TLR4 mRNA Expression Research Articles

Impact of an anti-Salmonella. Typhimurium Bacteriophage on intestinal microbiota and immunity status of laying hens.

Typhoid is a distinct gastrointestinal disease that largely affects the public by consumption of inadequately or partially cooked eggs from contaminated laying hen farms. This has led the research on laying hens to focus on controlling the contamination by an effective anti-Salmonella spp. agent in the intestine. The treatments included, control, without challenge; PC, Salmonella typhimurium challenged (STC); BP5, 5ppm bacteriophage/kg+STC; BP10, 10ppm bacteriophage/kg+STC, on Salmonella shedding, body organs inflammatory reactions, and expression of toll-like receptor (TLR), pro-inflammatory cytokines, and heat shock protein (HSP) in the jejunum, liver,and thigh muscle in the STC laying hens. The RT-PCR method was used to enumerate the number of Salmonella typhimurium in the organs. The birds in the STC groups exhibited the increased population of Salmonella spp. in the excreta (p<.01). In the STC groups, the BP5 and BP10 laying hens exhibited a lower (p<.01) population of Salmonella spp. in the excreta at d 7 after STC. Supplementation of bacteriophage significantly decreased (p<.01) the colonization of S. Typhimurium in the spleen, oviduct, caecum and excreta. Among the STC treatments, the BP10 laying hens showed lower (p<.01) mRNA expression of interferon-γ (IFNγ) and TLR-4 in the jejunum compared with the PC treatment. After the STC, dietary supplementation with BP5 or BP10 decreased (p<.01) the mRNA expressions of IFNγ, HSP-27 and tumour necrosis factor-α in the liver compared with the PC treatment. These results suggest that bacteriophage can be used as an effective agent to decrease S. Typhimurium contamination in laying hens and possibly lower S. Typhimurium transfer to foods.

Hydroxyl functionalized multi-walled carbon nanotubes modulate immune responses without increasing 2009 pandemic influenza A/H1N1 virus titers in infected mice

Growing use of carbon nanotubes (CNTs) have garnered concerns regarding their association with adverse health effects. Few studies have probed how CNTs affect a host's susceptibility to pathogens, particularly respiratory viruses. We reported that exposure of lung cells and mice to pristine single-walled CNTs (SWCNTs) leads to significantly increased influenza virus H1N1 strain A/Mexico/4108/2009 (IAV) titers in concert with repressed antiviral immune responses. In the present study, we investigated if hydroxylated multi-walled CNTs (MWCNTs), would result in similar outcomes. C57BL/6 mice were exposed to 20 μg MWCNTs on day 0 and IAV on day 3 and samples were collected on day 7. We investigated pathological changes, viral titers, immune-related gene expression in lung tissue, and quantified differential cell counts and cytokine and chemokine levels in bronchoalveolar lavage fluid. MWCNTs alone caused mild inflammation with no apparent changes in immune markers whereas IAV alone presented typical infection-associated inflammation, pathology, and titers. The co-exposure (MWCNTs + IAV) did not alter titers or immune cell profiles compared to the IAV only but increased concentrations of IL-1β, TNFα, GM-CSF, KC, MIPs, and RANTES and inhibited mRNA expression of Tlr3, Rig-i, Mda5, and Ifit2. Our findings suggest MWCNTs modulate immune responses to IAV with no effect on the viral titer and modest pulmonary injury, a result different from those reported for SWCNT exposures. This is the first study to show that MWCNTs modify cytokine and chemokine responses that control aspects of host defenses which may play a greater role in mitigating IAV infections.

HMGB1 and inflammatory cytokines in experimental acute lung injury induced in rabbits

ABSTRACT The aim of this work was to measure HMGB1, TNF-alpha, and IL-8 in bronchoalveolar lavage (BAL), serum and TLR2 and TLR4mRNA expression in lung tissue of rabbits with two grades of acute lung injury (ALI). The animals were randomly assigned to groups with severe (S) and mild/moderate (MM) ALI, induced with warm saline, and a control group. HMGB1, TNF-alpha, IL-8, TLR2mRNA and TLR4mRNA were measured after ALI induction. The results showed increased levels of IL-8, TNF-alpha, HMGB1 and TLR4mRNA in the ALI groups. HMGB1, IL-8 and TNF-alpha concentrations in BAL were higher in S compared MM. Increased TLR4mRNA was observed in S and MM versus control. The results suggest an early participation of HMGB1 in ALI together with IL-8 and TNF-alpha and association with severity. TLR4 has early expression and role in ALI pathophysiology but is not associated with severity.

Single immunoglobulin and Toll‑interleukin‑1 receptor domain containing molecule protects against severe acute pancreatitis invitro by negatively regulating the Toll‑like receptor‑4 signaling pathway: A clinical and experimental study.

