Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence.

Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.

E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis.

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

Properdin provides protection from Citrobacter rodentium-induced intestinal inflammation in a C5a/IL-6-dependent manner.

Citrobacter rodentium is an attaching and effacing mouse pathogen that models enteropathogenic and enterohemorrhagic Escherichia coli in humans. The complement system is an important innate defense mechanism; however, only scant information is available about the role of complement proteins during enteric infections. In this study, we examined the impact of the lack of properdin, a positive regulator of complement, in C. rodentium-induced colitis. Following infection, properdin knockout (P(KO)) mice had increased diarrhea and exacerbated inflammation combined with defective epithelial cell-derived IL-6 and greater numbers of colonizing bacteria. The defect in the mucosal response was reversed by administering exogenous properdin to P(KO) mice. Then, using in vitro and in vivo approaches, we show that the mechanism behind the exacerbated inflammation of P(KO) mice is due to a failure to increase local C5a levels. We show that C5a directly stimulates IL-6 production from colonic epithelial cells and that inhibiting C5a in infected wild-type mice resulted in defective epithelial IL-6 production and exacerbated inflammation. These outcomes position properdin early in the response to an infectious challenge in the colon, leading to complement activation and C5a, which in turn provides protection through IL-6 expression by the epithelium. Our results unveil a previously unappreciated mechanism of intestinal homeostasis involving complement, C5a, and IL-6 during bacteria-triggered epithelial injury.

Mast cells protect against Pseudomonas aeruginosa-induced lung injury.

Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals. Maintaining the integrity of the respiratory epithelium is critical for an effective host response to P. aeruginosa. Given the close spatial relationship between mast cells and the respiratory epithelium, and the importance of tightly regulated epithelial permeability during lung infections, we examined whether mast cells influence airway epithelial integrity during P. aeruginosa lung infection in a mouse model. We found that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice displayed greatly increased epithelial permeability, bacterial dissemination, and neutrophil accumulation compared with wild-type animals after P. aeruginosa infection; these defects were corrected on reconstitution with mast cells. An in vitro Transwell co-culture model further demonstrated that a secreted mast cell factor decreased epithelial cell apoptosis and tumor necrosis factor production after P. aeruginosa infection. Together, our data demonstrate a previously unrecognized role for mast cells in the maintenance of epithelial integrity during P. aeruginosa infection, through a mechanism that likely involves prevention of epithelial apoptosis and tumor necrosis factor production. Our understanding of mechanisms of the host response to P. aeruginosa will open new avenues for the development of successful preventative and treatment strategies.

Dietary Supplementation of Haskap Berry (Lonicera caerulea L.) Anthocyanins and Probiotics Attenuate Dextran Sulfate Sodium-Induced Colitis: Evidence from an Experimental Animal Model.

Haskap berry (Lonicera caerulea L.) is a rich dietary source of anthocyanins with potent anti-inflammatory properties. In this study, isolated haskap berry anthocyanins were encapsulated in maltodextrin and inulin (3:1) by freeze-drying to improve stability and bioavailability. The structural properties of microcapsules, encapsulation yield, efficiency, recovery, and powder retention were evaluated. The microcapsules that exhibited the highest encapsulation efficiency (60%) and anthocyanin recovery (89%) were used in the dextran sulfate sodium (DSS)-induced acute colitis in mice. Thirty-five BALB/c male mice of seven weeks old were divided into seven dietary supplementation groups (n = 5) to receive either free anthocyanins, encapsulated anthocyanins (6.2 mg/day), or probiotics (1 × 109 CFU/day) alone or as combinations of anthocyanin and probiotics. As observed by clinical data, free anthocyanin and probiotic supplementation significantly reduced the severity of colitis. The supplementary diets suppressed the DSS-induced elevation of serum inflammatory (interleukin (IL)-6 and tumor necrosis factor) and apoptosis markers (B-cell lymphoma 2 and Bcl-2-associated X protein) in mice colon tissues. The free anthocyanins and probiotics significantly reduced the serum IL-6 levels. In conclusion, the dietary supplementation of haskap berry anthocyanins and probiotics protects against DSS-induced colitis possibly by attenuating mucosal inflammation, and this combination has the potential as a health-promoting dietary supplement and nutraceutical.

