Inter-kingdom relationships in Crohn's disease explored using a multi-omics approach.

The etiology of Crohn's disease (CD) is multifactorial. Bacterial and fungal microbiota are involved in the onset and/or progression of the disease. A bacterial dysbiosis in CD patients is accepted; however, less is known about the mycobiome and the relationships between the two communities. We investigated the interkingdom relationships, their metabolic consequences, and the changes in the fungal community during relapse and remission in CD.Two cohorts were evaluated: a British cohort (n = 63) comprising CD and ulcerative colitis patients, and controls. The fungal and bacterial communities of biopsy and fecal samples were analyzed, with the fecal volatiles; datasets were also integrated; and a Dutch cohort (n = 41) comprising CD patients and healthy controls was analyzed for stability of the gut mycobiome.A dysbiosis of the bacterial community was observed in biopsies and stool. Results suggest Bacteroides is likely key in CD and may modulate Candida colonization. A dysbiosis of the fungal community was observed only in the Dutch cohort; Malassezia and Candida were increased in patients taking immunosuppressants. Longitudinal analysis showed an increase in Cyberlindnera in relapse. Saccharomyces was dominant in all fecal samples, but not in biopsies, some of which did not yield fungal reads; amino acid degradation was the main metabolic change associated with CD and both bacteria and fungi might be implicated.We have shown that Bacteroides and yeasts may play a role in CD; understanding their role and relationship in the disease would shed new light on the development and treatment of CD.

DNA extraction and amplicon production strategies deeply inf luence the outcome of gut mycobiome studies.

Microbial ecology studies are often performed through extraction of metagenomic DNA followed by amplification and sequencing of a marker. It is known that each step may bias the results. These biases have been explored for the study of bacterial communities, but rarely for fungi. Our aim was therefore to evaluate methods for the study of the gut mycobiome. We first evaluated DNA extraction methods in fungal cultures relevant to the gut. Afterwards, to assess how these methods would behave with an actual sample, stool from a donor was spiked with cells from the same cultures. We found that different extraction kits favour some species and bias against others. In terms of amplicon sequencing, we evaluated five primer sets, two for ITS2 and one for ITS1, 18S and 28S rRNA. Results showed that 18S rRNA outperformed the other markers: it was able to amplify all the species in the mock community and to discriminate among them. ITS primers showed both amplification and sequencing biases, the latter related to the variable length of the product. We identified several biases in the characterisation of the gut mycobiome and showed how crucial it is to be aware of these before drawing conclusions from the results of these studies.

Combined oral and rectal mesalazine for the treatment of mild-to-moderately active ulcerative colitis: rapid symptom resolution and improvements in quality of life.

BACKGROUND AND AIMS: Mesalazine (5-aminosalicylic acid) is the standard first-line therapy for mild-to-moderate ulcerative colitis. In the PINCE study, remission rates were significantly greater with combined oral/enema vs. oral/placebo treatment at 8 weeks (64% vs. 43%, respectively; p=0.030). In this analysis, we explored early response, mucosal healing rates, cessation of rectal bleeding, and quality of life in PINCE. METHODS: Patients with extensive mild-to-moderately active ulcerative colitis received 8weeks of oral mesalazine 4 g/day, plus 4 weeks of daily active (1g mesalazine) or placebo enema. Early response was assessed using the abbreviated ulcerative colitis disease activity index. Mucosal healing was assessed by disease activity index endoscopic mucosal appearance score. Cessation of bleeding (patient diaries), quality of life (EQ-5D), and patient acceptability (questionnaire) were also assessed. RESULTS: Combined mesalazine oral/enema treatment achieved a significantly higher rate of improvement in abbreviated ulcerative colitis disease activity index (score decrease ≥ 2) within 2 weeks, compared with oral-only treatment (p = 0.032). Bleeding ceased significantly more quickly with combination vs. oral therapy (p = 0.003). More patients showed mucosal healing (disease activity index endoscopic mucosal appearance score 0/1) with combination vs. oral therapy, which was significantly different between groups at week 4 (p = 0.052). Both groups showed quality of life improvements, with a significant benefit for combination vs. oral therapy at week 4 in multiple domains. Most patients reported finding the treatment acceptable. CONCLUSIONS: Rapid cessation of symptoms was seen with combination therapy, which is particularly important to patients and may improve quality of life.

