disease Virus Causes Structural Changes and Alters Innate Immune Sensing.

Recombinant Newcastle disease viruses (rNDV) have been used as bivalent vectors for vaccination against multiple economically important avian pathogens. NDV-vectored vaccines expressing the immunogenic H5 hemagglutinin (rNDV-H5) are considered attractive candidates to protect poultry from both highly pathogenic avian influenza (HPAI) and Newcastle disease (ND). However, the impact of the insertion of a recombinant protein, such as H5, on the biological characteristics of the parental NDV strain has been little investigated to date. The present study compared a rNDV-H5 vaccine and its parental NDV LaSota strain in terms of their structural and functional characteristics, as well as their recognition by the innate immune sensors. Structural analysis of the rNDV-H5 demonstrated a decreased number of fusion (F) and a higher number of hemagglutinin-neuraminidase (HN) glycoproteins compared to NDV LaSota. These structural differences were accompanied by increased hemagglutinating and neuraminidase activities of rNDV-H5. During in vitro rNDV-H5 infection, increased mRNA expression of TLR3, TLR7, MDA5, and LGP2 was observed, suggesting that the recombinant virus is recognized differently by sensors of innate immunity when compared with the parental NDV LaSota. Given the growing interest in using NDV as a vector against human and animal diseases, these data highlight the importance of thoroughly understanding the recombinant vaccines’ structural organization, functional characteristics, and elicited immune responses.

Dietary oregano essential oil supplementation improves intestinal functions and alters gut microbiota in late-phase laying hens

BackgroundDietary essential oil (EO) supplementation can exert favorable effects on gut health in broilers. However, it is unknown whether EO could improve intestinal functions, consequently beneficial for egg performance and quality in late-phase laying hens. This study was aimed to investigate the potential effects of EO on production performance, egg quality, intestinal health and ileal microbiota of hens in the late phase of production. A total of 288 60-week-old Hy-line Brown laying hens were randomly divided into 4 groups and fed a basal diet (control) or basal diets supplemented with oregano EO at 100, 200 and 400 mg/kg (EO100, EO200 and EO400).ResultsDietary EO supplementation resulted in a quadratic decrease (P < 0.05) in feed conversion ratio with lower (P < 0.05) feed conversion ratio in EO200 group than the control during weeks 9–12 and 1–12 of the trial. Compared to the control, EO addition resulted in higher (P < 0.05) eggshell thickness at the end of week. 4, 8 and 12 and higher (P < 0.05) chymotrypsin activity. There was a quadratic elevation (P < 0.05) in ileal chymotrypsin and lipase activity, along with a linear increase in villus height to crypt depth ratio. Quadratic declines (P < 0.05) in mRNA expression of IL-1β, TNF-α, IFN-γ and TLR-4, concurrent with a linear and quadratic increase (P < 0.05) in ZO-1 expression were identified in the ileum with EO addition. These favorable effects were maximized at medium dosage (200 mg/kg) of EO addition and intestinal microbial composition in the control and EO200 groups were assessed. Dietary EO addition increased (P < 0.05) the abundances of Burkholderiales, Actinobacteria, Bifidobacteriales, Enterococcaceae and Bacillaceae, whereas decreased Shigella abundance in the ileum.ConclusionsDietary EO addition could enhance digestive enzyme activity, improve gut morphology, epithelial barrier functions and modulate mucosal immune status by altering microbial composition, thus favoring feed efficiency and eggshell quality of late-phase laying hens.

Per- and post-remote ischemic conditioning attenuates ischemic brain injury via inhibition of the TLR4/MyD88 signaling pathway in aged rats.