Single immunoglobulin and Toll-interleukin-1 receptor domain-containing molecule (SIGIRR) is a specific inhibitor of IL-1R and Toll-like receptor (TLR) signaling and considered a potential target for the treatment of inflammatory diseases. Pathogenic mechanisms associated with the TLR4 signaling pathway have a critical role in the development of severe acute pancreatitis (SAP). The aim of the present study was to determine the role of SIGIRR in the regulation of TLR4 signaling during the progression of SAP. Pancreatitis-associated ascitic fluid (PAAF) was collected from patients with SAP. Murine RAW264.7 macrophages were transfected with a SIGIRR overexpression plasmid and co-cultured with the PAAF from the donors in order to evaluate the effect of SIGIRR in vitro. The mRNA expression of TLR4, SIGIRR and other key downstream signaling molecules was quantified using semi-quantitative PCR with agarose gel electrophoresis. Furthermore, the levels of pro-inflammatory cytokines in the culture supernatant were detected using ELISA. In contrast to SIGIRR, the mRNA expression levels of TLR4, myeloid differentiation factor 88 (MyD88), IL-1R-associated kinase-1 (IRAK-1) and TNF receptor-associated factor-6 (TRAF-6) were significantly increased in RAW264.7 cells following treatment with PAAF. Furthermore, TLR4, MyD88, IRAK-1 and TRAF-6 mRNA levels were significantly downregulated following SIGIRR overexpression and PAAF treatment in RAW264.7 cells. The levels of IL-2, IL-12, IL-17 and IFN-γ in the culture supernatant were also significantly decreased, while IL-10 levels were increased. Overall, SIGIRR negatively regulated the TLR4 signaling pathway to protect against the development of SAP in an in vitro model. Therefore, SIGIRR may represent a promising therapeutic target for SAP.

Effects of extracellular Hsp70, lipopolysaccharide and lipoteichoic acid on human monocyte-derived macrophages and differentiated THP-1 cells

Abstract Extracellular Hsp70 (eHsp70) is elevated in blood of patients with inflammatory diseases (e.g. chronic obstructive pulmonary disease), prone to bacterial infections. It stimulates pro-inflammatory effects in cells via TLR2 and TLR4. The main hypothesis was that eHsp70 induces inflammation and alters inflammatory responses to lipopolysaccharide (LPS) and lipoteichoic acid (LTA). We assessed pro-inflammatory cytokines concentrations, TLR2, TLR4 and Hsp70 mRNA expressions, NF-κB and MAPKs activation, and viability after treatment of monocyte-derived macrophages (MDMs) and THP-1 cells with recombinant human (rh) Hsp70, LPS and LTA. rhHsp70, LPS, LTA and their combinations induced IL-6, IL-8 and TNF-α from MDMs. LPS, LTA, and combinations with rhHsp70 increased TLR2 and decreased TLR4 in both cells. rhHsp70 increased TLR2 and TLR4 in THP-1 cells, and TLR2 in MDMs. rhHsp70, LPS and their combinations up-regulated Hsp70 in MDMs, while down-regulated it in THP-1. LPS and its combinations with rhHsp70 lowered cell viability in THP-1 cells and activated caspases-8 and -9. Involvement of NF-κB and MAPKs in inflammatory and survival mechanisms stimulated by rhHsp70 was demonstrated. eHsp70 alone and in combination with bacterial components could provoke inflammation and affect viability of MDMs and THP-1 cells leading to disturbed inflammatory responses in diseases accompanied with bacterial colonisations or infections.

Therapeutic effects of bone marrow mesenchymal stem cells via modulation of TLR2 and TLR4 on renal ischemia-reperfusion injury in male Sprague-Dawley rats.

Introduction: Acute kidney injury (AKI) induced by renal ischemia-reperfusion (I/R) injury is a pro-inflammatory process that activates toll-like receptors (TLRs). Stem cell therapy holds a great promise for kidney repair. Therefore, we investigated the immunomodulatory role of bone marrow stromal cells (BMSCs) on TLR2 and TLR4 expression in AKI in male Sprague-Dawley rats. Methods: BMSCs were isolated from the bone marrow of male rats, cultured in DMEM, and characterized using appropriate markers before transplantation. Renal I/R was induced by 45 minutes bilateral ischemia followed by 24 hours of reperfusion. Rats received intraperitoneal injections of BMSCs (1.5 × 106 cells, i.p, per rat) immediately after termination of renal ischemia. Serum samples were collected pre-and post-stem cells injection for assessment of blood urea nitrogen (BUN) and creatinine (Cr) levels. The kidneys were harvested after 24 hours of reperfusion for structural and molecular analysis. Results: Renal I/R caused severe tissue injuries and increased the level of BUN (166.5 ± 12.9 vs. 18.25 ± 1.75) and Cr (3.7 ± 0.22 vs. 0.87 ± 0.06) compared to the sham group. In addition, mRNA expression of TLR2 and TLR4 elevated in the renal I/R group. Administration of BMSCs improved the functional and structural state of the kidney induced by I/R and down-regulated TLR2 and TLR4 gene expression. Conclusion: The results showed a highly significant renoprotection by BMSCs that indicates their therapeutic potential in I/R injures. These effects are most likely associated with the TLR2/4 signaling pathway via modulation of the inflammatory response cascades.