Peroxisomal cholesterol metabolism regulates yap-signaling, which maintains intestinal epithelial barrier function and is altered in Crohn's disease.

Intestinal epithelial cells line the luminal surface to establish the intestinal barrier, where the cells play essential roles in the digestion of food, absorption of nutrients and water, protection from microbial infections, and maintaining symbiotic interactions with the commensal microbial populations. Maintaining and coordinating all these functions requires tight regulatory signaling, which is essential for intestinal homeostasis and organismal health. Dysfunction of intestinal epithelial cells, indeed, is linked to gastrointestinal disorders such as irritable bowel syndrome, inflammatory bowel disease, and gluten-related enteropathies. Emerging evidence suggests that peroxisome metabolic functions are crucial in maintaining intestinal epithelial cell functions and intestinal epithelium regeneration and, therefore, homeostasis. Here, we investigated the molecular mechanisms by which peroxisome metabolism impacts enteric health using the fruit fly Drosophila melanogaster and murine model organisms and clinical samples. We show that peroxisomes control cellular cholesterol, which in turn regulates the conserved yes-associated protein-signaling and contributes to intestinal epithelial structure and epithelial barrier function. Moreover, analysis of intestinal organoid cultures derived from biopsies of patients affected by Crohn's Disease revealed that the dysregulation of peroxisome number, excessive cellular cholesterol, and inhibition of Yap-signaling are markers of disease and could be novel diagnostic and/or therapeutic targets for treating Crohn's Disease. Our studies provided mechanistic insights on peroxisomal signaling in intestinal epithelial cell functions and identified cholesterol as a novel metabolic regulator of yes-associated protein-signaling in tissue homeostasis.

Human colonic epithelial cells detect and respond to C5a via apically expressed C5aR through the ERK pathway.

Intestinal epithelial cells (IECs) exhibit numerous adaptations to maintain barrier function as well as play sentinel roles by expressing receptors for microbial products and antimicrobial peptides. The complement system is another important innate sensing and defense mechanism of the host against bacteria and increasing evidence shows that complement plays a role in colitis. The split component C5a is a potent proinflammatory molecule, and the C5a receptor (C5aR) CD88 has been reported on multiple cell types. Here, we examined the question of whether human colonic cell lines can detect activated complement via C5aR and what signaling pathway is critical in the subsequent responses. T84, HT29, and Caco2 cell lines all possessed mRNA and protein for C5aR and the decoy receptor C5L2. Polarized cells expressed the proteins on the apical cell membrane. C5a binding to the C5aR on human IECs activates the ERK pathway, which proved critical for a subsequent upregulation of IL-8 mRNA, increased permeability of monolayers, and enhanced proliferation of the cells. The fact that human IECs are capable of detecting complement activation in the lumen via this anaphylatoxin receptor highlights the potential for IECs to detect pathogens indirectly through complement activation and be primed to amplify the host response through heightened inflammatory mediator expression to further recruit immune cells.

Neutrophil transintestinal epithelial migration to CXCR2 ligands is regulated by adenosine.

OBJECTIVES: Polymorphonuclear leukocytes (PMN) feature prominently in the mucosa, including in crypt abscesses, of patients with inflammatory bowel disease, yet the mediators that are responsible for this migration are unknown. We discovered that CXCR2 chemokines (reportedly elevated in the mucosa) have reduced potency recruiting PMN across epithelial cell monolayers versus acellular filters, so the objective was to determine what molecules modify transepithelial PMN migration to CXCR2 chemokines. METHODS: Transwells with T84 colon carcinoma monolayers or no epithelium were used with adolescent patient peripheral blood PMN and CXCL8 (interleukin-8 [IL-8], binds CXCR1 and CXCR2), CXCL5 (epithelial-derived neutrophil chemoattractant-78 [ENA-78]), or CXCL1 (Gro-α, both bind CXCR2) as chemoattractants. RESULTS: IL-8 was equally potent at recruiting PMN across filters and T84 monolayers growing on the filters. In contrast, ENA-78 and Gro-α were significantly less potent at recruiting PMN across monolayers than across bare filters. Blocking CXCR1 reduced PMN migration across monolayers to IL-8. We ruled out superoxide radicals possibly enhancing migration to IL-8 by using PMN from a patient with chronic granulomatous disease. PMN constitutively produce adenosine, so we added adenosine deaminase to the transwell assays and observed increased migration to ENA-78 across T84 monolayers. The level of migration was further enhanced by pretreating PMN with adenosine before adding the cells to the assay in the presence of the deaminase. CONCLUSIONS: PMN migration mediated by CXCR2 through the epithelium is regulated by adenosine. Adenosine appears to reduce transepithelial migration by influencing ß2 integrin use on the PMN.