Remote ischemic conditioning (RIC), as an emerging protective method, might be used clinically to prevent ischemia-reperfusion injury (IRI) in ischemic stroke. In this study, we aim to investigate whether RIC performed either during brain ischemia or after reperfusion has a protective effect and further explore the mechanistic basis for the protective effects of RIC against IRI in an aged rat model. We investigated brain IRI in 16-18months old SD rats. Animals underwent: (i) sham laparotomy, (ii) brain IRI, (iii) brain IRI + RIC during ischemia (IRI + RIperC), or (iv) brain IRI + RIC after reperfusion (IRI + RIpostC). RIC consists of three cycles of 10min of hind limb ischemia followed by 10min reperfusion. After 24h of reperfusion, the infarct size, neurological deficit scores and brain oedema were assessed in all groups. The levels of IL-1β, IL-6, TNF-α were measured by ELISA. The mRNA and protein expressions of TLR4, MyD88, TRAF6 and NF-κB were detected by RT-PCR and western blot. Both RIperC and RIpostC treatment attenuated the IRI-induced neuronal injury, reflected by reductions in the infarct size, neurological deficit scores and brain oedema. RIperC and RIpostC also can decrease the concentration of IL-1β, IL-6, TNF-α in IRI. From the results of RT-PCR and western blot, we found that RIC decreased the mRNA and protein expression of TLR4, MyD88, TRAF6 and NF-κB compared to that in the IRI group. The present study suggested that RIC protected aged rats against IRI, and this protective effect might be mediated by inhibiting the TLR-4/MyD88/TRAF-6/NF-κB signaling pathway.

Paroxetine effect on expression of TLR2 and TLR4 genes in PBMCs of mentally normal individuals

Background: Paroxetine is one of the well-known antidepressants. Recent studies have focused on paroxetine’s probable immuno-modulatory effects, since findings have indicated inflammation’s role in the pathophysiology of depression. Therefore, in the present study, TLR2 and TLR4 mRNA genes expression was assessed in paroxetine-treated peripheral blood mononuclear cells (PBMCs). Methods: Venous blood samples were drawn from five healthy men (20-40 years old). Peripheral blood mononuclear cells (PBMCs) were isolated from samples and were cultured. After the first incubation for 24h, phytohemagglutinin plus lipopolysaccharide were added to the cells and then were incubated for 24h. Thereafter, cells were treated with different concentrations of paroxetine in the presence or absence of inhibitors of 5-HT2 and 5-HT7 receptors. After incubation for 48h, RNA was extracted and cDNA was synthesized. Using the real-Time PCR technique, TLR2 and TLR4 genes mRNA expression were evaluated. Statistical analysis of data were carried out using GraphPad Prism 7. Results: TLR2 and TLR4 mRNA expression were significantly increased in response to paroxetine at all concentrations. Furthermore, the co-culture of cells with the drug and the 5-HT2R and 5HT7R inhibitor simultaneously revealed that paroxetine’s immuno-modulatory effects viaTLR2 are dependent on serotonin, while it is independent of serotonin in the case of TLR4. Conclusion: Considering paroxetine’s effect in modulating immune responses via increasing TLR2 and TLR4 expression, paroxetine could have therapeutic potentials in diseases with a deficiency in these receptors.

TLR2 and TLR4 mRNA expression levels in liver transplant patients with acute rejection

Toll-like receptors (TLRs) have important role in transplant outcomes by activating the innate immune system and production of pro-inflammatory cytokines, leading to graft rejection. We assessed the expression level of TL2 and TLR4 in acute rejection (AR) on the 1st and 7th-day post-transplantation. TLR2 and TLR4 expressions were evaluated by real-time PCR in both the AR group (n = 50) and non-AR (n = 50), compared with the control group. Also, the correlation of the expression levels of TLRs between both the 1st and 7th day was analyzed. ROC curve analysis was used to determine the cut-off value for TLRs expression. TLR4 mRNA expression was significantly up-regulated in AR patients vs. the controls on the 1st day (p ≤ 0.05) and it was down-regulated in non-AR vs. controls on the 1st day (p ≤ 0.05). Also, TLR4 expression had decreased in both AR and non-AR groups vs. control on the 7th day (p ≤ 0.05). Both TLR2 and TLR4 expression in comparison to non-AR had increased in the AR group on the 7th day (p ≤ 0.05). TLR2 expression positively correlated between 1st and 7th day in AR (r = 0.3, (p ≤ 0.05) and non-AR group (r = 0.2, p ≤ 0.05). ROC curve analysis showed a cut-off value of TLR2 up to 0.98 with sensitivity 71.05 (95%CI = 54.1–84.6) and specificity 63.27 (95%CI = 48.3–76.6) that could distinguish between AR and non-AR group (p ≤ 0.05). The data support that both TLR2 and TLR4 expression have an effective role in AR after liver transplantation and could be used as possible biomarkers for AR to choose better therapeutic strategies based on immunological aspects.

