Intestinal Toll-like Receptor Research Articles

Effects of Shu Bu Wenshen Guchang recipe on intestinal injury and the TLR4/NF-κB signaling pathways in mice with irinotecan-induced delayed-type diarrhea.

BackgroundIrinotecan (also known as CPT-11) is a topoisomerase I inhibitor that is primarily used for the treatment of advanced colorectal cancer. CPT-11 and its active metabolite SN-38 can directly damage intestinal mucosal cells. In addition, CPT-11 can activate the Toll-like receptor 4 (TLR4) inflammasome/nuclear factor kappa-B p65 (NF-κB p65) pathway, ultimately leading to intestinal inflammation-related injury. Shu Bu Wenshen Guchang recipe (SBWGR) has the spleen and kidneys. Herein, we investigated the effects of SBWGR on intestinal injury and the TLR4/NF-κB signaling pathways in mice with CPT-11-induced delayed-type diarrhea, aiming to provide evidence for the treatment of CPT-11-induced delayed-type diarrhea.MethodsThirty tumor-bearing mice were divided into normal control, model control, octreotide, low dose SBWGR, and high dose SBWGR groups, with 6 mice in each group. After successful modelling of delayed diarrhea, the normal and model control groups were given equal amounts of saline for 5 consecutive days, and the other three groups gave the corresponding intra-drug administration. Body weight, tumor size, Chiu score, intestinal ischemia and reperfusion injury, and disease activity index (DAI) were recorded in each group. The levels of intestinal interleukin-1β (IL-1β), IL-18, and tumor necrosis factor-α (TNF-α) were measured by an enzyme-linked immunosorbent assay (ELISA). Intestinal TLR4 and NF-κB p65 levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and protein blotting.ResultsThe weight of octreotide and kidney was higher than the control group (P<0.05); The tumor volume comparison of the model control group, octreotide group, warm kidney intestine low dose group, and warm kidney intestine high dose group were not significantly different (P>0.05). Octreotide group, intestinal Chiu score, diarrhea score, DAI level, intestinal inflammatory cytokines, IL-1β, IL-18 and TNF-α intestinal level, intestinal TLR4, NF-κB p65 mRNA protein expression levels were significantly lower than those of the model control group (P<0.05), and the amount of the treatment group was increased (P<0.05).ConclusionsSBWGR exerts a prominent protective effect on intestinal damage caused by CPT-11-induced delayed-type diarrhea, which may be achieved by inhibiting the activation of the intestinal TLR4/NF-κB signaling pathway.

Intestinal TLR4 deletion exacerbates acute pancreatitis through gut microbiota dysbiosis and Paneth cells deficiency

ABSTRACT Toll-like receptor 4 (TLR4) has been identified as a potentially promising therapeutic target in acute pancreatitis (AP). However, the role of intestinal TLR4 in AP and AP-associated gut injury remains unclear. This study aimed to explore the relationship between intestinal TLR4 and gut microbiota during AP. A mouse AP model was establish by intraperitoneal injection of L-arginine. Pancreatic injury and intestinal barrier function were evaluated in wild-type and intestinal epithelial TLR4 knockout (TLR4ΔIEC) mice. Gut microbiota was analyzed by 16S rRNA sequencing. Quadruple antibiotics were applied to induce microbiota-depleted mice. Differentially expressed genes in gut were detected by RNA sequencing. L. reuteri treatment was carried out in vivo and vitro study. Compared with wild-type mice, AP and AP-associated gut injury were exacerbated in TLR4ΔIEC mice in a gut microbiota-dependent manner. The relative abundance of Lactobacillus and number of Paneth cells remarkably decreased in TLR4ΔIEC mice. The KEGG pathway analysis derived from RNA sequencing suggested that genes affected by intestinal TLR4 deletion were related to the activation of nod-like receptor pathway. Furthermore, L. reuteri treatment could significantly improve the pancreatic and intestinal injury in TLR4ΔIEC mice through promoting Paneth cells in a NOD2-dependent manner. Loss of intestinal epithelial TLR4 exacerbated pancreatic and intestinal damage during AP, which might be attributed to the gut microbiota dysbiosis especially the exhausted Lactobacillus. L. reuteri might maintain intestinal homeostasis and alleviate AP via Paneth cells modulation. Abbreviations: AP Acute pancreatitis, TLR4 Toll-like receptor 4, IL-1β Interleukin-1β, IL-6 Interleukin-6, TNF-α Tumor necrosis factor-α, SIRS Systematic inflammatory response syndrome, LPS Lipopolysaccharides, SPF Specific pathogen-free, ZO-1 Zonula occludens-1, CON Control, H&E Hematoxylin and eosin, FISH Fluorescence in situ hybridization, DAPI 4′,6-diamidino-2-phenylindole, PCoA Principal co-ordinates analysis, SCFA Short chain fatty acid, LEfSe Linear discriminant analysis Effect Size, ANOVA Analysis of variance, F/B Firmicutes/Bacteroidetes, PCA Principal component analysis, NOD2 Nod-like receptor 2, ABX antibiotics, PCNA proliferating cell nuclear antigen

