Anti-Inflammatory Effects of Idebenone Attenuate LPS-Induced Systemic Inflammatory Diseases by Suppressing NF-κB Activation.

Inflammation is a natural protective process through which the immune system responds to injury, infection, or irritation. However, hyperinflammation or long-term inflammatory responses can cause various inflammatory diseases. Although idebenone was initially developed for the treatment of cognitive impairment and dementia, it is currently used to treat various diseases. However, its anti-inflammatory effects and regulatory functions in inflammatory diseases are yet to be elucidated. Therefore, this study aimed to investigate the anti-inflammatory effects of idebenone in cecal ligation puncture-induced sepsis and lipopolysaccharide-induced systemic inflammation. Murine models of cecal ligation puncture-induced sepsis and lipopolysaccharide-induced systemic inflammation were generated, followed by treatment with various concentrations of idebenone. Additionally, lipopolysaccharide-stimulated macrophages were treated with idebenone to elucidate its anti-inflammatory effects at the cellular level. Idebenone treatment significantly improved survival rate, protected against tissue damage, and decreased the expression of inflammatory enzymes and cytokines in mice models of sepsis and systemic inflammation. Additionally, idebenone treatment suppressed inflammatory responses in macrophages, inhibited the NF-κB signaling pathway, reduced reactive oxygen species and lipid peroxidation, and normalized the activities of antioxidant enzyme. Idebenone possesses potential therapeutic application as a novel anti-inflammatory agent in systemic inflammatory diseases and sepsis.

The Effect of the Mixed Extract of Kalopanax pictus Nakai and Achyranthes japonica Nakai on the Improvement of Degenerative Osteoarthritis through Inflammation Inhibition in the Monosodium Iodoacetate-Induced Mouse Model.

Osteoarthritis is a chronic inflammatory disease, and, due to the lack of fundamental treatment, the main objective is to alleviate pain and prevent cartilage damage. Kalopanax pictus Nakai and Achyranthes japonica Nakai are herbal plants known for their excellent anti-inflammatory properties. The objective of this study is to confirm the potential of a mixture extract of Kalopanax pictus Nakai and Achyranthes japonica Nakai as a functional raw material for improving osteoarthritis through anti-inflammatory effects in macrophages and MIA-induced arthritis experimental animals. In macrophages inflamed by lipopolysaccharide (LPS), treatment of Kalopanax pictus Nakai and Achyranthes japonica Nakai mixture inhibits NF-κB and mitogen-activated protein kinase (MAPK) activities, thereby inhibiting inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), inflammatory factors PGE2, MMP-2, and MMP-9, and nitric oxide (NO) was reduced. In addition, in an animal model of arthritis induced by MIA (monosodium iodoacetate), administration of Kalopanax pictus Nakai and Achyranthes japonica Nakai mixture reduced blood levels of inflammatory cytokines TNF-α and IL-6, inflammatory factors prostaglandin E2(PGE2), matrix metalloproteinase-2(MMP-2), and NO. Through these anti-inflammatory effects, MIA-induced pain reduction (recovery of clinical index, increase in weight bearing, and increase in area and width of the foot), recovery of meniscus damage, loss of cartilage tissue or inflammatory cells in tissue infiltration reduction, and recovery of the proteglycan layer were confirmed. Therefore, it is considered that Kalopanax pictus Nakai and Achyranthes japonica Nakai mixture has the potential as a functional raw material that promotes joint health.

Potential Effect of Enzymatic Porcine Placental Hydrolysate (EPPH) to Improve Alcoholic Liver Disease (ALD) by Promoting Lipolysis in the Liver.