Peripheral leucocyte molecular indicators of inflammation and oxidative stress are altered in dairy cows with embryonic loss

Objective of experiment was to determine whether oxidative stress (OS) and inflammation altered embryonic loss in dairy cows. Blood samples were collected at days 0, 16, 32 and 60 after timed (AI) from 200 Holstein cows to determine embryonic loss based on interferon-stimulated gene-15 (ISG15) mRNA expression (day 16) and ultrasound at day 32 and day 60. Leucocyte expressions of mRNA TLR2, TLR4, TNF-α, IL1B, IL10, STAT3 (inflammation), PTGS2, PTGES (prostaglandin synthesis), and PLA2G4A and ALOX5AP (eicosanoid metabolism) at days 0 and 16 were determined. Plasma redox status for antioxidant enzymatic activities of glutathione peroxidase (GPX), superoxide dismutase (SOD), total antioxidant capacity (TAC), and concentrations of malondialdehyde (MDA) were determined at days 0, 16, 32 and 60. All antioxidant-redox responses were beneficially significant in pregnant cows diagnosed pregnant at day16 and sustained pregnancy to day 60 compared to non-pregnant cows at day16 or pregnant at day16 and lost embryos by days 32 or 60. The leucocyte mRNA expressions of TLR2, TLR4, STAT 3, IL1B, PTGS2, PLA2G4A and ALOX5AP were greater and PTGES was lower at day16 in pregnant cows that lost embryos early (P < 0.05). In conclusion peripheral leucocyte molecular indicators of inflammation and plasma indicators of OS were altered in pregnant cows undergoing embryonic losses compared to cows with a sustained pregnancy.

Amomum tsaoko fruit extract exerts anticonvulsant effects through suppression of oxidative stress and neuroinflammation in a pentylenetetrazol kindling model of epilepsy in mice.

BackgroundChronic epilepsy is a multifaceted common brain disorder with manifold underlying factors. Epilepsy affects around 70 million peoples worldwide. Amomum tsaoko is a perennial herbaceous plant that is extensively cultivated in many provinces of China reported to exert immense biological activities.ObjectiveThis research work was aimed to reveal the therapeutic actions of ethanolic extract of A.tsaoko fruits (EE-ATF) against the pentylenetetrazol (PTZ)-provoked convulsive seizures in the mice.MethodologyThe convulsive seizures were provoked to the animals via administering 70 mg/kg of PTZ through intraperitoneally to trigger the convulsive seizures then treated with the EE-ATF at 50, 75, and 100 mg/kg orally 30 min prior to PTZ challenge. After the 30 min of PTZ challenge, animals closely monitored for signs of convulsion, generalized clonic and tonic convulsion durations, and mortality. A sub-convulsive dose 35 mg/kg of PTZ was used to provoke the kindling and seizure stages were examined using standard method. The levels of dopamine, GABA, glutamate, and Na + K + ATPase and Ca + ATPase activities in the brain tissues were studied using marker specific assay kits. The oxidative stress and antioxidant markers studied using standard methods. The mRNA expressions of COX-2, TNF-α, NF-κB, TLR-4, and IL-1β in the brain tissues were studied using RT-PCR analysis. The brain tissues were examined histologically.ResultsEE-ATF treatment remarkably decreased the onset and duration of convulsion and suppressed the seizure severity and mortality in the PTZ animals. EE-ATF treatment appreciably ameliorated the PTZ triggered modifications in the GABA, glutamate, dopamine levels and Ca + 2ATPase and Na + K + ATPase activities in the brain tissues. EE-ATF suppressed the mRNA expressions of NF-κB, IL-1β, TLR-4, TNF-α, and COX-2. The status of antioxidants were elevated by the EE-ATF. Histological findings also demonstrated the curative actions of EE-ATF.ConclusionOur findings evidenced that the EE-ATF substantially ameliorated the PTZ-provoked convulsive seizures in the mice.

Zhuifeng Tougu capsules improve rheumatoid arthritis symptoms in rats by regulating the toll-like receptor 2/4-nuclear factor kappa-B signaling pathway.