Gut microbiota is causally associated with poststroke cognitive impairment through lipopolysaccharide and butyrate

BackgroundPoststroke cognitive impairment (PSCI) is prevalent in stroke patients. The etiology of PSCI remains largely unknown. We previously found that stroke induces gut microbiota dysbiosis which affects brain injury. Hereby, we aimed to investigate whether the gut microbiota contributes to the pathogenesis of PSCI.Methods83 stroke patients were recruited and their cognitive function were measured by Montreal Cognitive Assessment (MoCA) scores 3 months after stroke onset. The peripheral inflammatory factor levels and gut microbiota compositions of the patients were analyzed. Fecal microbiota transplantation from patients to stroke mice was performed to examine the causal relationship between the gut microbiota and PSCI. The cognitive function of mice was evaluated by Morris water maze test.Results34 and 49 stroke patients were classified as PSCI and non-PSCI, respectively. Compared with non-PSCI patients, PSCI patients showed significantly higher levels of gut Enterobacteriaceae, lipopolysaccharide (LPS) and peripheral inflammation markers. Consistently, stroke mice that received microbiota from PSCI patients (PSCI mice) presented a higher level of Enterobacteriaceae, intestinal Toll-like receptor-4 (TLR4) expression, circulating LPS, LPS-binding protein (LBP) and inflammatory cytokines, and a lower level of fecal butyrate, severer intestine destruction and cognitive impairment than mice that received microbiota from nPSCI patients (nPSCI mice). In addition, we observed exacerbations in blood–brain barrier (BBB) integrity, microglial activation, neuronal apoptosis in the CA1 region of the hippocampus, and Aβ deposition in the thalamus of PSCI mice in comparison with nPSCI mice. Intraperitoneal injection of LPS after stroke caused similar pathology to those seen in PSCI mice. Supplementation with sodium butyrate (NaB) via drinking water rescued these detrimental changes in PSCI mice.ConclusionsOur data indicate a cause–effect relationship between gut microbiota and PSCI for the first time, which is likely mediated by inflammation-regulating metabolites including LPS and butyrate.

Lactoferrin modulates gut microbiota and Toll-like receptors (TLRs) in mice with dysbiosis induced by antibiotics.

Background: Antibiotic administration can result in gut microbiota and immune system alterations that impact health. Bovine lactoferrin is a milk protein with anticancer, anti-inflammatory, antimicrobial and immune modulatory activities. The aim was to study the ability of native and iron-saturated lactoferrin to reverse the effects of clindamycin on gut microbiota and intestinal Toll-like receptor (TLR) expression in a murine model. Methods: Male C57BL/6 mice were treated with vehicle, clindamycin (Clin), native bovine lactoferrin (nLf), nLf + clindamycin (nLf_Clin), iron-saturated bovine lactoferrin (sLf) and sLf + clindamycin (sLf_Clin). Fecal samples of each group were collected, and bacterial DNA was extracted. Sequencing of 16s rRNA V4 hypervariable gene regions was conducted to assess the microbial composition. mRNA expression levels of TLRs (1-9) were determined in mouse colon by qPCR. Pearson's correlation test was carried out between bacteria showing differences in abundance among samples and TLR2, TLR8 and TLR9. Results: Beta-diversity analysis showed that the microbial community of the vehicle was different from the communities of Clin, nLf_Clin and sLf_Clin. At the family level, Bacteroidaceae, Prevotellaceae and Rikenellaceae decreased in the Clin group, and treatment with nLf or sLf reverted these effects. Clin reduced the expression of TLR2, TLR8 and TLR9 and sLf reverted the decrease in the expression of these receptors. Finally, TLR8 was positively correlated with Rikenellaceae abundance. Conclusion: In a situation of intestinal dysbiosis induced by clindamycin, lactoferrin restores the normal levels of some anti-inflammatory bacteria and TLRs and, therefore, could be a good ingredient to be added to functional foods.

Expression of Toll-Like Receptors 2, 4 and 5 in Relation to Gut Microbiota in Colon Neoplasm Patients with and without Inflammatory Bowel Disease.