Phyllathin From Phyllanthus Amarus Ameliorates Epileptic Convulsion and Kindling Associated Post-Ictal Depression in Mice via Inhibition of NF-κB/TLR-4 Pathway.

Background:Epilepsy is a chronic, complex, unprovoked, and recurrent disorder of the nervous system that affected several people worldwide. Phyllanthus amarus (PA) has been documented to have neuroprotective potential.Aim:To evaluate the potential of standardized extract of PA and its possible mechanism of action against the Pentylenetetrazol (PTZ)-induced convulsion and kindling associated post-ictal depression in experimental mice.Materials and Methods:Phyllathin was isolated from methanolic extract of PA and well-characterized using HPTLC, ESI-MS/MS, and LC/MS. Phyllathin containing a standardized extract of PA (50, 100, and 200 mg/kg) was administered in convulsed and kindled mice, followed by an assessment of various parameters.Results:The spectral analysis confirmed the molecular formula and weight of phyllanthin as C24H34O6 and 418.2342 Da. PA (100 and 200 mg/kg) significantly ameliorated PTZ-induced (p < 0.05) duration, onset of tonic-clonic convulsion, and mortality in mice. It also significantly attenuated (p < 0.05) PTZ-induced kindling in mice. Alteration in brain GABA, dopamine, and glutamate, Na+K+ATPase, Ca+2-ATPase activities, and oxido-nitrosative stress in kindled mice was significantly restored (p < 0.05) by PA treatment. It also significantly (p < 0.05) down-regulated brain mRNA expressions of NF-κB, TNF-α, IL-1β, COX-2, and TLR-4. Histological aberrations induced by PTZ in the brain of a kindled rat was significantly (p < 0.05) ameliorated by PA.Conclusion:Phyllanthin containing a standardized extract of PA exerts its antiepileptic potential via balancing excitatory (glutamate) and inhibitory (GABA) brain monoamines, voltage-gated ion channels (Na+K+/Ca+2-ATPase) and inhibition of NF-κB/TLR-4 pathway to ameliorate neuroinflammation (TNF-α, IL-1β, and COX-2) in experimental mice.

Stress Response Simulated by Continuous Injection of ACTH Attenuates Lipopolysaccharide-Induced Inflammation in Porcine Adrenal Gland.

On modern farms, animals are at high risk of bacterial invasion due to environmental stress factors. The adrenal gland is the terminal organ of the stress response. The crosstalk between adrenal endocrine stress and innate immune response is critical for the maintenance of immune homeostasis during inflammation. Thus, it's important to explore whether stresses play a pivotal role in lipopolysaccharide (LPS)-induced inflammatory response in the porcine adrenal gland. Thirty-days-old Duroc × Landrace × Large White crossbred piglets (12 ± 0.5 kg) were randomly allocated into four groups in a 2 × 2 factorial arrangement of treatments, including ACTH pretreatment (with or without ACTH injection) and LPS challenge (with or without LPS injection). Each group consisted of six male piglets. The results showed that our LPS preparation alone induced mRNA expressions of IL-1β, IL-6, TNF-α, IL-10, COX-2, TLR2, TLR4, and GR (P < 0.05). ACTH pretreatment downregulated the TLR2 mRNA and IL-6 protein level induced by our LPS preparation significantly (P < 0.05) by one-way ANOVA analysis. Treatment with LPS alone extremely significantly decreased ssc-miR-338 levels (P < 0.01). Interaction of ACTH × LPS was significant for cNOS level (P = 0.011) and ssc-miR-338 expression (P = 0.04) by two-way ANOVA analysis. The LPS treatment significantly downregulated cNOS levels (P < 0.01), which was significantly attenuated by ACTH pretreatment (P < 0.05). Lipopolysaccharide alone did not affect ssc-miR-146b expression levels compared to that in the vehicle group. However, ACTH pretreatment in combination with LPS significantly increased this micro-RNA expression (P < 0.05). TLRs 1–10 were all expressed in adrenal tissue. The LPS challenge alone induced remarkable compensatory mitochondrial damages at the ultrastructural level, which was alleviated by ACTH pretreatment. Accordingly, ACTH pretreatment was able to block LPS-induced secretion of local adrenal cortisol (P < 0.05). Taken together, our results demonstrate that ACTH pretreatment seems to attenuate LPS-induced mitochondria damage and inflammation that decreased cNOS activity in the adrenal gland and ultimately returned local adrenal cortisol to basal levels at 6 h post LPS injection.