To investigate the efficacy of Zhuifeng tougu capsules (, ZFTG) in the treatment of rheumatoid arthritis (RA) in rats and study its mechanism, focusing on the toll-like receptor 2/4-nuclear factor kappa-B (TLR2/4-NF-κB) signaling pathway. Type Ⅱ collagen and an artificial climate box were used to construct the rat model of collagen-induced arthritis with wind-cold-dampness arthralgia syndrome. The rats were divided randomly into a control group, wind-cold-dampness syndrome model group, and high-, medium-, and low-dose ZFTG groups. The methotrexate (MTX) control group was treated with the corresponding drug intervention for 28 d. The joint temperature, pain threshold, joint swelling degree, and arthritis index (AI) score were measured. The production of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and rheumatoid factors (RFs) in the blood was detected by enzyme-linked immunosorbent assay. The protein expression of TLR2, TLR4, and NF-κB in synovial tissues was detected by Western blotting, and the mRNA expression of TLR2, TLR4, and NF-κB was detected by real-time polymerase chain reaction. Compared with the model group, the joint temperature in each treatment group, the MTX control group, and MTX group recovered, the degree of foot swelling, pain threshold, AI score decreased, serum CRP, ESR, RF level and the levels of TLR2, TLR4, and NF-κB in synovial tissue were decreased (P < 0.05). Among them, the curative effect in the medium-dose and MTX groups was more evident (P < 0.01). ZFTG has a significant effect on RA in rats, and its mechanism may involve regulating CRP levels, the ESR, and RFs via the TLR2/4-NF-κB signaling pathway.

Toll-like receptor 4 methylation grade is linked to depressive symptom severity

This study explores potential associations between the methylation of promoter-associated CpG sites of the toll-like receptor (TLR)-family, plasma levels of pro-inflammatory proteins and depressive symptoms in young female psychiatric patients. Ratings of depressive symptoms and blood samples were obtained from 92 young women seeking psychiatric care. Methylation of 32 promoter-associated CpG sites in TLR1 to TLR10 was analysed using the Illumina Infinium Methylation EPIC BeadChip. Expression levels of 91 inflammatory proteins were determined by proximity extension assay. Statistical correlations between depressive state, TLR1-10 methylation and inflammatory proteins were investigated. Four additional cohorts were studied to evaluate the generalizability of the findings. In the discovery cohort, methylation grade of cg05429895 (TLR4) in blood was inversely correlated with depressive symptoms score in young adults. After correction for multiple testing, plasma levels of macrophage inflammatory protein 1β (MIP-1β/CCL4) were associated with both TLR4 methylation and depressive symptom severity. A similar inverse association between TLR4 methylation in blood and affective symptoms score was also found in a cohort of 148 both males and females (<40 years of age) from the Danish Twin Registry. These findings were not, however, replicated in three other external cohorts; which differed from the first two cohorts by a higher age and mixed ethnicities, thus limiting the generalizability of our findings. However, TLR4 methylation inversely correlated with TLR4 mRNA expression in the Danish Twin Study indicating a functional significance of methylation at this particular CpG. Higher depression scores in young Scandinavian adults was associated with decreased methylation of TLR4 in blood.

Artemisinin derivative SM934, influences the activation, proliferation, differentiation and antibody-secreting capacity of β-cells in systemic lupus erythematosus mice via inhibition of TLR7/9 signaling pathway

Purpose: To study the influence of artemisinin derivative, SM934 on activation, proliferation, differentiation and antibody-secreting capacity of B cells of systemic lupus erythematosus (SLE) mice, and the underlying mechanism.&#x0D; Methods: Female MRL/lpr mice (n = 60) were randomly assigned to four groups of 15 mice each: SLE, 2.5 mg/kg SM934; 5 mg/kg SM934, and 10 mg/kg SM934 groups. Serum levels of interleukins 6, 10, 17 and 21 (IL-6, IL-17, IL-10 and IL-21) were determined. The secretions of immunoglobulins G and M (IgG and IgM) by B cells were determined. The population of B lymphocyte subtypes was determined flow cytometrically. The expressions of Blimp-1 and Bcl-6, Toll-like receptors 7 and 9 (TLR7 and TLR9) mRNAs were determined.&#x0D; Results: SLE-induced upregulation of serum IL-10, IL-6, IL-17 and IL-21 was significantly and dosedependently reduced following a 2-month treatment with SM934 (p &lt; 0.01). Treatment with SM934 significantly and dose-dependently accentuated B cell germinal center B cell populations, but significantly and dose-dependently decreased the populations of plasma and activated B cells (p &lt; 0.01). The splenic levels of IgG and IgM were decreased in a dose-dependent fashion after 8 weeks of treatment (p &lt; 0.01). Artemisinin derivative SM934 decreased the expression of Blimp-1, and upregulated the expression of Bcl-6, both in a dose-dependent manner (p &lt; 0.01). Moreover, SM934 decreased the mRNA expressions of TLR7 and TLR9 in a dose-based manner (p &lt; 0.01).&#x0D; Conclusion: Artemisinin derivative SM934 mitigates LSE syndromes by suppressing the TLR-induced B-cell stimulation and plasma cell generation