Background:Toll-Like Receptors (TLRs) are the critical mediators of inflammatory routs in the gut, which play an essential role in regulating the immune responses towards various ligands derived from pathogenic bacteria. Also, TLR signaling has been implicated in the development of Inflammatory Bowel Disease (IBD), Adenomatous Polyp (AP), and Colorectal Cancer (CRC). Here, we aimed to examine the expression of some TLRs concerning certain fecal bacteria in AP and CRC patients with and without IBD.Methods:This case-control study collected fecal and colonic tissue samples from 93 patients versus Normal Controls (NC) via colonoscopy. Fecal samples were used for DNA extraction, and the abundance of selected fecal bacteria was determined by absolute real-time PCR. Also, the gene expression of TLR2, 4, and 5 was analyzed using RT-PCR on the colonic tissues of participants.Results:Compared to NC individuals, in AP and CRC patients, the mRNA expressions of TLR4 and TLR2 were significantly increased while TLR5 was decreased. A meaningful association between TLRs mRNA expression levels and the abundance of some selected fecal bacteria was detected. Also, there was a significant relationship between participant’s food regimes, smoking habit and intestinal TLRs expression.Conclusion:Our study proposed the important role of TLRs during adenomatous and CRC formation. Alterations in TLRs expression associated with certain gut bacteria may contribute to disease development.

Long-chain PUFA ameliorate enterotoxigenic Escherichia coli-induced intestinal inflammation and cell injury by modulating pyroptosis and necroptosis signaling pathways in porcine intestinal epithelial cells.

This study was aimed to investigate whether EPA and arachidonic acid (ARA), the representative n-3 or n-6 PUFA, could alleviate enterotoxigenic Escherichia coli (ETEC) K88-induced inflammation and injury of intestinal porcine epithelial cells 1 (IPEC-1) by modulating pyroptosis and necroptosis signalling pathways. IPEC-1 cells were cultured with or without EPA or ARA in the presence or absence of ETEC K88. EPA and ARA reduced ETEC K88 adhesion and endotoxin content in the supernatant. EPA and ARA increased transepithelial electrical resistance, decreased permeability of fluorescein isothiocyanate-labelled dextran, increased membrane protein expression of occludin, ZO-1 and claudin-1 and relieved disturbed distribution of these proteins. EPA and ARA also reduced cell necrosis ratio. EPA or ARA reduced mRNA and concentration of TNF-α, IL-6 and IL-8 and decreased mRNA abundances of intestinal toll-like receptors 4 and its downstream signals. Moreover, EPA and ARA downregulated mRNA expression of nod-like receptor protein 3 (NLRP3), caspase 1 and IL-18 and inhibited protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D and caspase-1. Finally, EPA and ARA reduced mRNA expression of fas-associated death domain protein, caspase 8, receptor-interacting protein kinase (RIP) 1, mixed lineage kinase-like protein (MLKL), phosphoglycerate mutase 5 (PGAM5), motility-related protein 1 (Drp1) and high mobility protein 1 (HMGB1) and inhibited protein expression of phosphorylated-RIP1, p-RIP3, p-MLKL and HMGB1. These data demonstrate that EPA and ARA prevent ETEC K88-induced cell inflammation and injury, which is partly through inhibiting pyroptosis and necroptosis signalling pathways.

Intestinal toll-like receptor 4 knockout alters the functional capacity of the gut microbiome following irinotecan treatment.

Irinotecan can cause high levels of diarrhea caused by toxic injury to the gastrointestinal microenvironment. Toll-like receptor 4 (TLR4) and the gut microbiome have previously been implicated in gastrointestinal toxicity and diarrhea; however, the link between these two factors has not been definitively determined. We used a tumor-bearing, intestinal epithelial cell (IEC) TLR4 knockout model (Tlr4ΔIEC) to assess microbiome changes following irinotecan treatment. We then determined if a fecal microbiota transplant (FMT) between Tlr4ΔIEC and wild-type (WT) mice altered irinotecan-induced gastrointestinal toxicity. MC-38 colorectal cancer cells were injected into WT and Tlr4ΔIEC mice. Fecal samples were collected prior to tumor inoculation, prior to irinotecan treatment and at cull. 16S rRNA gene sequencing was used to assess changes in the microbiome. Next, FMT was used to transfer themicrobiome phenotype between Tlr4ΔIEC and WT mice prior to irinotecan treatment. Gastrointestinal toxicity symptoms were assessed. In study 1, there were no compositional differences in the microbiome between Tlr4ΔIEC and WT mice at baseline. However, predicted functional capacity of the microbiome was different between WT and Tlr4ΔIEC at baseline and post-irinotecan. In study 2, Tlr4ΔIEC mice were protected from grade 3 diarrhea. Additionally, WT mice who did not receive FMT had more colonic damage in the colon compared to controls (P = 0.013). This was not seen in Tlr4ΔIEC mice or WT mice who received FMT (P > 0.05). Tlr4ΔIEC and WT had no baseline compositional microbiome differences, but functional differences at baseline and following irinotecan. FMT altered some aspects of irinotecan-induced gastrointestinal toxicity.