Alcoholic liver disease is associated with the production of highly reactive free radicals by ethanol and its metabolites. Free radicals not only induce liver oxidation and damage tissues, but also stimulate an inflammatory response in hepatocytes, leading to severe liver disease. In order to improve alcoholic liver disease, enzymatic porcine placenta hydrolysate was studied by exploring various materials. Enzymatic porcine placenta hydrolysate (EPPH) contains various amino acids, peptides, and proteins, and is used as a useful substance in the body. In this study, changes were confirmed in indicators related to the antioxidant efficacy of EPPH in vitro and in vivo. EPPH inhibits an EtOH-induced decrease in superoxide dismutase and catalase activity through inhibition of free radicals without endogenous cytotoxicity. EPPH has been observed to have a partial effect on common liver function factors such as liver weight, ALT, AST, ALP, and GGT. In addition, EPPH affected changes in fat regulators and inflammatory cytokines in blood biochemical assays. It was confirmed that EPPH was involved in fat metabolism in hepatocytes by regulating PPARα in an alcoholic liver disease animal model. Therefore, EPPH strongly modulates Bcl-2 and BAX involved in apoptosis, thereby exhibiting cytochrome P450 (CYP)-inhibitory effects in alcoholic liver disease cells. As a result, this study confirmed that EPPH is a substance that can help liver health by improving liver disease in an alcoholic liver disease animal model.

Antimicrobial Activity and Action Mechanisms of Arg-Rich Short Analog Peptides Designed from the C-Terminal Loop Region of American Oyster Defensin (AOD).

American oyster defensin (AOD) was previously purified from acidified gill extract of the American oyster, Crassostrea virginica. AOD is composed of 38 amino acids with three disulfide bonds and exhibits strong antimicrobial activity against Gram-positive bacteria as well as significant activity against Gram-negative bacteria. Here, to develop promising peptides into antibiotic candidates, we designed five arginine-rich analogs (A0, A1, A2, A3, and A4), predicted their loop and extended strand/random structures-including nine amino acids and a disulfide bond derived from the C-terminus of AOD-and described their antimicrobial and cytotoxic effects, as well as their modes of action. In our experimental results, the A3 and A4 analogs exhibited potent antimicrobial activity against all test organisms-including four Gram-positive bacteria, six Gram-negative bacteria, and Candida albicans-without cell toxicity. A sequence of experiments, including a membrane permeabilization assay, DNA binding study, and DNA polymerization inhibition test, indicated that the two analogs (A3 and A4) possibly did not act directly on the bacterial membrane but instead interacted with intracellular components such as DNA or DNA amplification reactions. AOD analogs also showed strong bacterial inhibition activity in the plasma environment. In addition, analog-treated microbial cells clearly exhibited membrane disruption, damage, and leakage of cytoplasmic contents. Collectively, our results suggest that two analogs, A3 and A4, have potent antimicrobial activity via DNA interaction and have the potential for development into novel antimicrobial agents.

Intestinal anti-inflammatory activity of Ulva ohnoi oil in DSS-induced experimental mouse model.

This study was conducted to examine the physiological activity of Ulva ohnoi, some of which may be used for food or natural products but could disturbing coastal ecosystems due to large scale green-tide, to check values of U. ohnoi oil through experimental results. U. ohnoi oil was extracted from bulk of Ulva biomass to confirm its antioxidant and antibacterial activity, and the efficacy of U. ohnoi oil in the state of inflammation was confirmed through animal experiments. To confirm the anti-inflammatory effect, a mouse model induced with DSS was used. As a result of measuring NO using plasma after induction of inflammation, the amount of NO produced in the U. ohnoi oil group was decreased compared to the control group. Expression of inflammatory cytokines TNF-α, IL-6, and IL-1ß was decreased compared to the control group. As a result of observing H&E staining, lower crypt loss and inflammatory cell infiltration were found in the U. ohnoi oil group compared to the control group. Consequently, U. ohnoi oil appears to have great anti-inflammatory properties.

The Mechanism of Action of Ursolic Acid as a Potential Anti-Toxoplasmosis Agent, and Its Immunomodulatory Effects.