IL-13 promotes collagen accumulation in Crohn's disease fibrosis by down-regulation of fibroblast MMP synthesis: a role for innate lymphoid cells?

BACKGROUND: Fibrosis is a serious consequence of Crohn's disease (CD), often necessitating surgical resection. We examined the hypothesis that IL-13 may promote collagen accumulation within the CD muscle microenvironment. METHODS: Factors potentially modulating collagen deposition were examined in intestinal tissue samples from fibrotic (f) CD and compared with cancer control (C), ulcerative colitis (UC) and uninvolved (u) CD. Mechanisms attributable to IL-13 were analysed using cell lines derived from uninvolved muscle tissue and tissue explants. RESULTS: In fCD muscle extracts, collagen synthesis was significantly increased compared to other groups, but MMP-2 was not co-ordinately increased. IL-13 transcripts were highest in fCD muscle compared to muscle from other groups. IL-13 receptor (R) α1 was expressed by intestinal muscle smooth muscle, nerve and KIR(+) cells. Fibroblasts from intestinal muscle expressed Rα1, phosphorylated STAT6 in response to IL-13, and subsequently down-regulated MMP-2 and TNF-α-induced MMP-1 and MMP-9 synthesis. Cells with the phenotype KIR(+)CD45(+)CD56(+/-)CD3(-) were significantly increased in fCD muscle compared to all other groups, expressed Rα1 and membrane IL-13, and transcribed high levels of IL-13. In explanted CD muscle, these cells did not phosphorylate STAT6 in response to exogenous IL-13. CONCLUSIONS: The data indicate that in fibrotic intestinal muscle of Crohn's patients, the IL-13 pathway is stimulated, involving a novel population of infiltrating IL-13Rα1(+), KIR(+) innate lymphoid cells, producing IL-13 which inhibits fibroblast MMP synthesis. Consequently, matrix degradation is down-regulated and this leads to excessive collagen deposition.

Identification and characterisation of an iron-responsive candidate probiotic.

BACKGROUND: Iron is an essential cofactor in almost all biological systems. The lactic acid bacteria (LAB), frequently employed as probiotics, are unusual in having little or no requirement for iron. Iron in the human body is sequestered by transferrins and lactoferrin, limiting bacterial growth. An increase in the availability of iron in the intestine by bleeding, surgery, or under stress leads to an increase in the growth and virulence of many pathogens. Under these high iron conditions, LAB are rapidly out-competed; for the levels of probiotic bacteria to be maintained under high iron conditions they must be able to respond by increasing growth rate to compete with the normal flora. Despite this, iron-responsive genera are poorly characterised as probiotics. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that a panel of probiotics are not able to respond to increased iron availability, and identify an isolate of Streptococcus thermophilus that can increase growth rate in response to increased iron availability. The isolate of S. thermophilus selected was able to reduce epithelial cell death as well as NF-κB signalling and IL-8 production triggered by pathogens. It was capable of crossing an epithelial cell barrier in conjunction with E. coli and downregulating Th1 and Th17 responses in primary human intestinal leukocytes. CONCLUSIONS/SIGNIFICANCE: We propose that an inability to compete with potential pathogens under conditions of high iron availability such as stress and trauma may contribute to the lack of efficacy of many LAB-based probiotics in treating disease. Therefore, we offer an alternative paradigm which considers that probiotics should be able to be competitive during periods of intestinal bleeding, trauma or stress.

Repertoire of the alpha beta T-cell receptor in the intestine.