Toll-like receptor 4-mediated enteric glia loss is critical for the development of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants, whose pathogenesis remains incompletely understood, although activation of the Gram-negative bacterial receptor Toll-like receptor 4 (TLR4) on the intestinal epithelium plays a critical role. Patients with NEC typically display gastrointestinal dysmotility before systemic disease is manifest, suggesting that dysmotility could drive NEC development. Both intestinal motility and inflammation are governed by the enteric nervous system, a network of enteric neurons and glia. We hypothesized here that enteric glia loss in the premature intestine could lead to dysmotility, exaggerated TLR4 signaling, and NEC development. We found that intestinal motility is reduced early in NEC in mice, preceding the onset of intestinal inflammation, whereas pharmacologic restoration of intestinal motility reduced NEC severity. Ileal samples from mouse, piglet, and human NEC revealed enteric glia depletion, and glia-deficient mice (Plp1ΔDTR, Sox10ΔDTR, and BdnfΔDTR) showed increased NEC severity compared with wild-type mice. Mice lacking TLR4 on enteric glia (Sox10-Tlr4ko) did not show NEC-induced enteric glia depletion and were protected from NEC. Mechanistically, brain-derived neurotrophic factor (BDNF) from enteric glia restrained TLR4 signaling on the intestine to prevent NEC. BDNF was reduced in mouse and human NEC, and BDNF administration reduced both TLR4 signaling and NEC severity in enteric glia–deficient mice. Last, we identified an agent (J11) that enhanced enteric glial BDNF release, inhibited intestinal TLR4, restored motility, and prevented NEC in mice. Thus, enteric glia loss might contribute to NEC through intestinal dysmotility and increased TLR4 activation, suggesting enteric glia therapies for this disorder.

Review of the contribution of genetic factors to hyperbilirubinemia and kernicterus risk in neonates: a targeted update

: The genesis of neonatal hyperbilirubinemia is characterized by etiologic heterogeneity, environmental modulation, and the interaction of multiple gene loci. In addition to inherited hemolytic conditions, common icterogenic gene variants may act as modifiers of hyperbilirubinemia and kernicterus risk. The current review targets the effect of biologic sex, uridine diphosphate glucuronosyltransferase isoenzyme UGT1A1 gene variants of Gilbert syndrome, including their role in breastmilk jaundice, and the co-expression of icterogenic alleles have on potentiating hyperbilirubinemia risk in neonates. Notably, evidence accrued during the past two decades, from around the globe, confirm that breastmilk jaundice is a prevalent Gilbert syndrome phenotype in neonates. Moreover, novel data from humanized murine models suggest an important repressive effect of breastmilk oligosaccharides on intestinal (as opposed to hepatic) UGT1A1 expression in driving breast milk jaundice risk. More specifically, human milk oligosaccharides block intestinal Toll-like receptor activation and downstream IĸB kinase phosphorylation. This in turn represses newborn intestinal UGT1A1 activity. Formula feeding, by contrast, activates IĸB and induces intestinal (but not hepatic) UGT1A1 activity thereby lowering the total serum bilirubin (TSB). Whether this phenomenon is operative in human neonates is unclear. Although UGT1A1 is expressed in adult intestine, there are no comparable developmental data on intestinal UGT1A1 expression in the human fetus or neonate, a knowledge gap that is ripe for clinical investigation.

Surfactant protein A reduces TLR4 and inflammatory cytokine mRNA levels in neonatal mouse ileum

Levels of intestinal toll-like receptor 4 (TLR4) impact inflammation in the neonatal gastrointestinal tract. While surfactant protein A (SP-A) is known to regulate TLR4 in the lung, it also reduces intestinal damage, TLR4 and inflammation in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. We hypothesized that SP-A-deficient (SP-A−/−) mice have increased ileal TLR4 and inflammatory cytokine levels compared to wild type mice, impacting intestinal physiology. We found that ileal TLR4 and proinflammatory cytokine levels were significantly higher in infant SP-A−/− mice compared to wild type mice. Gavage of neonatal SP-A−/− mice with purified SP-A reduced ileal TLR4 protein levels. SP-A reduced expression of TLR4 and proinflammatory cytokines in normal human intestinal epithelial cells (FHs74int), suggesting a direct effect. However, incubation of gastrointestinal cell lines with proteasome inhibitors did not abrogate the effect of SP-A on TLR4 protein levels, suggesting that proteasomal degradation is not involved. In a mouse model of experimental NEC, SP-A−/− mice were more susceptible to intestinal stress resembling NEC, while gavage with SP-A significantly decreased ileal damage, TLR4 and proinflammatory cytokine mRNA levels. Our data suggests that SP-A has an extrapulmonary role in the intestinal health of neonatal mice by modulating TLR4 and proinflammatory cytokines mRNA expression in intestinal epithelium.