Epigenetic regulation of TLR2‐mediated periapical inflammation

To determinethe methylation pattern of TLR2 gene promoter and its association with the transcriptional regulation of periapical inflammatory and angiogenic responses in symptomatic and asymptomatic forms of apical periodontitis. In this cross-sectional study, apical lesions were obtained from volunteers with asymptomatic apical periodontitis (AAP) (n=17) and symptomatic apical periodontitis (SAP) (n=17) scheduled for tooth extraction, and both total RNA and DNA were extracted. DNA was bisulfite-treated, a region of CpG island within the TLR2 gene was amplified by qPCR and the products were sequenced. Additionally, the mRNA expression of TLR2, TLR4, IL-6, IL-12, TNFalpha, IL-23, IL-10, TGFbeta, VEGFA and CDH5 was analysed by qPCR. The data were analysed with chi-square tests, Mann-Whitney or unpaired t-tests, and Spearman´s correlation; variable adjustments were performed using multiple linear regression (P<0.05). TLR2 depicted a hypomethylated DNA profile at the CpG island in SAPwhen compared with AAP, along with upregulated expression of TLR2, with pro-inflammatory cytokines IL-6 and IL-23, and the angiogenesis marker CDH5 (P<0.05). TLR2 methylation percentage negatively correlated with mRNA levels of IL-23 and CDH5 in apical periodontitis. Lower methylation frequencies of single CpG dinucleotides -8 and -10 localized in close proximity to nuclear factor κB (NFκB) binding within the TLR2 promoter were identified in SAP versus AAP (P<0.05). Finally, unmethylated -10 and -8 single sites demonstrated up-regulation of IL-23, IL-10 and CDH5 transcripts compared to their methylated counterparts (P<0.05). TLR2 gene promoter hypomethylation was linked to transcriptional activity of pro-inflammatory cytokines and angiogenic markers in exacerbated periapical inflammation. Moreover, unmethylated single sites in close proximity to NFκB binding were involved in active transcription of IL-23, IL-10 and CDH5.

Cotinine inhibits TLR4/NF-κB signaling pathway and improves deep vein thrombosis in rats.

Background: The present study was designed to explore the regulatory mechanisms and influences of cotinine on deep vein thrombosis (DVT) in rats via the toll-like receptor 4/nuclear factor κ binding (TLR-4/NF-κB) pathway. Methods: In this experimental study, 30 SD rats were randomly assigned to control group, sham operation group, model group, cotinine (10 μg/kg) group, and model + cotinine (10 μg/kg) group. The thromboxane B2 (TXB2), 6-keto-PGF1α, plasminogen activator inhibitor (PAI), tissue plasminogen activator (t-PA), TLR4, NF-κB, and p65 mRNA and protein expression and tissue changes were analyzed by ELISA, Hematoxylin–Eosin (HE) staining, RT-PCR, and Western blot. Results: There was no significant difference between the control and sham operation groups (P>0.05). The model and cotinine groups showed significantly higher mRNA and protein levels of TXB2, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), PAI, TLR-4, and NF-κB, and significantly lower levels of 6-keto-PGF1α and t-PA than the control and sham operation groups (P<0.05), and the model + cotinine group showed significantly higher mRNA and protein levels of TXB2, IL-6 and TNF-α, PAI, TLR-4, and NF-κB and significantly lower levels of 6-keto-PGF1α and t-PA than the model group (P<0.05). Conclusion: Cotinine can aggravate thrombus and inflammation in rats with DVT, and the mechanism may be associated with the activation of the TLR-4/NF-κB inflammatory signaling pathway.