MLH1 Deficiency Down-Regulates TLR4 Expression in Sporadic Colorectal Cancer.

Patients with mismatch repair (MMR)-deficient colorectal cancer (CRC) have a more favorable prognosis than patients with tumors with intact MMR. In order to obtain further insights on the reasons for this different outcome, we investigated the interplay between MMR genes and TLR4/MyD88 signaling. The cancer genome atlas (TCGA) databases were selected to predict the differential expression of TLR4 in colon cancer and its correlation with MMR genes. Moreover, the expression of MMR genes and TLR4 was evaluated by immunohistochemistry in 113 CRC samples and a cohort of 63 patients was used to assess TLR4 mRNA expression and MLH1 epigenetic silencing status. In vitro, the effect of MLH1 knockdown on TLR4 expression was quantified by Real Time PCR. TLR4 expression resulted dependent on MMR status and directly correlated to MLH1 expression. In vitro, MLH1 silencing decreased TLR4 expression. These observations may reflect the better prognosis and the chemoresistance of patients with CRC and MMR defects.

Hepatic steatosis precedes pregnancy in the BPH/5 preeclampsia‐like mouse model

BPH/5 mice represent a superimposed model of preeclampsia that spontaneously develop maternal and fetal characteristics of preeclamptic pregnancies. In women, the development of preeclampsia is associated with pre-existing dysregulation of lipid metabolism including hepatic steatosis. The study objective was tocharacterize the basal hepatic health of the female BPH/5 mouse to determine if hepatic steatosis precedes the preeclampsia-like syndrome. We hypothesized that non-pregnant female BPH/5 mice have an excess of hepatocellular lipid that promotes intrahepatic pro-inflammatory cytokine production. Blood and liver samples were collected from 8–10-week-old BPH/5 non-pregnant females (n=10) and 8–10-week-old C57Bl/6 non-pregnant females (n=6). Liver and body weight were recorded. Biochemical markers of hepatocellular function (albumin, ALT, BUN, and bilirubin) were quantified. Hepatic RNA was isolated, and qPCR was used to evaluate pro-inflammatory markers. H&E staining was used to evaluate hepatic histomorphology and lipid accumulation. There was no significant difference in albumin, ALT, BUN, or bilirubin between BPH/5 and C57Bl/6 mice. There was significantly higher hepatic CXCL-2, CXCL-10, IL-1ß, TLR2, and TLR7 mRNA expression in BPH/5 versus C57Bl/6 mice (p<0.05). There was a significantly greater liver weight in BPH/5 mice compared to C57Bl/6 mice (p=0.05). The histologic microsteatosis score was significantly increased in the BPH/5 mice (2.5) compared to C57Bl/6 mice (0.98, p=0.025). The BPH/5 mouse has basal hepatic steatosis and a pro-inflammatory hepatic phenotype that precedes pregnancy. Dysregulated lipid metabolism in the liver may contribute to the development of preeclampsia.

