Xiaolun Sun

Microbial Colonization Induces Dynamic Temporal and Spatial Patterns of NF-κB Activation in the Zebrafish Digestive Tract

BACKGROUND & AIMS The nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) transcription factor pathway is activated in response to diverse microbial stimuli to regulate expression of genes involved in immune responses and tissue homeostasis. However, the temporal and spatial activation of NF-κB in response to microbial signals have not been determined in whole living organisms, and the molecular and cellular details of these responses are not well understood. We used in vivo imaging and molecular approaches to analyze NF-κB activation in response to the commensal microbiota in transparent gnotobiotic zebrafish. METHODS We used DNA microarrays, in situ hybridization, and quantitative reverse transcription polymerase chain reaction analyses to study the effects of the commensal microbiota on gene expression in gnotobiotic zebrafish. Zebrafish PAC2 and ZFL cells were used to study the NF-κB signaling pathway in response to bacterial stimuli. We generated transgenic zebrafish that express enhanced green fluorescent protein under transcriptional control of NF-κB, and used them to study patterns of NF-κB activation during development and microbial colonization. RESULTS Bacterial stimulation induced canonical activation of the NF-κB pathway in zebrafish cells. Colonization of germ-free transgenic zebrafish with a commensal microbiota activated NF-κB and led to up-regulation of its target genes in intestinal and extraintestinal tissues of the digestive tract. Colonization with the bacterium Pseudomonas aeruginosa was sufficient to activate NF-κB, and this activation required a functional flagellar apparatus. CONCLUSIONS In zebrafish, transcriptional activity of NF-κB is spatially and temporally regulated by specific microbial factors. The observed patterns of NF-κB-dependent responses to microbial colonization indicate that cells in the gastrointestinal tract respond robustly to the microbial environment.

Gnotobiotic IL-10−/−; NF-κBEGFP Mice Develop Rapid and Severe Colitis Following Campylobacter jejuni Infection

Limited information is available on the molecular mechanisms associated with Campylobacter jejuni (C. jejuni) induced food-borne diarrheal illnesses. In this study, we investigated the function of TLR/NF-κB signaling in C. jejuni induced pathogenesis using gnotobiotic IL-10−/−; NF-κBEGFP mice. In vitro analysis showed that C. jejuni induced IκB phosphorylation, followed by enhanced NF-κB transcriptional activity and increased IL-6, MIP-2α and NOD2 mRNA accumulation in infected-mouse colonic epithelial cells CMT93. Importantly, these events were blocked by molecular delivery of an IκB inhibitor (Ad5IκBAA). NF-κB signalling was also important for C.jejuni-induced cytokine gene expression in bone marrow-derived dendritic cells. Importantly, C. jejuni associated IL-10−/−; NF-κBEGFP mice developed mild (day 5) and severe (day 14) ulcerating colonic inflammation and bloody diarrhea as assessed by colonoscopy and histological analysis. Macroscopic analysis showed elevated EGFP expression indicating NF-κB activation throughout the colon of C. jejuni associated IL-10−/−; NF-κBEGFP mice, while fluorescence microscopy revealed EGFP positive cells to be exclusively located in lamina propria mononuclear cells. Pharmacological NF-κB inhibition using Bay 11-7085 did not ameliorate C. jejuni induced colonic inflammation. Our findings indicate that C. jejuni induces rapid and severe intestinal inflammation in a susceptible host that correlates with enhanced NF-κB activity from lamina propria immune cells.

Campylobacter jejuni Induces Colitis through Activation of Mammalian Target of Rapamycin Signaling

BACKGROUND & AIMS Campylobacter jejuni is the worldwide leading cause of bacterial-induced enteritis. The molecular and cellular events that lead to campylobacteriosis are poorly understood. We identify mammalian target of rapamycin (mTOR) as a signaling pathway that leads to C jejuni-induced intestinal inflammation. METHODS Germ-free (control) or conventionally derived Il10(-/-) mice that express enhanced green fluorescent protein (EGFP) under the control of nuclear factor κB (Il10(-/-); NF-κB(EGFP) mice) were infected with C jejuni (10(9) colony-forming units/mouse) for 12 days; their responses were determined using histologic, semiquantitative reverse-transcription polymerase chain reaction, fluorescence in situ hybridization, transmission electron microscopy, and tissue culture analyses. mTOR signaling was blocked by daily intraperitoneal injections of the pharmacologic inhibitor rapamycin (1.5 mg/kg). CD4(+) T cells were depleted by intraperitoneal injections of antibodies against CD4 (0.5 mg/mouse every 3 days). Bacterial survival in splenocytes was measured using a gentamycin killing assay. RESULTS C jejuni induced intestinal inflammation, which correlated with activation of mTOR signaling and neutrophil infiltration. The inflamed intestines of these mice had increased levels of interleukin-1β, Cxcl2, interleukin-17a, and EGFP; C jejuni localized to colons and extraintestinal tissues of infected Il10(-/-); NF-κB(EGFP) mice compared with controls. Rapamycin, administered before or after introduction of C jejuni, blocked C jejuni-induced intestinal inflammation and bacterial accumulation. LC3II processing and killing of C jejuni were increased in splenocytes incubated with rapamycin compared with controls. CONCLUSIONS mTOR signaling mediates C jejuni-induced colitis in Il10(-/-) mice, independently of T-cell activation. Factors involved in mTOR signaling might be therapeutic targets for campylobacteriosis.