This study was performed to investigate the mechanism of action of ursolic acid in terms of anti-Toxoplasma gondii effects, including immunomodulatory effects. We evaluated the anti-T. gondii effects of ursolic acid, and analyzed the production of nitric oxide (NO), reactive oxygen species (ROS), and cytokines through co-cultured immune cells, as well as the expression of intracellular organelles of T. gondii. The subcellular organelles and granules of T. gondii, particularly rhoptry protein 18, microneme protein 8, and inner membrane complex sub-compartment protein 3, were markedly decreased when T. gondii was treated with ursolic acid, and their expressions were effectively inhibited. Furthermore, ursolic acid effectively increased the production of NO, ROS, interleukin (IL)-10, IL-12, granulocyte macrophage colony stimulating factor (GM-CSF), and interferon-ß, while reducing the expression of IL-1ß, IL-6, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta 1 (TGF-ß1) in T. gondii-infected immune cells. These results demonstrate that ursolic acid not only causes anti-T. gondii activity/action by effectively inhibiting the survival of T. gondii and the subcellular organelles of T. gondii, but also induces specific immunomodulatory effects in T. gondii-infected immune cells. Therefore, this study indicates that ursolic acid can be effectively utilized as a potential candidate agent for developing novel anti-toxoplasmosis drugs, and has immunomodulatory activity.

Evaluation of Anti-Toxoplasma gondii Effect of Ursolic Acid as a Novel Toxoplasmosis Inhibitor.

This study was carried out to evaluate the anti-parasitic effect of ursolic acid against Toxoplasma gondii (T. gondii) that induces toxoplasmosis, particularly in humans. The anti-parasitic effects of ursolic acid against T. gondii-infected cells and T. gondii were evaluated through different specific assays, including immunofluorescence staining and animal testing. Ursolic acid effectively inhibited the proliferation of T. gondii when compared with sulfadiazine, and consistently induced anti-T. gondii activity/effect. In particular, the formation of parasitophorous vacuole membrane (PVM) in host cells was markedly decreased after treating ursolic acid, which was effectively suppressed. Moreover, the survival rate of T. gondii was strongly inhibited in T. gondii group treated with ursolic acid, and then 50% inhibitory concentration (IC50) against T. gondii was measured as 94.62 μg/mL. The T. gondii-infected mice treated with ursolic acid indicated the same survival rates and activity as the normal group. These results demonstrate that ursolic acid causes anti-T. gondii action and effect by strongly blocking the proliferation of T. gondii through the direct and the selective T. gondii-inhibitory ability as well as increases the survival of T. gondii-infected mice. This study shows that ursolic acid has the potential to be used as a promising anti-T. gondii candidate substance for developing effective anti-parasitic drugs.

Elucidation of the anti-hyperammonemic mechanism of Lactobacillus amylovorus JBD401 by comparative genomic analysis.

BACKGROUND: Recent experimental evidence showed that lactobacilli could be used as potential therapeutic agents for hyperammonemia. However, lack of understanding on how lactobacilli reduce blood ammonia levels limits application of lactobacilli to treat hyperammonemia. RESULTS: We report the finished and annotated genome sequence of L. amylovorus JBD401 (GenBank accession no. CP012389). L. amylovorus JBD401 reducing blood ammonia levels dramatically was identified by high-throughput screening of several thousand probiotic strains both within and across Lactobacillus species in vitro. Administration of L. amylovorus JBD401 to hyperammonemia-induced mice reduced the blood ammonia levels of the mice to the normal range. Genome sequencing showed that L. amylovorus JBD401 had a circular chromosome of 1,946,267 bp with an average GC content of 38.13%. Comparative analysis of the L. amylovorus JBD401 genome with L. acidophilus and L. amylovorus strains showed that L. amylovorus JBD401 possessed genes for ammonia assimilation into various amino acids and polyamines Interestingly, the genome of L. amylovorus JBD401 contained unusually large number of various pseudogenes suggesting an active stage of evolution. CONCLUSIONS: L. amylovorus JBD401 has genes for assimilation of free ammonia into various amino acids and polyamines which results in removal of free ammonia in intestinal lumen to reduce the blood ammonia levels in the host. This work explains the mechanism of how probiotics reduce blood ammonia levels.