Neutrophil infiltration of the colon is independent of the FPR1 yet FPR1 deficient mice show differential susceptibilities to acute versus chronic induced colitis.

BACKGROUND: The receptor for formylated peptides, formyl peptide receptor 1 (FPR1), potently activates and serves as a chemoattractant receptor for neutrophils. AIM: Given the abundance of neutrophils in the inflamed colon, our aim was to determine if the FPR1 mediates colonic neutrophil migration, using the dextran sodium sulfate (DDS)-induced model of colitis. METHODS: Formyl peptide receptor 1 gene-deficient mice were administered DDS in drinking water for a single 5-day period (acute) or in two 5-day periods separated by 16 days (chronic). At the end of the treatment their colons were excised, measured, and prepared for histological evaluation. RESULTS: FPR1(-/-) mice experienced less severe acute colonic pathology than C57BL/6 (wildtype) mice. The opposite was observed following the second colitis cycle, with FPR1(-/-) mice developing worse pathology than wildtype mice. Both strains had similar numbers of infiltrating neutrophils in ulcerated areas of the colon after a single DSS cycle, but FPR1(-/-) mice had significantly more neutrophils in the ulcerated mucosa after two cycles. There was no difference in the capacity of neutrophils from each strain to migrate to chemoattractants. Since the FPR1(-/-) mice had larger ulcers compared to the wildtype mice, we propose that the FPR1(-/-) mice failed to recover at the same rate as wildtype mice. This apparent difference in restitution could not be attributed to observable differences in annexin A1. CONCLUSIONS: We conclude that neutrophil migration into the inflamed mouse colon does not depend on FPR1 but that FPR1 contributes in other pathological mechanisms that are harmful during acute inflammation but protective during chronic inflammation.

Calcineurin Aα Contributes to IgE-Dependent Mast-Cell Mediator Secretion in Allergic Inflammation.

Mast cells (MCs) are key mediators of allergic inflammation through the activation of cross-linked immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcϵRI) on the cell surface, leading to the release of biologically potent mediators, either from preformed granules or newly synthesized. Pharmacological inhibitors have been developed to target a key signaling protein phosphatase in this pathway, calcineurin, yet there is a lack of genetic and definitive evidence for the various isoforms of calcineurin subunits in FcϵRI-mediated responses. In this study, we hypothesized that deficiency in the calcineurin Aα isoform will result in a decreased allergic immune response by the MCs. In a model of passive cutaneous anaphylaxis, there was a reduction in vascular permeability in MC-deficient mouse tissues reconstituted with calcineurin subunit A (CnAα) gene-knockout (CnAα-/-) MCs, and in vitro experiments identified a significant reduction in release of preformed mediators from granules. Furthermore, released levels of de novo synthesized cytokines were reduced upon FcϵRI activation of CnAα-/- MCs in vitro. Characterizing the mechanisms associated with this deficit response, we found a significant impairment of nuclear factor of kappa light polypeptide gene enhancer in B cell phosphorylation and impaired nuclear factor kappa-light-chain-enhancer of activated B-cell inhibitor alpha (NF-κB) activation. Thus, we concluded that CnAα contributes to the release of preformed mediators and newly synthesized mediators from FcϵRI-mediated activation of MCs, and this regulation includes NF-κB signaling.

Mitochondrial damage-associated molecular patterns trigger arginase-dependent lymphocyte immunoregulation.