Epithelial-Specific TLR4 Knockout Challenges Current Evidence of TLR4 Homeostatic Control of Gut Permeability

Introduction: Toll-like receptor 4 (TLR4) is a highly conserved immunosurveillance protein of innate immunity, displaying well-established roles in homeostasis and intestinal inflammation. Current evidence shows complex relationships between TLR4 activation, maintenance of health, and disease progression; however, it commonly overlooks the importance of site-specific TLR4 expression. This omission has the potential to influence translation of results as previous evidence shows the differing and distinct roles that TLR4 exhibits are dependent on its spatiotemporal expression. Methods: An intestinal epithelial TLR4 conditional knockout (KO) mouse line (Tlr4^ΔIEC, n = 6–8) was utilized to dissect the contribution of epithelial TLR4 expression to intestinal homeostasis with comparisons to wild-type (WT) (n = 5–7) counterparts. Functions of the intestinal barrier in the ileum and colon were assessed with tissue resistance in Ussing chambers. Molecular and structural comparisons in the ileum and colon were assessed via histological staining, expression of tight junction proteins (occludin and zonular occludin 1 [ZO-1]), and presence of CD11b-positive immune cells. Results: There was no impact of the intestinal epithelial TLR4 KO, with no differences in (1) tissue resistance–ileum (mean ± standard error of mean [SEM]): WT 22 ± 7.2 versus Tlr4^ΔIEC 20 ± 5.6 (Ω × cm²) p = 0.831, colon WT 30.8 ± 3.6 versus Tlr4^ΔIEC 45.1 ± 9.5 p = 0.191; (2) histological staining (overall tissue structure); and (3) tight junction protein expression (% area stain, mean ± SEM)–ZO-1: ileum–WT 1.49 ± 0.155 versus Tlr4^ΔIEC 1.17 ± 0.07, p = 0.09; colon–WT 1.36 ± 0.26 versus Tlr4^ΔIEC 1.12 ± 0.18 p = 0.47; occludin: ileum–WT 1.07 ± 0.12 versus Tlr4^ΔIEC 0.95 ± 0.13, p = 0.53; colon–WT 1.26 ± 0.26 versus Tlr4^ΔIEC 1.02 ± 0.16 p = 0.45. CD11b-positive immune cells (% area stain, mean ± SEM) in the ileum were mildly decreased in WT mice: WT 0.14 ± 0.02 versus Tlr4^ΔIEC 0.09 ± 0.01 p = 0.04. However, in the colon, there was no difference in CD11b-positive immune cells between strains: WT 0.53 ± 0.08 versus Tlr4^ΔIEC 0.49 ± 0.08 p = 0.73. Conclusions: These data have 2 important implications. First, these data refute the assumption that epithelial TLR4 exerts physiological control of intestinal physiology and immunity in health. Second, and most importantly, these data support the use of the Tlr4^ΔIEC line in future models interrogating health and disease, confirming no confounding effects of genetic manipulation.

Small Bowel Resection Increases Paracellular Gut Barrier Permeability via Alterations of Tight Junction Complexes Mediated by Intestinal TLR4

BackgroundShort bowel syndrome resulting from small bowel resection (SBR) is associated with significant morbidity and mortality. Many adverse sequelae including steatohepatitis and bacterial overgrowth are thought to be related to increased bacterial translocation, suggesting alterations in gut permeability. We hypothesized that after intestinal resection, the intestinal barrier is altered via toll-like receptor 4 (TLR4) signaling at the intestinal level. MethodsB6 and intestinal-specific TLR4 knockout (iTLR4 KO) mice underwent 50% SBR or sham operation. Transcellular permeability was evaluated by measuring goblet cell associated antigen passages via two-photon microscopy. Fluorimetry and electron microscopy evaluation of tight junctions (TJ) were used to assess paracellular permeability. In parallel experiments, single-cell RNA sequencing measured expression of intestinal integral TJ proteins. Western blot and immunohistochemistry confirmed the results of the single-cell RNA sequencing. ResultsThere were similar number of goblet cell associated antigen passages after both SBR and sham operation (4.5 versus 5.0, P > 0.05). Fluorescein isothiocyanate–dextran uptake into the serum after massive SBR was significantly increased compared with sham mice (2.13 ± 0.39 ng/μL versus 1.62 ± 0.23 ng/μL, P < 0.001). SBR mice demonstrated obscured TJ complexes on electron microscopy. Single-cell RNA sequencing revealed a decrease in TJ protein occludin (21%) after SBR (P < 0.05), confirmed with immunostaining and western blot analysis. The KO of iTLR4 mitigated the alterations in permeability after SBR. ConclusionsPermeability after SBR is increased via changes at the paracellular level. However, these alterations were prevented in iTLR4 mice. These findings suggest potential protein targets for restoring the intestinal barrier and obviating the adverse sequelae of short bowel syndrome.