P1139EXPRESSION OF TOLL LIKE RECEPTORS, CELL ADHESION MOLECULES AND PRO-INFLAMMATORY CYTOKINES IN ESRD PATIENTS

Abstract Background and Aims CAPD is well established modality of treatment for patients with end stage renal disease (ESRD). Peritonitis is a leading cause of technique failure and death in patients on CAPD. Studies on expressions of host factors like TLRs, CAMs and their relationship with inflammatory cytokines (IL-6, IL-12, TNF-alpha and IL-1 beta) involved in peritonitis and other co-morbidity and functional status are lacking throughout the world. Hence the present study has to be done to determine the expressions of TLR2 and TLR4, and CAMs in ESRD patients. To compare the expression of TLR2, TLR4, ICAM 1 and Pro-inflammatory cytokines (IL-6, IL-12, TNF-alpha and IL-1-beta) in Peritonitis, CAPD and CRF group patients. Method A total of 85 ESRD patients recruited and sub-divided into 3 groups. Group1- CAPD patient (n=25), Group 2- Peritonitis patient (n=30), and Group 3- CRF (n=30 patients). mRNA expression of TLR-2, TLR-4 were examined at gene levels by RT PCR and cell adhesion molecule (ICAM-1) were examined at gene and protein levels by RT PCR and ELISA respectively in Serum and Pro-inflammatory cytokines level were also examined by ELISA in serum. We performed microbiological culture for bacterial and fungal pathogens using automated BACTEC culture system. Cell counts were routinely done on every dialysate. Results Out of 30 samples of peritonitis group 15 were culture positive and 15 were culture negative. We found that in peritonitis group the mRNA expression of TLR-2 and TLR-4 was higher as compared to CRF (4.183±2.857vs 3.633±2.41) (p=0.049), (4.314±2.91vs 4.14±1.99) (p=0.015) and CAPD (4.183±2.857vs3.683±2.85) (p=0.041), (4.314±2.91vs 3.88±1.91) (p=0.009) respectively. At gene and protein level ICAM-1 was higher in peritonitis patient compared to CAPD (mRNA expression 4.76±2.64vs 4.36±3.48) (level in sera 660±201.2vs 514±157) (p=0.003). The IL-6 and IL-12 expression was higher in Peritonitis group as compared to CAPD (66.87±64.51vs214.35±220.05) (p=0.04) and (230.17±153.45vs417.04±302.96) (p=0.028) respectively. The TNF-alpha and IL-1-beta expression was not significant among the groups. Conclusion TLRs activation by bacterial molecules leads to the induction of cytokines (IL-6 and IL-12) and chemokine through the activation of NF-ķB pathway and may be responsible for atherosclerosis, morbidity and mortality in ESRD patients. Elevated level of ICAM-1, IL-6 and IL-12 may be responsible for chronic inflammation in Peritonitis group patients.

Probiotic Bifidobacterium lactis V9 attenuates hepatic steatosis and inflammation in rats with non-alcoholic fatty liver disease

Both steatosis and inflammation are key pathological events in the progression of non-alcoholic fatty liver disease (NAFLD). Probiotics are beneficial for the prevention and treatment of NAFLD. Bifidobacterium animalis subsp. lactis V9 (V9) is a newly isolated strain with favorable probiotic properties. The study aims to evaluate the effects and mechanisms of V9 on the hepatic steatosis and inflammatory responses in a rat model of NAFLD induced by high-fat diets (HFD). Our results showed that administration of V9 significantly attenuated the HFD-induced increases in alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, resulting in alleviated hepatic steatosis. V9 supplementation reduced the accumulation of hepatic triglyceride and free fatty acid,while increasing the levels of glycogen. Serum levels of glucose were also decreased in HFD rats administrated with V9. Meanwhile, the transcription of SREBP-1c and FAS was reduced, and the hepatic expression of phosphorylated-AMPK and PPAR-α was restored after V9 administration. V9 suppressed the production of inflammatory cytokines (e.g. IL-6, IL-1β, and TNF-α) in HFD-fed rats. The anti-inflammatory effects of V9 was found to be associated with the inhibition of hepatic expression of TLR4, TLR9, NLRP3, and ASC mRNA. Furthermore, the activation of ERK, JNK, AKT and NF-κB were suppressed by V9 treatment. These results indicate that Bifidobacterium lactis V9 improves NAFLD by regulating de novo lipid synthesis and suppressing inflammation through AMPK and TLR-NF-κB pathways, respectively.

The TLRs and IFNs in patients with connective tissue diseases

Autoimmune connective tissue diseases (ACTD) are characterized by spontaneous stimulation of the immune system and the production of autoantibodies. Some autoantibodies may create an immune complex with DNA and/or RNA and promote tissue inflammation. Immune complexes that contain nucleic acids can act as ligands for endosomal Toll-like receptors (TLR), which activation induces secretion of the type I and type III interferons. The present study aimed to determine whether TLRs and IFNs genes could be considered as potential ACTD biomarkers. IFN-A and IFN-G showed a relationship with a predisposition to the development of SLE and MCTD, and IFN-B with a predisposition to the development of SLE. The TLR7 rs5743305 T allele and rs5743316 A allele may play a protective role against the development of MCTD, and the TLR7 rs1731479 T allele and TLR8 rs17256081 C allele may be predictors of MCTD development. mRNA expression of the IFN-α, IFN-β, IFN-γ, TLR3, TLR8 and TLR7 was significantly higher in patients with SLE compared to patients with MCTD and SSc. MCTD patients with anti-U1-70k (+) had higher IFN-γ and lower IFN-β serum levels than patients without this antibody. In patients with SLE, serum levels of IFN-α and IFN-γ correlate with the concentration of complement components, and serum levels of IFN-α with disease activity. The study confirmed that the TLR-IFN pathway may be considered as an important pathogenic mechanism for ACTD.

Effect of long-term consumption of tea (Camellia sinensis L.) flower polysaccharides on maintaining intestinal health in BALB/c mice.