Antimicrobial effect of the 60S ribosomal protein L29 (cgRPL29), purified from the gill of pacific oyster, Crassostrea gigas.

We purified an ∼6.4-kDa antimicrobial peptide from an acidified gill extract of the Pacific oyster, Crassostrea gigas, by cation-exchange and C18 reversed-phase high performance liquid chromatography (HPLC). The identified peptide was composed of 54 amino acids and had a molecular weight of 6484.6 Da. Comparison of the amino acid sequence and molecular weight with those of other known proteins or peptides revealed that the peptide had high identity with the 60S ribosomal protein L29, and so was named cgRPL29. The full-length cgRPL29 cDNA of the Pacific oyster comprised 325-bp, including a 5'-untranslated region (UTR) of 100-bp, a 3'-UTR of 57-bp, and an open reading frame of 168-bp encoding 55 amino acids, with a Met residue at the N-terminus. The cgRPL29 mRNA tissue distribution suggested that it is constitutively expressed in a non-tissue-specific manner. Secondary structural prediction and homology modeling indicated cgRPL29 have an unordered structure containing two partial α-helical regions. This is to our knowledge the first report of the antimicrobial effect of the 60S ribosomal protein L29 from marine invertebrates.

Hemerythrin-related antimicrobial peptide, msHemerycin, purified from the body of the Lugworm, Marphysa sanguinea.

A ∼1.7 kDa antimicrobial peptide was purified from the acidified body extract of the Lugworm, Marphysa sanguinea, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase high performance liquid chromatography (HPLC). The identified peptide is composed of 14 amino acids with the N-terminal acetylation. Comparison of the identified amino acid sequences and molecular weight of this peptide with those of other known proteins or peptides revealed that this peptide had high identity to the N-terminus of hemerythrin of marine invertebrates and named the msHemerycin. The full-length hemerythrin cDNA of Lugworm was contained 1027-bp, including a 5'-untranslated region (UTR) of 60-bp, a 3'-UTR of 595-bp, and an open reading frame of 372-bp encoding 123 amino acids including the msHemerycin at the N-terminus. Tissue distribution of the msHemerycin mRNA suggests that it is constitutively expressed as a non-tissue-specific manner, however, a relatively higher expression level was observed in muscle (6.8-fold) and brain (6.3-fold), and the lowest level in digestive gland. The secondary structural prediction and homology modeling studies indicate that the msHemerycin might form an unordered structure and might act via unconventional mechanism. Our results suggest that the msHemerycin might be an innate immune component related to the host defenses in the Lugworm. This is the first report on the antimicrobial function of the peptide derived from the N-terminus of hemerythrin in the Lugworm, Marphysa sanguinea.

Intestinal removal of free fatty acids from hosts by Lactobacilli for the treatment of obesity.

Recent findings on the association of gut microbiota with various diseases, including obesity, prompted us to investigate the possibility of using a certain type of gut bacteria as a safe therapeutic for obesity. Lactobacillus mutants with enhanced capacity in absorption of free fatty acids (FFAs) were isolated to show reduced absorption of FFAs by the administered host, attributing to inhibition of body weight gain and body fat accumulation as well as amelioration of blood profiles. Consequently, high throughput screening of natural FFAs-absorbing intestinal microbes led to the isolation of Lactobacillus reuteri JBD30 l. The administration of Lactobacillus JBD30l lowered the concentration of FFAs in the gut fluid content of small intestine, thus reducing intestinal absorption of FFAs whereas promoting fecal excretion of FFAs. Animal data also confirmed that the efficacy of Lactobacillus JBD30l on body weight similar to that of orlistat, an FDA-approved pharmaceutical for long-term use to treat obesity. In a subsequent random, double-blind, placebo-controlled clinical trial (KCT0000452 at Clinical Research Information Service of Korea), there was a statistically significant difference in the percentage change in body weight between the Lactobacillus JBD301 and the placebo group (P = 0.026) as well as in the BMI (P = 0.036) from the 0-week assessment to the 12-week assessment. Our results show that FFA-absorbing Lactobacillus JBD301 effectively reduces dietary fat absorption, providing an ideal treatment for obesity with inherent safety.