Effect of electroacupuncture on intestinal Toll-like receptor 4 and nuclear factor-kappa B in obese rats

To investigate the effect of electroacupuncture (EA) on intestinal Toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) in obese rats, so as to explore the mechanism of action of acupuncture in losing weight. A total of 50 male Wistar rats were randomly divided into control and model groups. High-fat feed was used to establish a rat model of obesity, and after modeling, the 24 rats were randomly divided into model group, TLR4 inhibitor group, and EA group, with 8 rats in each group. The rats in the EA group were given EA at "Guanyuan" (CV4), "Zhongwan "(CV12), "Zusanli" (ST36), and" Fenglong" (ST40), 10 minutes each time, 3 times a week, and those in the TLR4 inhibitor group were given intraperitoneal injection of TAK-242 three times a week; the course of treatment was 8 weeks for both groups. Body weight and blood glucose were measured every two weeks. Co-immunoprecipitation was used to observe the interaction between TLR4 and NF-κB p65 in the intestinal tissue; electrophoretic mobility shift assay was used to measure the activity of NF-κB p65; Western blot was used to measure the protein expression of TLR4, phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (p-IκBα), and NF-κB p65; quantitative real-time PCR was used to measure the mRNA expression of TLR4, NF-κB p65, and IκBα. Compared with the control group, the model group had significant increases in body weight, blood glucose, and protein and mRNA expression of TLR4 and NF-κB p65 (P<0.01, P<0.05), as well as significant enhancement in the interaction between TLR4 and NF-κB p65 and activity of NF-κB p65 (P<0.05，P<0.01). Compared with the model group, the EA group had a significant reduction in body weight (P<0.05), both of the EA group and the TLR4 inhibitor group had significant reductions in blood glucose, and protein and mRNA expression of TLR4, p-IκBα， and NF-κB p65 (P<0.05，P<0.01), as well as significant reductions in the activity of NF-κB p65 (P<0.01). EA can effectively regulate intestinal TLR4, inhibit the interaction between TLR4 and NF-κB p65, and reduce the activity of NF-κB p65, which may be a potential mechanism of EA in reducing body weight and blood glucose in obese rats.

Broad-spectrum antibiotics alter the microbiome, increase intestinal fxr, and decrease hepatic steatosis in zebrafish short bowel syndrome.

Short bowel syndrome (SBS) is associated with changes in the intestinal microbiome and marked local and systemic inflammation. There is also a late complication of SBS, intestinal failure associated liver disease (IFALD) in which hepatic steatosis progresses to cirrhosis. Most patients with SBS arrive at massive intestinal resection after a contaminating intraabdominal catastrophe and have a history of exposure to broad-spectrum antibiotics. We therefore investigated whether the administration of broad-spectrum antibiotics in conjunction with SBS in zebrafish (ZF) would replicate these systemic effects observed in humans to identify potentially druggable targets to aid in the management of SBS and resulting IFALD. In zebrafish with SBS, broad-spectrum antibiotics altered the microbiome, decreased inflammation, and reduced the development of hepatic steatosis. After two weeks of broad-spectrum antibiotics, these fish exhibited decreased alpha diversity, with less variation in microbial community composition between SBS and sham fish. Additionally, administration of broad-spectrum antibiotics was associated with decreased expression of intestinal toll-like receptor 4 (tlr4), increased expression of the intestinal gene encoding the Farnesoid X receptor (fxr), decreased expression of downstream hepatic cyp7a1, and decreased development of hepatic steatosis. SBS in zebrafish reproducibly results in increased epithelial surface area as occurs in human patients who demonstrate intestinal adaptation, but antibiotic administration in zebrafish with SBS reduced these gains with increased cell death in the intervillus pocket that contains stem/progenitor cells. These alternate states in SBS zebrafish might direct the development of future human therapies.NEW & NOTEWORTHY In a zebrafish model that replicates a common clinical scenario, systemic effects of the administration of broad-spectrum antibiotics in a zebrafish model of SBS identified two alternate states that led to the establishment of fat accumulation in the liver or its absence. Broad-spectrum antibiotics given to zebrafish with SBS over 2 wk altered the intestinal microbiome, decreased intestinal and hepatic inflammation, and decreased hepatic steatosis.

Intestinal expression of toll-like receptor gene changes early after gastric bypass surgery and association with type 2 diabetes remission.