Tissue damage leads to loss of cellular and mitochondrial membrane integrity and release of damage-associated molecular patterns, including those of mitochondrial origin (mitoDAMPs). Here, we describe the lymphocyte response to mitoDAMPs. Using primary cells from mice and human donors, we demonstrate that natural killer (NK) cells and T cells adopt regulatory phenotypes and functions in response to mitoDAMPs. NK cell-mediated cytotoxicity, interferon gamma (IFN-γ) production, T cell proliferation, and in vivo anti-viral T cell activation are all interrupted in the presence of mitoDAMPs or mitoDAMP-rich irradiated cells in in vitro and in vivo assays. Mass spectrometry analysis of mitoDAMPs demonstrates that arginase and products of its enzymatic activity are prevalent in mitoDAMP preparations. Functional validation by arginase inhibition and/or arginine add-back shows that arginine depletion is responsible for the alteration in immunologic polarity. We conclude that lymphocyte responses to mitoDAMPs reflect a highly conserved mechanism that regulates inflammation in response to tissue injury.

Effects of CNS Injury-Induced Immunosuppression on Pulmonary Immunity.

Patients suffering from stroke, traumatic brain injury, or other forms of central nervous system (CNS) injury have an increased risk of nosocomial infections due to CNS injury-induced immunosuppression (CIDS). Immediately after CNS-injury, the response in the brain is pro-inflammatory; however, subsequently, local and systemic immunity is suppressed due to the compensatory release of immunomodulatory neurotransmitters. CIDS makes patients susceptible to contracting infections, among which pneumonia is very common and often lethal. Ventilator-acquired pneumonia has a mortality of 20-50% and poses a significant risk to vulnerable patients such as stroke survivors. The mechanisms involved in CIDS are not well understood. In this review, we consolidate the evidence for cellular processes underlying the pathogenesis of CIDS, the emerging treatments, and speculate further on the immune elements at play.

The Production and Function of Endogenous Interleukin-10 in Intestinal Epithelial Cells and Gut Homeostasis.

The healthy gut is achieved and maintained through a balanced relationship between the mucosal immune system, microbial communities resident in the lumen, and the intestinal epithelium. The intestinal epithelium plays an exceptionally important role in harmonizing the interaction between the host immunity and the luminal residents, as this selectively permeable barrier separates but also allows interchange between the 2 environments. Interleukin (IL)-10 has been well established to play an important role in maintaining gut homeostasis by imparting diverse effects on a variety of cell types in this relationship. In the intestine, the source and the target of IL-10 include leukocytes and epithelial cells. Given that both the epithelium and IL-10 are essential players in supporting homeostasis, we discuss the relationship between these 2 factors, focusing on epithelial sources of IL-10 and the effects of IL-10 on the intestinal epithelium. Insight into this relationship reveals an important aspect of the innate immune function of intestinal epithelial cells.

Gut bacterial gene changes following pegaspargase treatment in pediatric patients with acute lymphoblastic leukemia.

Treatment of pediatric acute lymphoblastic leukemia (ALL) with pegaspargase exploits ALL cells dependency on asparagine. Pegaspargase depletes asparagine, consequentially affecting aspartate, glutamine and glutamate. The gut as a confounding source of these amino acids (AAs) and the role of gut microbiome metabolism of AAs has not been examined. We examined asparagine, aspartate, glutamine and glutamate in stool samples from patients over pegaspargase treatment. Microbial gene-products, which interact with these AAs were identified. Stool asparagine declined significantly, and 31 microbial genes changed over treatment. Changes were complex, and included genes involved in AA metabolism, nutrient sensing, and pathways increased in cancers. While we identified changes in a gene (iaaA) with limited asparaginase activity, it lacked significance after correction leaving open other mechanisms for asparagine decline, possibly including loss from gut to blood. Understanding pathways that change AA availability, including by microbes in the gut, could be useful in optimizing pegaspargase therapy.

Therapeutic effect of blocking CXCR2 on neutrophil recruitment and dextran sodium sulfate-induced colitis.