Locoregional effects of microbiota in a preclinical model of colon carcinogenesis

Inflammation and microbiota are critical components of intestinal tumorigenesis. To dissect how the microbiota contributes to tumor distribution, we generated germ-free (GF) ApcMin/+ and ApcMin/+ ;Il10-/- mice and exposed them to specific-pathogen-free (SPF) or colorectal cancer-associated bacteria. We found that colon tumorigenesis significantly correlated with inflammation in SPF-housed ApcMin/+ ;Il10-/- , but not in ApcMin/+ mice. In contrast, small intestinal neoplasia development significantly correlated with age in both ApcMin/+ ;Il10-/- and ApcMin/+ mice. GF ApcMin/+ ;Il10-/- mice conventionalized by an SPF microbiota had significantly more colon tumors compared with GF mice. Gnotobiotic studies revealed that while Fusobacterium nucleatum clinical isolates with FadA and Fap2 adhesins failed to induce inflammation and tumorigenesis, pks+Escherichia coli promoted tumorigenesis in the ApcMin/+ ;Il10-/- model in a colibactin-dependent manner, suggesting colibactin is a driver of carcinogenesis. Our results suggest a distinct etiology of cancers in different locations of the gut, where colon cancer is primarily driven by inflammation and the microbiome, while age is a driving force for small intestine cancer. Cancer Res; 77(10); 2620-32. ©2017 AACR.

Phosphatidylinositol 3-Kinase-γ Signaling Promotes Campylobacter jejuni–Induced Colitis through Neutrophil Recruitment in Mice

Crypt abscesses caused by excessive neutrophil accumulation are prominent features of human campylobacteriosis and its associated pathology. The molecular and cellular events responsible for this pathological situation are currently unknown. We investigated the contribution of PI3K-γ signaling in Campylobacter jejuni–induced neutrophil accumulation and intestinal inflammation. Germ-free and specific pathogen-free Il10−/− and germ-free Il10−/−;Rag2−/− mice were infected with C. jejuni (109 CFU/mouse). PI3K-γ signaling was manipulated using either the pharmacological PI3K-γ inhibitor AS252424 (i.p. 10 mg/kg daily) or genetically using Pi3k-γ−/− mice. After up to 14 d, inflammation was assessed histologically and by measuring levels of colonic Il1β, Cxcl2, and Il17a mRNA. Neutrophils were depleted using anti-Gr1 Ab (i.p. 0.5 mg/mouse/every 3 d). Using germ-free Il10−/−;Rag2−/− mice, we observed that innate immune cells are the main cellular compartment responsible for campylobacteriosis. Pharmacological blockade of PI3K-γ signaling diminished C. jejuni–induced intestinal inflammation, neutrophil accumulation, and NF-κB activity, which correlated with reduced Il1β (77%), Cxcl2 (73%), and Il17a (72%) mRNA accumulation. Moreover, Pi3k-γ−/− mice pretreated with anti–IL-10R were resistant to C. jejuni–induced intestinal inflammation compared with Wt mice. This improvement was accompanied by a reduction of C. jejuni translocation into the colon and extraintestinal tissues and by attenuation of neutrophil migratory capacity. Furthermore, neutrophil depletion attenuated C. jejuni–induced crypt abscesses and intestinal inflammation. Our findings indicate that C. jejuni–induced PI3K-γ signaling mediates neutrophil recruitment and intestinal inflammation in Il10−/− mice. Selective pharmacological inhibition of PI3K-γ may represent a novel means to alleviate severe cases of campylobacteriosis, especially in antibiotic-resistant strains.