The anti-tubercular activity of Melia azedarach L. and Lobelia chinensis Lour. and their potential as effective anti-Mycobacterium tuberculosis candidate agents.

OBJECTIVE: To evaluate the anti-mycobacterial activity of Melia azedarach L. (M. azedarach) and Lobelia chinensis Lour. (L. chinensis) extracts against the growth of Mycobacterium tuberculosis (M. tuberculosis). METHODS: The anti-M. tuberculosis activity of M. azedarach and L. chinensis extracts were evaluated using different indicator methods such as resazurin microtiter assay (REMA) and mycobacteria growth indicator tube (MGIT) 960 system assay. The M. tuberculosis was incubated with various concentrations (50-800 µg/mL) of the extracts for 5 days in the REMA, and for 4 weeks in MGIT 960 system assay. RESULTS: M. azedarach and L. chinensis extracts showed their anti-M. tuberculosis activity by strongly inhibiting the growth of M. tuberculosis in a concentration-dependent manner in the REMA and the MGIT 960 system assay. Particularly, the methanol extract of M. azedarach and n-hexane extract of L. chinensis consistently exhibited their effects by effectively inhibiting the growth of M. tuberculosis in MGIT 960 system for 4 weeks with a single-treatment, indicating higher anti-M. tuberculosis activity than other extracts, and their minimum inhibitory concentrations were measured as 400 µg/mL and 800 µg/mL, respectively. CONCLUSIONS: These results demonstrate that M. azedarach and L. chinensis extracts not only have unique anti-M. tuberculosis activity, but also induce the selective anti-M. tuberculosis effects by consistently inhibiting or blocking the growth of M. tuberculosis through a new pharmacological action. Therefore, this study suggests the potential of them as effective candidate agents of next-generation for developing a new anti-tuberculosis drug, as well as the advantage for utilizing traditional medicinal plants as one of effective strategies against tuberculosis.

Chitinase 3-like 1 synergistically activates IL6-mediated STAT3 phosphorylation in intestinal epithelial cells in murine models of infectious colitis.

BACKGROUND: Chitinase 3-like 1 (CHI3L1) is an inducible molecule on intestinal epithelial cells during the development of inflammatory bowel disease. METHODS: To investigate the role of CHI3L1 in bacterial infectious colitis, we orally inoculated pathogenic Salmonella typhimurium and potentially pathogenic adherent-invasive Escherichia coli (AIEC) LF82 virulent strain into C57Bl/6 wild-type mice or CHI3L1 knockout (KO) mice. RESULTS: Both S. typhimurium and AIEC LF82 were found to efficiently induce severe intestinal inflammation in wild-type mice but not in CHI3L1 KO mice. These bacteria-infected CHI3L1 KO mice exhibit decreased cellular infiltration, bacterial translocation, and production of interleukin (IL)-6 and IL-22, as compared with those of wild-type mice. More importantly, CHI3L1 KO mice displayed aberrant STAT3 activation after bacterial infections. Co-stimulation of CHI3L1 and IL-6, but not IL-22, synergistically activates STAT3 signaling pathway in intestinal epithelial cells in an NF-κB/MAPK-dependent manner. CONCLUSIONS: CHI3L1 promotes the onset of selected gram-negative bacterial infectious colitis through IL-6/STAT3 pathway.