Polysaccharides have various health-promoting functions. However, dietary polysaccharides cannot be digested by the human alimentary tract, thus the gut is the most important location where polysaccharides play their role. The effect of polysaccharides from tea (Camellia sinensis L.) flower (TFPS) on intestinal health was investigated in the present study. TFPS with the molecular weight of 1,316.29kDa was prepared, and twenty 6-week-old BALB/c male mice were randomly allotted to a chow diet (normal control group, NC group) or with 200mg/kg (body weight)/day of TFPS for 13 weeks (n = 10 each). Histomorphology observation of jejunum and colons showed that TFPS maintained the adequate gut barrier. qPCR analysis revealed that the expression of colonic tight junction proteins of claudin1 (1.29±0.15compared with 1.00±0.13, P <0.05) and claudin5 (2.91±0.44compared with 1.00±0.27, P <0.01) at mRNA level with a significant difference between TFPS supplement or not, while the expression of TLR4 and TNF-α mRNA was not changed statistically. 16S rDNA amplicons sequencing was applied to measure the compositions of gut microbiota from feces of mice. TFPS treatment exhibited similar relative abundances in Bacteroidetes and Firmicutes; however, it decreased the relative abundance of Akkermansia and increased that of Lactobacillus compared with the NC group. The contents of short-chain fatty acids after TFPS supplementation, both in cecal contents and feces, were significantly higher than those of the NC group. Besides, TFPS significantly increased IgA production. These results suggest that TFPS is beneficial to intestinal health and can improve intestinal adaptive immune tolerance. PRACTICAL APPLICATION: Dietary polysaccharides improve human intestinal health. Understanding the effect of TFPS, safe and healthy food components, on gut health increases the likelihood that TFPS will be developed as a functional food.

Traditional Chinese Medicine Li-Zhong-Tang accelerates the healing of indomethacin-induced gastric ulcers in rats by affecting TLR-2/MyD88 signaling pathway

Ethnopharmacological relevanceLi-Zhong-Tang (LZT) is a well-known Chinese herbal formulation first described in one of traditional Chinese medicine (TCM) scriptures, Treatise on Febrile Diseases. LZT has been commonly prescribed for the treatment of various gastrointestinal diseases for over 1800 years, and has demonstrated pronounced therapeutic effects on patients with gastric ulcers. Aim of the studyThe present study aimed to scientifically evaluate protective effects of LZT on indomethacin (IND)-induced gastric injury in rats and to elucidate whether LZT exerts its gastro-protective effects via enhancing mucosal immunity by regulating TLR-2/MyD88 signaling pathway. Material and methodsGastric ulcers were induced in male Sprague-Dawley (SD) rats with a single oral dose of 150 mg/kg IND. Ulcer index (UI) and curative index (CI) were evaluated. Histopathological examinations were performed and microscopic score (MS) was macroscopically calculated. The volume of gastric juice, free acidity, total acidity, and gastric pH was measured. The gastroprotective and inflammatory biomarkers including levels of nitric oxide (NO), tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and malondialdehyde (MDA) were determined. Expression levels of TLR-2 and MyD88 mRNA were assessed by qRT-PCR. The expression, distribution, and co-localization of TLR-2 and MyD88 protein were determined by Western blot, immunohistochemistry, and immunofluorescence, respectively. ResultsInduction of gastric ulcers in rats resulted in very significantly increased UI and elevated volume and acidity of gastric juice, which were markedly attenuated by LZT treatment. Microscopic examinations of the IND-induced gastric ulcers revealed severe gastric hemorrhagic necrosis, submucosal edema, and destruction of epithelial cells, which were significantly attenuated in LZT-treated rats. Moreover, treatment with LZT remarkably increased gastric mucosal levels of PGE2 and NO, and lowered highly elevated levels of TNF-α and MDA in gastric ulcerative rats. Mechanistically, LZT inhibited mRNA and protein expression of TLR-2 and MyD88 and enhanced immune function in gastric mucosa. Immunohistochemical analyses and immunofluorescent detection further confirmed a markedly decreased co-localization of TLR-2 and MyD88 protein in the gastric mucosa of LZT-treated rats as compared to that of gastric ulcerative rats. ConclusionsThese findings indicate that LZT alleviates serious gastric mucosal ulcerations induced by IND. Protective effects of LZT on gastric ulcers are believed to be associated with the intensification of the anti-oxidative defense system, mitigation of proinflammatory cytokines, stimulation of the production of cytoprotective mediators, and improvement of the mucosal immunity through TLR-2/MyD88 signaling pathway.

Characterization and strong risk association of TLR2 del -196 to -174 polymorphism and Helicobacter pylori and their influence on mRNA expression in gastric cancer.