A Brief Review of Biomarkers for Preventing and Treating Cardiovascular Diseases

Cardiovascular diseases are the most prominent circulation disorders around the world. Biomarkers are characteristic biological properties that can be objectively measured as an indicator to evaluate a variety of health or disease characteristics. Cardiac biomarkers are a valuable tool for assessing the pathogenesis and diagnosis of cardiovascular diseases. In this review, we will focus on the major biomarkers used in recent clinical research for the diagnosis of cardiovascular diseases, which include mean platelet volume, hyperhomocysteinemia, serum magnesium, microalbuminuria, and prolongation of QT interval and dispersion. We also highlight the key findings of clinical case report based studies presented in this issue of JCDR.

Nucleotide-Binding Oligomerization Domain–Containing Protein 2 Controls Host Response to Campylobacter jejuni in Il10−/− Mice

Innate signaling-induced antimicrobial response represents a key protective host feature against infectious microorganisms such as Campylobacter species. In this study, we investigated the role of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in Campylobacter jejuni-induced intestinal inflammation. Specific-pathogen-free Il10(-/-), Nod2(-/-), and Il10(-/-); Nod2(-/-) mice were infected with C. jejuni (10(9) colony-forming units/mouse) 24 hours after a 7-day course of antibiotic treatment. Three weeks later, host responses were determined. The nitric oxide (NO) donor sodium nitroprusside was injected intraperitoneally (2 mg/kg daily) to supplement NO. Although healthy in specific-pathogen-free conditions, Il10(-/-); Nod2(-/-) mice developed severe intestinal inflammation following C. jejuni infection, compared with Nod2(-/-) and Il10(-/-) mice. The onset of colitis was associated with elevated neutrophil accumulation, crypt abscesses, and expression of the endogenous proinflammatory mediators Il-1β, Tnfα, and Cxcl1. Fluorescence in situ hybridization and culture assay showed enhanced C. jejuni invasion into the colon and mesenteric lymph nodes in Il10(-/-); Nod2(-/-) mice, compared with Il10(-/-) mice. C. jejuni-induced bactericidal NO production was reduced in peritoneal macrophages from Il10(-/-); Nod2(-/-) mice, compared with Il10(-/-) mice. Importantly, sodium nitroprusside attenuated C. jejuni-induced colitis in Il10(-/-); Nod2(-/-) mice. Our findings suggest that NOD2 signaling is critical to control campylobacteriosis in Il10(-/-) mice, a process involving NOD2-mediated bactericidal responses.

Bacterial mediated gastrointestinal inflammation.

Mouse models have proven to be a key approach in our understanding of the etiology and physiology underlying bacterial mediated gastrointestinal inflammation. Generally, these models are based on the inoculation of genetically susceptible mice with either commensal or pathogenic bacteria to elicit an inflammatory response. Here, we describe models of acute and chronic gastrointestinal inflammation using interleukin 10-deficient (Il10 (-/-)) mice colonized with the pathogenic Campylobacter jejuni strain 81-176 or the commensal Escherichia coli strain NC 101.

Natural Products Targeting on Oxidative Stress and Inflammation: Mechanisms, Therapies, and Safety Assessment

Substantial evidence suggests that overproduction of reactive oxygen species (ROS) and its associated oxidative stress and inflammatory responses play a crucial role in the pathogenesis of various human chronic diseases including diabetes, cardiovascular diseases, obesity, neurodegenerative diseases, and cancer. Enhanced ROS production can cause oxidative damage to the cellular macromolecules including lipids, proteins, and DNA. In addition, oxidative stress activates nuclear factor kappa B (NF-?B), nuclear factor-erythroid 2-related factor 2 (Nrf2), and antioxidant-responsive elements (ARE) to regulate anti-inflammatory and antioxidative genes involved in inflammatory responses and cellular defense mechanisms. Recent studies have showed that certain natural compounds and/or their derived small molecules have the ability to protect cells from oxidative stress and ameliorate various oxidative stress-related diseases. However, a large number of natural products are underutilized and unexploited because their bioactivities are unknown and the molecular mechanisms involved are yet to be identified. Understanding the mechanisms of actions of natural products would shed further light into the application of these compounds in the prevention and treatment of oxidative stress-related diseases in humans. This special issue includes two invited reviews and twelve original research articles that are focused on the antioxidative/anti-inflammatory effects of various natural compounds and their safety in vitro and in vivo. Further, these studies identified the cellular and molecular mechanisms underlying the bioactivities of these natural compounds.