Dextran sodium sulfate (DSS)-induced colitis in mice is characterized by polymorphonuclear neutrophil (PMN) infiltration into the colonic mucosa and lumen. The mechanism by which this occurs is unclear. To begin to understand the mechanism, we determined the role of the PMN chemokine receptor, CXCR2, in DSS-induced colitis by using CXCR2(-/-) mice or by neutralizing CXCR2. DSS was administered through drinking water to CXCR2(-/-) and BALB/c mice for 5 days followed by regular water for 1 day. In the neutralization study, mice were injected with control serum or goat anti-CXCR2 antiserum. BALB/c mice receiving DSS and control serum-injected mice receiving DSS lost weight and showed considerable clinical illness. Histological observation revealed submucosal edema, PMN infiltration into the submucosa and mucosa, extensive crypt damage with abscesses, and ulceration. In contrast, both the CXCR2(-/-) and anti-CXCR2 antiserum-treated mice gained weight and had significantly lower symptom scores. Histology of these mice showed submucosal edema but relatively intact crypt architecture and very few ulcers. Significantly fewer PMNs were found in the mucosa in anti-CXCR2 anti-serum compared with control serum-injected inflamed mice, but no significant difference in eosinophil infiltration was observed between the groups. Our experiments identify a role for CXCR2 in DSS-induced colitis and suggest that antagonizing CXCR2 provides some therapeutic efficacy, possibly by impeding PMN recruitment into the mucosa. Antagonizing CXCR2 may form the basis for therapeutic drugs directed at controlling colitis.

Opinion: Are Organoids the End of Model Evolution for Studying Host Intestinal Epithelium/Microbe Interactions?

In the pursuit to understand intestinal epithelial cell biology in health and disease, researchers have established various model systems, from whole animals (typically rodents) with experimentally induced disease to transformed human carcinomas. The obvious limitation to the ex vivo or in vitro cell systems was enriching, maintaining, and expanding differentiated intestinal epithelial cell types. The popular concession was human and rodent transformed cells of mainly undifferentiated cells, with a few select lines differentiating along the path to becoming goblet cells. Paneth cells, in particular, remained unculturable. The breakthrough came in the last decade with the report of conditions to grow mouse intestinal organoids. Organoids are 3-dimensional ex vivo "mini-organs" of the organ from which the stem cells were derived. Intestinal organoids contain fully differentiated epithelial cells in the same spatial organization as in the native epithelium. The cells are suitably polarized and produce and secrete mucus onto the apical surface. This review introduces intestinal organoids and provide some thoughts on strengths and weaknesses in the application of organoids to further advance our understanding of the intestinal epithelial-microbe relationship.

Neutrophil transepithelial migration in response to the chemoattractant fMLP but not C5a is phospholipase D-dependent and related to the use of CD11b/CD18.

In Crohn's disease and ulcerative colitis patients, the numbers of neutrophils recovered from stool directly correlates with the severity of disease, implying that neutrophils in the lumen contribute to the tissue destruction; therefore, it is important to understand the mechanisms behind transintestinal epithelial migration. Neutrophil transintestinal epithelial migration to fMLP is appreciated to be CD11b/CD18 integrin (Mac-1)-dependent, while we recently reported that migration to C5a is Mac-1-independent. Here, we investigated whether phospholipase D (PLD), a signaling molecule linked to chemoattractant activation of neutrophils, is necessary for both Mac-1-dependent and Mac-1-independent migration. Both fMLP and C5a increased neutrophil expression of the Mac-1 activation epitope, indicating PLD was activated. This up-regulation was dose-dependently prevented by incubation of neutrophils in 1-butanol, an inhibitor of PLD activity. Despite this effect on Mac-1, 1-butanol did not prevent neutrophil migration across acellular filters. Incubation in 1-butanol did inhibit fMLP but not C5a-mediated migration across intestinal epithelial cell monolayers, showing that transepithelial migration to fMLP but not C5a is dependent on PLD. The addition of phosphatidic acid, a reaction product of PLD, partially restored fMLP-mediated transepithelial migration in the presence of 1-butanol but not the migration of Mac-1-deficient neutrophil-differentiated HL-60 cells. Thus PLD control over expression of the Mac-1 activation epitope is critical for neutrophil migration to fMLP but not C5a. Moreover, as PLD controls other neutrophil functions, such as the oxidative response, degranulation, and protease release, we could exclude these functions as being important in neutrophil transepithelial migration to C5a.

The anaphylatoxin C3a primes model colonic epithelial cells for expression of inflammatory mediators through Gαi.