Novel methylxanthine derivative-mediated anti-inflammatory effects in inflammatory bowel disease.

Family 18 chitinases have a binding capacity with chitin, a polymer of N-acetylglucosamine. Recent studies strongly suggested that chitinase 3-like 1 (CHI3L1, also known as YKL-40) and acidic mammalian chitinase, the two major members of family 18 chitinases, play a pivotal role in the pathogenesis of inflammatory bowel disease (IBD), bronchial asthma and several other inflammatory disorders. Based on the data from high-throughput screening, it has been found that three methylxanthine derivatives, caffeine, theophylline, and pentoxifylline, have competitive inhibitory effects against a fungal family 18 chitinase by specifically interacting with conserved tryptophans in the active site of this protein. Methylxanthine derivatives are also known as adenosine receptor antagonists, phosphodiesterase inhibitors and histone deacetylase inducers. Anti-inflammatory effects of methylxanthine derivatives have been well-documented in the literature. For example, a beneficial link between coffee or caffeine consumption and type 2 diabetes as well as liver cirrhosis has been reported. Furthermore, theophylline has a long history of being used as a bronchodilator in asthma therapy, and pentoxifylline has an immuno-modulating effect for peripheral vascular disease. However, it is still largely unknown whether these methylxanthine derivative-mediated anti-inflammatory effects are associated with the inhibition of CHI3L1-induced cytoplasmic signaling cascades in epithelial cells. In this review article we will examine the above possibility and summarize the biological significance of methylxanthine derivatives in intestinal epithelial cells. We hope that this study will provide a rationale for the development of methylxanthine derivatives, in particular caffeine, -based anti-inflammatory therapeutics in the field of IBD and IBD-associated carcinogenesis.

ß-Sitosterol attenuates high-fat diet-induced intestinal inflammation in mice by inhibiting the binding of lipopolysaccharide to toll-like receptor 4 in the NF-κB pathway.

SCOPE: ß-Sitosterol, a common phytosterol, has been shown to exhibit anti-inflammatory effects. Here, we investigated the effect of ß-sitosterol on high-fat diet (HFD) induced colitis in mice and on LPS-stimulated mouse intestinal macrophages. METHODS AND RESULTS: C57BL/6J mice were maintained on an LFD (10 kcal% fat), an HFD (60 kcal% fat), or an HFD with ß-sitosterol (20 mg/kg) administration for 8 weeks. The increased levels of body weight and epididymal fat pad weight as well as the concentrations of circulating proinflammatory cytokines and LPS in HFD mice compared with LFD mice were decreased by oral administration of ß-sitosterol. The HFD-induced colonic inflammation evidenced by the increased expression of proinflammatory cytokines and the activation of nuclear factor kappa B (NF-κB) in the colon was also inhibited by ß-sitosterol. In LPS-stimulated intestinal macrophages, ß-sitosterol inhibited the production of proinflammatory cytokines and inflammatory enzymes as well as NF-κB activation. In addition, ß-sitosterol significantly prevented the binding of LPS to intestinal as well as peritoneal macrophages. Furthermore, ß-sitosterol potently inhibited the interaction between LPS and toll-like receptor 4 in intestinal macrophages transfected with control siRNA or MyD88 siRNA. CONCLUSION: These findings indicate that ß-sitosterol ameliorates HFD-induced colitis by inhibiting the binding of LPS to toll-like receptor 4 in the NF-κB pathway.

Oral caffeine administration ameliorates acute colitis by suppressing chitinase 3-like 1 expression in intestinal epithelial cells.