β-1,3-Glucan from Euglena gracilis as an immunostimulant mediates the antiparasitic effect against Mesanophrys sp. on hemocytes in marine swimming crab (Portunus trituberculatus)

β-1,3-glucans, natural polysaccharide groups, exert immunomodulatory effects to improve the innate response and disease resistance in aquatic species and mammals. However, this β-glucan stimulant is yet to be assayed in swimming crab (Portunus trituberculatus) hemocytes. In this study, we explored the immunomodulatory effect of β-1,3-glucans (derived from Euglena gracilis) via in vitro 24 h stimulation assays in swimming crab hemocytes. We found that this algal β-1,3-glucans in crab hemocytes significantly elevated cellular enzymes related parameters, including phenoloxidase (PO), lysozyme, acid phosphatase (ACP) activities, and superoxide anion generation (O2−) rate both at intracellular (P < 0.05) and extracellular (P < 0.05) levels. Besides, alkaline phosphatase (AKP) in hemocytes exhibited no significant differences across the groups (P > 0.05). β-glucan significantly influenced (P < 0.05) the activities of the antioxidant enzyme, superoxide dismutase (SOD) in hemocytes. Moreover, the relative mRNA expression of numerous immune-related genes, including proPO, TLR-2, Alf-1, NOX, Lysozyme, Crustin-1, and Cuznsod, was significantly higher stimulated hemocytes than in control (P < 0.05). We also reported the dose-dependent antiparasitic activity against Mesanophyrs sp., in stimulated hemocytes than in the control (P < 0.05). The present study collectively demonstrated that β-glucan potentially stimulates innate immunity by elevating cellular enzyme responses and up-regulating the mRNA expression of genes associated with crab innate immunity. Thus, β-glucan is a promising immunostimulant for swimming crab farming in crustaceans aquaculture.

Shenling Baizhu Powder Inhibits RV-SA11-Induced Inflammation and Rotavirus Enteritis via TLR4/MyD88/NF-κB Signaling Pathway.

Rotavirus enteritis (RVE) is a common acute intestinal infectious disease caused by rotavirus infection. It is an important cause of death in children younger than 5 years worldwide. Shenling baizhu powder (SBP), a classic traditional Chinese formulation, is one of the most popularly prescribed medicines for digestive diseases. Clinical studies have revealed the protective effects of SBP on RVE. However, the potential mechanism is still unclear. In this study, we aimed to evaluate the anti-rotavirus effect of SBP and its mechanism, focusing on the TLR4/MyD88/NF-κB signaling pathway. Our results demonstrated that, based on the inhibition of the virus-induced cytopathic effect in Caco-2 cells, the concentration for 50% of maximal effect (EC50) and selectivity index (SI) of SBP for RV-SA11 in the serum were 5.911% and 11.63, respectively. A total of 219 active compounds with oral bioavailability ≥30% and drug-likeness ≥ 0.18 were selected from the 10 ingredients present in the formulation of SBP, which acted on 471 potential targets. A total of 226 target genes of RVE were obtained from the GeneCards database. The protein-protein interaction (PPI) network showed that there was a close interaction between 44 common targets of SBP and RVE. The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that SBP acted on RVE through various inflammatory pathways and the intestinal immune network. Subsequently, we investigated the effect of SBP on TLR4/MyD88/NF-κB signaling pathway in vitro. After infection with RV- SA11, the expression of TLR4, MyD88, and NF-κB mRNA and protein increased significantly, which could be abolished by SBP treatment. In addition, the IL-1β, TNF-α, IL-6, and IFN-β levels increased markedly in Caco-2 cells infected with RV-SV11. Treatment with SBP partly reversed the changes of IL-1β, TNF-α, and IL-6, while further increased the level of IFN-β. In conclusion, our study revealed that SBP can significantly inhibit rotavirus replication and proliferation in vitro. The antiviral effect may be related to the regulation of the TLR4/MyD88/NF-κB signaling pathway, followed by the down regulation of inflammatory cytokines and up regulation of IFN-β induced by rotavirus.

Inhibitory effects of lactoferrin on pulmonary inflammatory processes induced by lipopolysaccharide by modulating the TLR4-related pathway