Abnormal activation of toll-like receptors (TLRs) is observed in obese rodents and is correlated with local dysbiosis and increased gut permeability. These purported changes trigger systemic inflammation associated with obesity-related comorbidities, including type 2 diabetes (T2D). Roux-en-Y gastric bypass (RYGB) surgery is an effective treatment for severe obesity and known to induce changes in the gut microbiota and decrease systemic inflammation in humans. This study examined the intestinal expression of TLR-encoding genes in obese women (n=20) treated with RYGB surgery and the relationship of these genes with T2D remission (T2Dr METHODS: Intestinal biopsies were performed before and 3 months after RYGB surgery. Partial and complete T2Dr after 1 year was assessed using the American Diabetes Association criteria. Affymetrix Human GeneChip 1.0 ST array (microarray) and TaqMan assay (real-time quantitative polymerase chain reaction) were used to analyze intestinal gene expression, and associations with systemic markers of energy homeostasis were examined. Patients experienced significant weight loss (P < 0.001) and altered gut TLR gene expression 3 months after surgery. The main effects were a reduction in jejunal TLR4 expression in patients with complete and partial T2Dr (P < 0.05). There was a postoperative decrease in jejunal TLR7 expression in patients with complete T2Dr that correlated inversely with high-density lipoprotein cholesterol and positively with triglyceride concentrations, but not with weight loss. RYGB-induced weight loss-independent changes in the expression of intestinal TLR-encoding genes in obese women and complete T2Dr that was correlated with systemic markers of energy homeostasis. The modulation of intestinal TLRs may mediate inflammatory mechanisms linked to T2Dr after RYGB surgery.

Toll-like receptor distribution in colonic epithelium and lamina propria is disrupted in HIV viremic, immune success, and failure.

Since intestinal immunity and the microbiome are disrupted in HIV disease, we studied the abundance of innate immune sensors, Toll-like receptors (TLRs), in the mucosa of participants with viremia, prior to antiretroviral therapy (ART), immune success (>500 CD4 T cells/μl after 2 years of ART; suppressed viremia), and immune failure (<350 CD4 T cells/μl after 2 years of ART; suppressed viremia). We hypothesized that disruption of intestinal TLR abundance and location provides a mechanism behind persistent inflammation. Immunofluorescence for TLR3, TLR4, and TLR9 on paraffin embedded biopsies from uninfected, viremic, immune success, and immune failure colons was imaged by deconvolution microscopy and quantified with MetaMorph software. Plasma levels of C-reactive protein, IL-6, and intestinal fatty-acid binding protein (I-FABP) were correlated with TLR expression. Viremic participants have significantly higher levels of TLR3 and TLR9 on surface epithelium and in crypts when compared with uninfected controls. TLR3 is further elevated in immune failure and immune success. TLR9 abundance remains elevated in immune failure and is normalized in immune success. TLR9 expression in the crypt and lamina propria positively associates with C-reactive protein and IL-6 and negatively with I-FABP. TLR4 is significantly lower on surface epithelium and higher in crypts in viremic. Its expression in the lamina propria positively correlates with IL-6 and negatively correlates with I-FABP. Mucosal TLR imbalance and deregulation, and the resulting mucosal TLR desensitization and hypervigilance, remain after suppressive ART, in the presence or absence of T-cell recovery, likely contributing to chronic systemic inflammation.

Intestinal Dysbiosis and the Developing Lung: The Role of Toll-Like Receptor 4 in the Gut-Lung Axis.

BackgroundIn extremely premature infants, postnatal growth restriction (PNGR) is common and increases the risk of developing bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH). Mechanisms by which poor nutrition impacts lung development are unknown, but alterations in the gut microbiota appear to play a role. In a rodent model, PNGR plus hyperoxia causes BPD and PH and increases intestinal Enterobacteriaceae, Gram-negative organisms that stimulate Toll-like receptor 4 (TLR4). We hypothesized that intestinal dysbiosis activates intestinal TLR4 triggering systemic inflammation which impacts lung development.MethodsRat pups were assigned to litters of 17 (PNGR) or 10 (normal growth) at birth and exposed to room air or 75% oxygen for 14 days. Half of the pups were treated with the TLR4 inhibitor TAK-242 from birth or beginning at day 3. After 14 days, pulmonary arterial pressure was evaluated by echocardiography and hearts were examined for right ventricular hypertrophy (RVH). Lungs and serum samples were analyzed by western blotting and immunohistochemistry.ResultsPostnatal growth restriction + hyperoxia increased pulmonary arterial pressure and RVH with trends toward increased plasma IL1β and decreased IκBα, the inhibitor of NFκB, in lung tissue. Treatment with the TLR4 inhibitor attenuated PH and inflammation.ConclusionPostnatal growth restriction induces an increase in intestinal Enterobacteriaceae leading to PH. Activation of the TLR4 pathway is a promising mechanism by which intestinal dysbiosis impacts the developing lung.

The effect of dietary supplementation with Clostridium butyricum on the growth performance, immunity, intestinal microbiota and disease resistance of tilapia (Oreochromis niloticus).