Multiple studies have identified that complement becomes activated during inflammation of the intestines yet it is unclear what roles the split complement molecules play. The epithelium, in particular, may be impacted and accordingly, we first discovered that colonic cell lines indeed possess the C5aR. Here we examined whether these cells also possess the C3aR. We determined that T84, HT-29 and Caco2 all possess C3aR mRNA and protein; T84 and HT29 were used to further explore the consequence of C3a binding the C3aR. C3a led to increased mRNA for CXCL2, CXCL8 and CXCL11. Polarized T84 monolayers responded to apically applied C3a with increased CXCL8 mRNA more rapidly than if the C3a was applied basolaterally. Polarized monolayers also increased permeability when treated with C3a. ERK1/2 was activated by C3a and the increase in CXCL8 mRNA was ERK-dependent in both T84 and HT-29. C3a resulted in activation of Gαi, determined by the ERK1/2 signal showing sensitivity to pertussis toxin. The transmembrane signal was further mapped to include Ras and c-Raf. Finally, we show that the C3aR is expressed by primary cells in mouse enteroids. We conclude that complement activation will contribute to the epithelial response during inflammation through C3a binding to the C3aR including by priming the cells to upregulate mRNA for selected chemokines.

Activation of NF-kappaB following detachment delays apoptosis in intestinal epithelial cells.

We reported earlier that IL-1beta, an NF-kappaB-regulated cytokine, was made by intestinal epithelial cells during detachment-induced apoptosis (anoikis) and that IL-1 was antiapoptotic for detached cells. Since surviving anoikis is a prerequisite for cancer progression and metastases, we are further exploring the link between anoikis and cytokines. Here we determined that multiple genes are expressed following detachment including a number of NF-kappaB-regulated products and therefore aimed to determine whether NF-kappaB signalling plays any role in regulating apoptosis. Using Western blotting, we detected that IkappaBalpha becomes phosphorylated immediately following detachment and that levels of phospho-IkappaBalpha peaked within 20 min. Phosphorylation of IkappaBalpha was followed by Rel A (p65) nuclear translocation. Increased NF-kappaB activity following detachment was confirmed using the detection of NF-kappaB-promoted luciferase gene expression delivered by adenovirus infection. Infection of cells with adenovirus expressing a super-repressor IkappaBalpha protein and pharmacological inhibitors of NF-kappaB resulted in the failure to phosphorylate IkappaBalpha, a more rapid activation of caspases and earlier apoptosis. We also detected that IkappaB kinase alpha (IKKalpha) and not IKKbeta became phosphorylated following detachment. Since IKKalpha is activated by NF-kappaB-inducing kinase (NIK), we overexpressed native NIK using an adenovirus vector that resulted in enhanced phospho-IkappaBalpha and nuclear p65 in detached cells compared to control detached cells but did not result in a significantly greater number of cells surviving to 24 h. We conclude that detachment directly activates NF-kappaB, which, in addition to launching an inflammatory cytokine wave, contributes to a delay in apoptosis in intestinal epithelial cells.

Early Changes in Microbial Community Structure Are Associated with Sustained Remission After Nutritional Treatment of Pediatric Crohn's Disease.

BACKGROUND: Clinical remission achieved by exclusive enteral nutrition (EEN) is associated with marked microbiome changes. In this prospective study of exclusive enteral nutrition, we employ a hierarchical model of microbial community structure to distinguish between pediatric Crohn's disease patients who achieved sustained remission (SR) and those who relapsed early (non-SR), after restarting a normal diet. METHODS: Fecal samples were obtained from 10 patients (age 10-16) and from 5 healthy controls (age 9-14). The microbiota was assessed via 16S rRNA sequencing. In addition to standard measures of microbial biodiversity, we employed Bayesian methods to characterize the hierarchical community structure. Community structure between patients who sustained remission (wPCDAI <12.5) up to their 24-week follow-up (SR) was compared with patients that had not sustained remission (non-SR). RESULTS: Microbial diversity was lower in Crohn's disease patients relative to controls and lowest in patients who did not achieve SR. SR patients differed from non-SR patients in terms of the structure and prevalence of their microbial communities. The SR prevalent community contained a number of strains of Akkermansia muciniphila and Bacteroides and was limited in Proteobacteria, whereas the non-SR prevalent community had a large Proteobacteria component. Their communities were so different that a model trained to discriminate SR and non-SR had 80% classification accuracy, already at baseline sampling. CONCLUSIONS: Microbial community structure differs between healthy controls, patients who have an enduring response to exclusive enteral nutrition, and those who relapse early on introduction of normal diet. Our novel Bayesian approach to these differences is able to predict sustained remission after exclusive enteral nutrition.