This study tested the ability of lactoferrin to modulate pulmonary inflammation. To construct in vitro and in vivo inflammatory lung models, cells from the human lung adenocarcinoma cell line (A549) were exposed to lipopolysaccharide (LPS, 1 µg/mL), and mice (CD-1) were intratracheally administered LPS [10 mg/kg of body weight (BW), tracheal lumen injection], respectively. The A549 cells were preincubated with lactoferrin (10 mg/mL), and the mice were intraperitoneally injected with lactoferrin (100 mg/kg of BW), followed by LPS treatment. The concentrations of proinflammatory cytokines (IL-1β and TNF-α) in culture medium of A549 cells and in bronchoalveolar lavage fluid of the mice were determined using enzyme-linked immunosorbent assays. The toll-like receptor 4-related pathway (TLR4/MyD88/IRAK1/TRAF6/NFκB) was determined at gene and protein expression levels in A549 cells and mouse lung tissue. Results showed that LPS treatment significantly elevated the concentrations of IL-1β and TNF-α in the A549 cell culture medium and in bronchoalveolar lavage fluid of the mice; it also elevated both the mRNA and protein expressions of TLR4 and the TLR4 downstream factors in A549 cells and mouse lung tissue. Nevertheless, lactoferrin apparently depressed the releases of IL-1β and TNF-α from A549 cells and lung tissues stimulated by LPS, and significantly suppressed the TLR4 signaling pathway. Lactoferrin also promoted the enhancement of miR-146a expression in A549 cells and mouse lung tissue. Moreover, 100°C heating for 3 min caused total loss of the previously listed bioactivity of lactoferrin. Collectively, we proved that lactoferrin intervened in LPS-induced inflammation in the pulmonary cell model and in the mouse model, through inhibiting the TLR4-related pathway.

Seaweed polysaccharide mitigates intestinal barrier dysfunction induced by enterotoxigenic Escherichia coli through NF-κB pathway suppression in porcine intestinal epithelial cells.

This study aimed to investigate the protective effects and underlying mechanism of seaweed polysaccharide (SWP) on intestinal epithelial barrier dysfunction induced by E.coli in an IPEC-J2 model. A preliminary study was done to screen optimum SWP concentrations by cell viability, cytotoxicity, apoptosis and proliferation evaluation. The regular study was conducted to evaluate the protective effects of SWP against E.coli challenge via the analysis of transepithelial electrical resistance (TEER), tight junction proteins, NF-κB signalling pathway, proinflammatory cytokines and the E.coli adhesion and invasion. Our results show that 4h E.coli challenge down-regulated tight junction proteins expression, decreased TEER, activated NF-κB signalling pathway and increased proinflammatory response, which indicates that the E.coli infection model was well-established. Pre-treatment with 240μg/ml SWP for 24h alleviated the 4h E.coli -induced intestinal epithelial barrier dysfunction, as evidenced by the up-regulated expression of Occludin, Claudin-1 and ZO-1 at both mRNA and protein level and the increased TEER of IPEC-J2 cells. Pre-incubation with 240μg/ml SWP for 24h inhibited the activation of the NF-κB signalling pathway by 4h E.coli challenge, including the decreased mRNA expression of TLR-4, MyD88, IκBα, p-65, as well as the reduced ratio of protein expression of p-p65/p65. Also, pre-treatment with 240μg/ml SWP for 24h decreased proinflammatory response (IL-6 and TNF-α) induced by 4h E.coli challenge and decreased the E.coli adhesion and invasion. In conclusion, SWP mitigated intestinal barrier dysfunction caused by E.coli through NF-κB pathway in IPEC-J2 cells and 240μg/ml SWP exhibited better effect. Our results also provide a fundamental basis for SWP in reducing post-weaning diarrhoea of weaned piglets, especially under E.coli -infected or in-feed antibiotic-free conditions.

TLR4-NLRP3-GSDMD-Mediated Pyroptosis Plays an Important Role in Aggravated Liver Injury of CD38-/- Sepsis Mice.

Clinically, severe bacterial infection can cause septicemia and multiple organ dysfunction syndrome, especially liver injury. CD38 is closely related to many inflammatory pathways, but its role in liver injury caused by bacterial infection remains unclear. The purpose of this study is to discuss the specific role of CD38 in bacterial liver injury. Eight-week-old male C57BL/6 mice (WT, CD38−/− and CD38−/−TLR4mut) were used and stimulated with Escherichia coli (ATCC25922) or PBS, intraperitoneally. After 3 hours of bacterial stimulation, serum was collected to detect ALT and AST concentration, and liver tissue was harvested for hematoxylin and eosin staining and bacterial culture. The mRNA expressions of TLR4, NLRP3, IL-1β, IL-18, and GSDMD were quantitatively determined by RT-qPCR. The expressions of TLR4, MyD88, TRIF, NF-κB p65, NLRP3, GSDMD, and cytokines were detected by Western blot. The expression and localization of ERK1/2 were detected by immunohistochemistry and Western blot. The results showed that bacterial stimulation could upregulate the expression of inflammatory cytokines, leading to hepatic dysfunction. Moreover, bacterial stimulation of CD38-deficient mice can aggravate the inflammatory response, the expressions of TLR4, NF-κB, and ERK1/2 were significantly increased, and the biomarkers related to pyroptosis also manifested more obvious pyroptosis. However, TLR4 mutation significantly alleviated inflammation and pyroptosis in the liver caused by bacteria, on the basis of CD38 deficiency. Overall, CD38 knockout exacerbates bacteria-induced liver damage through TLR4-NLRP3-GSDMD-mediated pyroptosis.