BACKGROUNDToll-like receptor-2 (TLR2) is responsible for recognizing Helicobacter pylori (H. pylori) and activating the immune response. Polymorphisms in TLR2 may modulate gastric carcinogenesis.AIMTo evaluate whether the TLR2 19216T/C (rs3804099) and TLR2 -196 to -174 ins/del (rs111200466) polymorphisms contribute to gastric carcinogenesis in the Brazilian population, and to determine the influence of both polymorphisms and H. pylori infection on TLR2 mRNA expression.METHODSDNA was extracted from 854 peripheral blood leukocyte or gastric tissue samples [202 gastric cancer (GC), 269 chronic gastritis (CG), and 383 control/healthy (C)] and genotyped by allele-specific PCR or restriction fragment length polymorphism (RFLP)-PCR. Quantitative polymerase chain reaction by TaqMan® assay was used to quantify TLR2 mRNA levels in fresh gastric tissues (48 GC, 36 CG, and 14 C).RESULTSRegarding the TLR2 -196 to -174 polymorphism, the ins/del and del/del genotypes were associated with a higher risk of GC by comparison with the C in all of the analyzed inheritance models (codominant, dominant, recessive, overdominant and log-additive; P < 0.0001). Similarly, an increased risk was observed when comparing the GC and CG groups [codominant (P < 0.0001), dominant (P < 0.0001), recessive (P = 0.0260), overdominant (P < 0.0001) and log-additive (P < 0.0001)]. In contrast, TLR2 19216T/C was associated with a protective effect in the GC group compared to the C group [dominant (P = 0.0420) and log-additive (P = 0.0300)]. Regarding the association of polymorphisms with H. pylori infection, individuals infected with H. pylori and harboring the TLR2 -196 to -174 ins/del polymorphism had an increased risk of gastric carcinogenesis [codominant (P = 0.0120), dominant (P = 0.0051), overdominant (P = 0.0240) and log-additive (P = 0.0030)], while TLR2 19216T/C was associated with a protective effect [codominant (P = 0.0039), dominant (P < 0.0001), overdominant (P = 0.0097) and log-additive (P = 0.0021)]. TLR2 mRNA levels were significantly increased in the GC group (median RQ = 6.95) compared to the CG group (RQ = 0.84, P < 0.0001) and to the normal mucosa group (RQ = 1.0). In addition, both H. pylori infection (P < 0.0001) and the presence of the polymorphic TLR2 -196 to -174del (P = 0.0010) and TLR2 19216 C (P = 0.0004) alleles influenced TLR2 mRNA expression.CONCLUSIONThe TLR2 -196 to -174 ins/del and TLR2 19216 T/C polymorphisms are strongly associated with GC. TLR2 mRNA expression levels are upregulated in neoplastic tissues and influenced by both the presence of H. pylori and variant genotypes.

Antibacterial and immunomodulatory effects of Pheromonicin-NM on Escherichia coli-challenged bovine mammary epithelial cells

Mastitis affects cows in all regions of the world and Escherichia coli (E. coli) is by far the most common reason of mastitis. Now antibiotic therapy is still the preferred approach of treating mastitis. However, antibiotic usage is easy to lead to antibiotic resistance. There is an urgent need for developing efficacious alternative antimicrobials. Pheromonicin-NM (PMC-NM) is a new engineered bactericidal peptide consisting of colicin Ia and an anti-porin A antibody mimetic. It can lead to the dissipation of cellular energy and therefore kill the bacteria rapidly. The aim of the present study was to investigate the comparative effects of PMC-NM and antibiotic ceftiofur on antibacterial and innate immune responses of bovine mammary epithelial cells (BMEC) to E. coli infection. We found that E. coli growth was inhibited by PMC-NM from 0.5 h after treatment and was completely inhibited at 3 h, indicating a rapid antibacterial activity for PMC-NM. The mRNA expression of TLR2, IL-1β, IL-8, lactoferrin, LAP, TAP and DEFB1 was increased by PMC-NM treatment at 2 h after E. coli infection, suggesting the enhanced inflammatory responses induced by PMC-NM contribute to pathogens clearance at early phase. By contrast, in E. coli-infected BMECs, ceftiofur treatment upregulated TLR2 and NOD2 levels at 12 h, and extremely elevated transcription levels of TNF-α, IL-1β, IL-8, lactoferrin, LAP, TAP, BNBD5, DEFB1 at 6 h. The excessive expression of these genes at later phase can induce uncontrolled inflammatory responses and finally cause damage. Taken together, PMC-NM might be used as an ideal antibacterial agent against E. coli mastitis.

Selenium Deficiency via the TLR4/TRIF/NF-κB Signaling Pathway Leading to Inflammatory Injury in Chicken Spleen.