BACKGROUND: The initial trigger of inflammatory bowel disease (IBD) can be partly attributed towards the interaction and invasion of intestinal epithelial cells (IECs) and submucosal compartments. Identifying safe and economical methods to block these interactions may help prevent the onset of early colitis. Chitinase 3-like 1 (CHI3L1) is an inducible host protein that facilitates bacterial attachment and invasion on/into IECs. Therefore, we test the hypothesis of inhibiting CHI3L1 using the pan-chitinase inhibitor caffeine to reduce the likelihood of early colitis onset. METHODS: IEC lines were treated with caffeine (2.5 or 5 mM) and analyzed for CHI3L1 expression and the impact on bacterial invasion. In vivo, mice were treated with 2.5 mM caffeine and induced with 3.5 % dextran sulfate sodium (DSS)-mediated colitis and subsequently analyzed colitis development. RESULTS: In vitro, caffeine treatment in IEC lines down-regulated CHI3L1 mRNA expression, which resulted in the reduction of bacterial invasion in a caffeine dose-dependent manner. In vivo, mice treated with caffeine displayed a delayed response towards DSS-induced colitis, characterized by lower body weight loss, clinical and histological scores. Bacterial translocation into other organs and pro-inflammatory cytokines production were also reduced in the caffeine-treated mice with DSS-induced colitis. Caffeine treatment also resulted in the loss of CHI3L1-associated AKT signaling pathway activation both in vitro and in vivo. CONCLUSION: Development of acute colitis is reduced upon caffeine treatment. The mechanism involves the down-regulation of CHI3L1 expression and its associated bacterial interaction effect. Therefore, caffeine is proposed as a safe and economical candidate for successful IBD management.

Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection.

BACKGROUND & AIMS: Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. METHODS: We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) × Villin-cre) or CD11c(+) cells (Atg16l1(f/f) × CD11c-cre); these mice were used to assess cell type-specific antibacterial autophagy. All responses were compared with Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells. RESULTS: Autophagy was induced in small intestine and cecum after infection with S typhimurium, and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3ß (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) × Villin-cre mice. Atg16l1(f/f) × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1(f/f) × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. CONCLUSIONS: Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria.

Chitin-binding domains of Escherichia coli ChiA mediate interactions with intestinal epithelial cells in mice with colitis.

BACKGROUND & AIMS: Inducible chitinase 3-like-1 is expressed by intestinal epithelial cells (IECs) and adheres to bacteria under conditions of inflammation. We performed a structure-function analysis of the chitin-binding domains encoded by the chiA gene, which mediates the pathogenic effects of adherent invasive Escherichia coli (AIEC). METHODS: We created AIEC (strain LF82) with deletion of chiA (LF82-ΔchiA) or that expressed chiA with specific mutations. We investigated the effects of infecting different IEC lines with these bacteria compared with nonpathogenic E coli; chitinase activities were measured using the colloidal chitin-azure method. Colitis was induced in C57/Bl6 mice by administration of dextran sodium sulfate, and mice were given 10(8) bacteria for 15 consecutive days by gavage. Stool/tissue samples were collected and analyzed. RESULTS: LF82-ΔchiA had significantly less adhesion to IEC lines than LF82. Complementation of LF82-ΔchiA with the LF82 chiA gene, but not chiA from nonpathogenic (K12) E coli, increased adhesion. We identified 5 specific polymorphisms in the chitin-binding domain of LF82 chiA (at amino acids 362, 370, 378, 388, and 548) that differ from chiA of K12 and were required for LF82 to interact directly with IECs. This interaction was mediated by an N-glycosylated asparagine in chitinase 3-like-1 (amino acid 68) on IECs. Mice infected with LF82, or LF82-ΔchiA complemented with LF82 chiA, developed more severe colitis after administration of dextran sodium sulfate than mice infected with LF82-ΔchiA or LF82 that expressed mutant forms of chiA. CONCLUSIONS: AIEC adheres to an N-glycosylated chitinase 3-like-1 on IECs via the chitin-binding domain of chiA. This mechanism promotes the pathogenic effects of AIEC in mice with colitis.

Arctigenin ameliorates inflammation in vitro and in vivo by inhibiting the PI3K/AKT pathway and polarizing M1 macrophages to M2-like macrophages.