This study was conducted to assess the effects of dietary Clostridium butyricum on the growth, immunity, intestinal microbiota and disease resistance of tilapia (Oreochromis niloticus). Three hundreds of tilapia (56.21 ± 0.81 g) were divided into 5 groups and fed a diet supplemented with C. butyricum at 0, 1 x 104, 1 x 105, 1 x 106 or 1 x 107 CFU g-1 diet (denoted as CG, CB1, CB2, CB3 and CB4, respectively) for 56 days. Then 45 fish from each group were intraperitoneally injected with Streptococcus agalactiae, and the mortality was recorded for 14 days. The results showed that dietary C. butyricum significantly improved the specific growth rate (SGR) and feed intake in the CB2 group and decreased the cumulative mortality post-challenge with S. agalactiae in the CB2, CB3 and CB4 groups. The serum total antioxidant capacity and intestinal interleukin receptor-associated kinase-4 gene expression were significantly increased, and serum malondialdehyde content and diamine oxidase activity were significantly decreased in the CB1, CB2, CB3 and CB4 groups. Serum complement 3 and complement 4 concentrations and intestinal gene expression of tumour necrosis factor α, interleukin 8, and myeloid differentiation factor 88 were significantly higher in the CB2, CB3 and CB4 groups. Intestinal toll-like receptor 2 gene expression was significantly upregulated in the CB3 and CB4 groups. Dietary C. butyricum increased the diversity of the intestinal microbiota and the relative abundance of beneficial bacteria (such as Bacillus), and decreased the relative abundance of opportunistic pathogenic bacteria (such as Aeromonas) in the CB2 group. These results revealed that dietary C. butyricum at a suitable dose enhanced growth performance, elevated humoral and intestinal immunity, regulated the intestinal microbial components, and improved disease resistance in tilapia. The optimal dose was 1 x 105 CFU g-1 diet.

Intestinal Toll-like receptor 9 deficiency leads to Paneth cell hyperplasia and exacerbates kidney, intestine, and liver injury after ischemia/reperfusion injury

Intestinal Paneth cells play a critical role in ischemic acute kidney injury (AKI) by releasing interleukin 17A (IL-17A). Because Toll-like receptor 9 (TLR9) activation degranulates Paneth cells and necrotic tubular epithelial cells release several damage associated molecular patterns that target TLR9, we tested the hypothesis that intestinal TLR9 deficiency would protect against ischemic AKI and associated remote intestinal and hepatic dysfunction by decreasing Paneth cell degranulation. We generated mice lacking TLR9 in intestinal epithelia (TLR9fl/fl Villin Cre mice) and compared them to wild type (TLR9fl/fl) mice following right nephrectomy and left ischemia/reperfusion. To our surprise, mice lacking intestinal TLR9 had exacerbated kidney, liver, and small intestine injury after ischemia/reperfusion compared to wild type mice, characterized by increased kidney and intestinal inflammation, apoptosis, and necrosis as well as increased hepatic inflammation and apoptosis. Mice lacking intestinal TLR9 had larger Paneth cell granule size, pronounced intestinal macrophage infiltration, and higher intestinal crypt IL-17A expression. Administration of IL-17A neutralizing antibody prevented the exacerbation of ischemic AKI in mice lacking intestinal TLR9. These studies suggest that intestinal TLR9 activation protects against ischemic AKI and associated remote multi-organ dysfunction syndrome by regulating Paneth cell IL-17A synthesis.

Effect of single- and multi-strain probiotics on broiler breeder performance, immunity and intestinal toll-like receptors expression

ABSTRACT Effects of multi-strain (Lactofeed) and mono-strain (Pediguard) probiotics on broiler breeder performance and expression of toll-like receptors (TLR) were evaluated. Three hundred broiler breeder hens (Ross 308) at the age of 51 weeks were randomly allocated into 1 of 5 dietary treatments with 6 replicates in each in a completely randomized design. The dietary treatments included (1) the basal diet (control), (2) control + 0.1 g/kg Lactofeed, (3) control + 0.1 g/kg Pediguard, (4) control + 0.1 g/kg Lactofeed + 0.1 g/kg Pediguard and (5) control + 0.5 g/kg oxytetracycline antibiotic. Compared to the control group, treatments had no effect on hen-day egg production and body weight of broiler breeders (P > .05). The egg yolk cholesterol concentration of broiler breeders fed probiotic-supplemented diet was decreased (P < .05). There were no differences in the immune response to PHA-P injection, serum glutathione peroxidase activity, malondialdehyde and cholesterol concentration and blood haematology of broiler breeder among different dietary treatments (P > .05) while TLR2 and TLR4 mRNA expression up-regulated (P < .05). It can be concluded Lactofeed and Pediguard did not improve broiler breeder performance and T-cell-mediated immune response and are not advisable for breeder nutrition.