The aim of the present study was to investigate the effect of selenium (Se) deficiency on the expression of the toll-like receptor (TLR) signal transduction pathway in the spleen of chickens and explore the relationship between the TLR4/TRIF/NF-κB signaling pathway and inflammatory spleen injury. A total of 200 one-day-old healthy broilers were allocated to two groups. The experimental group was fed a self-made low-Se diet (0.004mg/kg) while the control group was fed a complete formula feed (0.2mg/kg) for 15, 25, 35, 45, and 55days, respectively. We observed histopathological changes in the chicken spleens. The messenger RNA(mRNA) expression levels of 8 kinds of ChTLRs, myeloid differential protein-88 (MyD88), toll-interleukine-1 receptor domain-containing adapter-inducing interferon-β (TRIF), nuclear factor-κB (NF-κB), and cytokine (IL-6, TNF-α, IL-2, and IFN-γ) were detected via quantitative real-time PCR. Western blotting was used to detect the protein expression level of TLR4. Then principal component analysis (PCA) was used to analyze the correlation between the ChTLRs, MyD88, TRIF, and NF-κB. The results showed that the boundary between red pulp and white pulp was unclear, the number of lymphocytes decreased, and the nucleus was fragmented and dissolved in the experimental group at 25-55days. At 15-45days, the relative expression of TLR4 mRNA was higher than in the control group, and the difference was extremely significant on day 15 (P < 0.01).The relative expression of TRIF mRNA in the experimental group was higher than in the control group at 25-55days, and the relative expression of NF-κB mRNA in the experimental group was higher than in the control group at 15-45days. The relative expression of IL-6 mRNA in the experimental group was higher than in the control group at 15-45days. The protein expression level of TLR4 in the experimental group was higher than in the control group at 15-45days. The PCA results showed that there was a strong correlation between TLR4, TRIF, and component 1. The results suggest that TLR4 plays an important role in regulating the expression of inflammatory cytokines in the spleens of Se-deficient chickens, and Se deficiency may cause inflammatory injury through the TLR4/TRIF/NF-κB signaling pathway in chicken spleen.

Differential gene analysis during the development of obliterative bronchiolitis in a murine orthotopic lung transplantation model: A comprehensive transcriptome-based analysis.

BackgroundObliterative bronchiolitis (OB) is a known issue during minor histocompatibility antigen (mHA) disparity during lung transplantation. This study evaluated gene expression in a murine orthotropic lung transplantation model using microarray analysis.MethodsLeft lungs from C57BL/10(H-2b) donor mice were transplanted into mHA-mismatched C57BL/6(H-2b) recipient mice. Three groups (OB, non-OB, and sham controls) were confirmed pathologically and analyzed. Gene expression changes in the lung grafts were determined by microarray and immunohistochemical staining, and genes were verified by quantitative PCR in the lungs and mediastinal lymph nodes (LNs).ResultsA total of 1343 genes were upregulated in the OB lungs compared to the sham group. Significant upregulation was observed for genes related to innate, e.g. Tlr2 and CCL3 and adaptive immunity, e.g. H2-ab1 and Il-21. Positive labeling for MHC class II antigen was observed in the bronchial epithelium of OB accompanied with B cells. We found increased Tlr2, Ccl3, H2-ab1, Il-21, Ighg3, Ifng, and Pdcd1 mRNA expression in the OB lung, and increased Il-21, Ighg3, and Pdcd1 expression in the OB LNs.ConclusionsAdaptive and innate immune reactions were involved in OB after lung transplantation, and genetic examination of related genes could be used for detection of OB.

Maternal dietary uridine supplementation reduces diarrhea incidence in piglets by regulating the intestinal mucosal barrier and cytokine profiles.

Nucleotides play an important role in the regulation of cellular energy and protein homeostasis, which facilitate the repair, recovery and repletion of tissue function. This study tested the effects of maternal uridine (UR) supplementation during late pregnancy and lactation of sows on the immune function of the small intestine in neonatal and suckling piglets. Results showed that compared to the control group, maternal dietary UR supplementation significantly decreased incidence of diarrhea in suckling piglets (P < 0.01); and increased both duodenal and ileal average villus height (P < 0.01) as well as villus height/crypt depth in ileum (P = 0.017) in neonatal piglets. RT-qPCR results showed that maternal UR supplementation decreased mRNA expression of claudin-1 in jejunum and ileum of neonatal piglets (P < 0.05), while significantly increased mRNA expression of claudin-1 in duodenum and jejunum of suckling piglets. Furthermore, in suckling piglets, maternal dietary UR supplementation increased mRNA expression of IL-6, IL-8 and IL-1β in duodenum, jejunum and ileum (P < 0.05), increased IL-10 expression in both jejunal and ileal mucosa (P < 0.05) and increased mRNA expression of IKB and TLR4 in ileal mucosa (P < 0.05). These results suggest that maternal dietary supplementation with UR contributed to reducing incidence of diarrhea by regulating cytokine secretion and intestinal mucosal barrier function in suckling piglets. © 2020 Society of Chemical Industry.