Anethole Attenuates Enterotoxigenic Escherichia coli-Induced Intestinal Barrier Disruption and Intestinal Inflammation via Modification of TLR Signaling and Intestinal Microbiota.

This study aimed to investigate the effects of dietary anethole supplementation on the growth performance, intestinal barrier function, inflammatory response, and intestinal microbiota of piglets challenged with enterotoxigenic Escherichia coli K88. Thirty-six weaned piglets (24 ± 1 days old) were randomly allocated into four treatment groups: (1) sham challenge (CON); (2) Escherichia coli K88 challenge (ETEC); (3) Escherichia coli K88 challenge + antibiotics (ATB); and (4) Escherichia coli K88 challenge + anethole (AN). On day 12, the piglets in the ETEC, ATB, and AN group were challenged with 10 mL E. coli K88 (5 × 109 CFU/mL), whereas the piglets in the CON group were orally injected with 10 mL nutrient broth. On day 19, all the piglets were euthanized for sample collection. The results showed that the feed conversion ratio (FCR) was increased in the Escherichia coli K88-challenged piglets, which was reversed by the administration of antibiotics or anethole (P < 0.05). The duodenum and jejunum of the piglets in ETEC group exhibited greater villous atrophy and intestinal morphology disruption than those of the piglets in CON, ATB, and AN groups (P < 0.05). Administration of anethole protected intestinal barrier function and upregulated mucosal layer (mRNA expression of mucin-1 in the jejunum) and tight junction proteins (protein abundance of ZO-1 and Claudin-1 in the ileum) of the piglets challenged with Escherichia coli K88 (P < 0.05). In addition, administration of antibiotics or anethole numerically reduced the plasma concentrations of IL-1β and TNF-α (P < 0.1) and decreased the mRNA expression of TLR5, TLR9, MyD88, IL-1β, TNF-α, IL-6, and IL-10 in the jejunum of the piglets after challenge with Escherichia coli K88 (P < 0.05). Dietary anethole supplementation enriched the abundance of beneficial flora in the intestines of the piglets. In summary, anethole can improve the growth performance of weaned piglets infected by ETEC through attenuating intestinal barrier disruption and intestinal inflammation.

Selenium Attenuates S. aureus-Induced Inflammation by Regulation TLR2 Signaling Pathway and NLRP3 Inflammasome in RAW 264.7 Macrophages.

This study aimed to investigate the effects of selenium (Se) on the expression of Toll-like receptor (TLR) 2 and pyrin domain-containing protein (NLRP)3 inflammasome in macrophages infected by Staphylococcus aureus (S. aureus). RAW 264.7 macrophages were treated with 2 μmol/L Na2SeO3 for 12 h before infection with S. aureus for 2 h. Through Western blot, qRT-PCR, and ELISA analysis, the core molecules of TLR2 signaling pathway and NLRP3 inflammasome in RAW 264.7 macrophages were detected. Results showed that Se significantly reduced the elevated mRNA expression of TLR2, myeloid differentiation factor-88 (Myd88), NLRP3, Caspase-recruitment domain (ASC), and Caspase-1 induced by S. aureus. Furthermore, compared with I group, the protein expression of TLR2, Myd88, NLRP3, ASC, and Caspase-1 were suppressed in T group. In addition, the mRNA and protein expression of interleukin-1 beta (IL-1β) induced by S. aureus were also decreased after Se treatment. In conclusion, Se inhibits S. aureus-induced inflammation by suppressing the activation of the TLR2 signaling pathway and NLRP3 inflammasome in RAW 264.7 macrophages.