Seeds of Arctium lappa, containing arctigenin and its glycoside arctiin as main constituents, have been used as a diuretic, anti-inflammatory and detoxifying agent in Chinese traditional medicine. In our preliminary study, arctigenin inhibited IKKß and NF-κB activation in peptidoglycan (PGN)- or lipopolysaccharide (LPS)-induced peritoneal macrophages. To understand the anti-inflammatory effect of arctigenin, we investigated its anti-inflammatory effect in LPS-stimulated peritoneal macrophages and on LPS-induced systemic inflammation as well as 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. Arctigenin inhibited LPS-increased IL-1ß, IL-6 and TNF-α expression in LPS-stimulated peritoneal macrophages, but increased LPS-reduced IL-10 and CD204 expression. Arctigenin inhibited LPS-induced PI3K, AKT and IKKß phosphorylation, but did not suppress LPS-induced IRAK-1 phosphorylation. However, arctigenin did not inhibit NF-κB activation in LPS-stimulated PI3K siRNA-treated peritoneal macrophages. Arctigenin suppressed the binding of p-PI3K antibody and the nucleus translocation of NF-κB p65 in LPS-stimulated peritoneal macrophages. Arctigenin suppressed blood IL-1ß and TNF-α level in mice systemically inflamed by intraperitoneal injection of LPS. Arctigenin also inhibited colon shortening, macroscopic scores and myeloperoxidase activity in TNBS-induced colitic mice. Arctigenin inhibited TNBS-induced IL-1ß, TNF-α and IL-6 expression, as well as PI3K, AKT and IKKß phosphorylation and NF-κB activation in mice, but increased IL-10 and CD204 expression. However, it did not affect IRAK-1 phosphorylation. Based on these findings, arctigenin may ameliorate inflammatory diseases, such as colitis, by inhibiting PI3K and polarizing M1 macrophages to M2-like macrophages.

High fat diet-induced gut microbiota exacerbates inflammation and obesity in mice via the TLR4 signaling pathway.

BACKGROUND & AIMS: While it is widely accepted that obesity is associated with low-grade systemic inflammation, the molecular origin of the inflammation remains unknown. Here, we investigated the effect of endotoxin-induced inflammation via TLR4 signaling pathway at both systemic and intestinal levels in response to a high-fat diet. METHODS: C57BL/6J and TLR4-deficient C57BL/10ScNJ mice were maintained on a low-fat (10 kcal % fat) diet (LFD) or a high-fat (60 kcal % fat) diet (HFD) for 8 weeks. RESULTS: HFD induced macrophage infiltration and inflammation in the adipose tissue, as well as an increase in the circulating proinflammatory cytokines. HFD increased both plasma and fecal endotoxin levels and resulted in dysregulation of the gut microbiota by increasing the Firmicutes to Bacteriodetes ratio. HFD induced the growth of Enterobecteriaceae and the production of endotoxin in vitro. Furthermore, HFD induced colonic inflammation, including the increased expression of proinflammatory cytokines, the induction of Toll-like receptor 4 (TLR4), iNOS, COX-2, and the activation of NF-κB in the colon. HFD reduced the expression of tight junction-associated proteins claudin-1 and occludin in the colon. HFD mice demonstrated higher levels of Akt and FOXO3 phosphorylation in the colon compared to the LFD mice. While the body weight of HFD-fed mice was significantly increased in both TLR4-deficient and wild type mice, the epididymal fat weight and plasma endotoxin level of HFD-fed TLR4-deficient mice were 69% and 18% of HFD-fed wild type mice, respectively. Furthermore, HFD did not increase the proinflammatory cytokine levels in TLR4-deficient mice. CONCLUSIONS: HFD induces inflammation by increasing endotoxin levels in the intestinal lumen as well as in the plasma by altering the gut microbiota composition and increasing its intestinal permeability through the induction of TLR4, thereby accelerating obesity.