The majority of T cells in the human and mouse intestine express the T-cell receptor (TCR) as an alphabeta heterodimer on their cell surface. As the major recognition element of antigens in the context of major histocompatibility complex-derived proteins, an examination of the structure of the alpha beta TCR in intestines has provided significant insights into the potential function of these cells and the major determinants that drive their selection. Studies in the human intestine have shown that the repertoires of intraepithelial lymphocytes (IELs), and likely lamina propria lymphocytes, are polyclonal before and shortly after birth, with the repertoire becoming oligoclonal in adults. Similarly, in adult mice the repertoire is oligoclonal, while in the newborn it is polyclonal. Investigations in mice have shown that some T cells may evade thymic selection. The population size and oligoclonality of IELs is influenced by the microbial content of the luminal microenvironment. This microenvironment probably directly determines the TCR repertoire. Studies in human inflammatory bowel disease (IBD) indicate that inflammation further skews the TCR repertoire. We speculate that dominant antigens associated with the pathogenesis of IBD are responsible for such skewing and that identifying the antigenic drivers may shed light on the environmental factors that trigger or potentiate human IBD.

A case-control study of drinking water and dairy products in Crohn's Disease--further investigation of the possible role of Mycobacterium avium paratuberculosis.

Similarities between Johne's disease in ruminants and Crohn's disease in humans have led to speculation that Mycobacterium avium paratuberculosis (MAP) might be a causative agent in Crohn's disease. However, evidence remains inconsistent. In this case-control study (1999-2004), the authors assessed the possible role of drinking water and dairy products potentially contaminated with MAP in the etiology of Crohn's disease. A total of 218 patients with Crohn's disease recruited from nine hospitals in England and 812 controls recruited from the community completed a short questionnaire for evaluation of proxy measures of potential exposure to MAP. Logistic regression showed no significant association with measures of potential contamination of water sources with MAP, water intake, or water treatment. Multivariate analysis showed that consumption of pasteurized milk (per kg/month: odds ratio (OR) = 0.82, 95% confidence interval (CI): 0.69, 0.97) was associated with a reduced risk of Crohn's disease. Meat intake (per kg/month: OR = 1.40, 95% CI: 1.17, 1.67) was associated with a significantly increased risk of Crohn's disease, whereas fruit consumption (per kg/month: OR = 0.78, 95% CI: 0.67, 0.92) was associated with reduced risk. This study does not support a role for water or dairy products potentially contaminated with MAP in the etiology of Crohn's disease. The observed association with meat and the negative association with pasteurized milk need further study.

The effect of weaning on the clonality of alpha beta T-cell receptor T cells in the intestine of GF and SPF mice.

In humans, intestinal antigen exposure during neonatal life influences the T-cell receptor (TCR) repertoire. To define the relative effects of bacteria and food antigens in early life, we examined TCR diversity in the intestine of SPF and GF mice. TCR repertoire was assessed at a single time point pre-, peri- and post-weaning in the small and large intestine of SPF and GF mice using spectratyping and/or TCR-beta-chain sequencing. There was good concordance of data obtained by the two techniques. In SPF mice, the repertoire was polyclonal shortly after birth in the small and large intestine. After weaning, there was a significant change towards an oligoclonal repertoire in the small intestine. There was some evidence that specific clones were shared between the small and large intestine. In contrast, in GF mice, the repertoire was oligoclonal after birth, and remained restricted. These data show: firstly, that under SPF conditions, the intestine is seeded with a diverse T-cell population that becomes oligoclonal around the time of weaning; secondly, that GF mice were oligoclonal at each time point.

Selective processing of gastrointestinal symptom-related stimuli in irritable bowel syndrome.

OBJECTIVES: We sought to determine whether irritable bowel syndrome (IBS) was associated with attentional bias toward symptom-related cues in IBS patients versus healthy controls, using a modified Stroop task to measure selective processing of gastrointestinal symptom-related cues. METHODS: Fifteen patients with a clinical diagnosis of IBS and 15 healthy controls were recruited into the study. All participants attended a single testing session, during which they completed a modified Stroop task using gastrointestinal symptom-related and neutral control words. RESULTS: Results indicated a significant main effect of word type (p = .013), with slower color-naming times for IBS-related compared with neutral words, and a significant main effect of exposure (p = .001), with slower color-naming times in the unmasked condition compared with the masked condition. The group x word type x exposure interaction was significant (p = .048). A series of post hoc tests indicated that among patients there was significant interference of symptom-related words in the masked condition but not in the unmasked condition, whereas among controls, the reverse was true. CONCLUSIONS: These results indicate that IBS patients selectively process gastrointestinal symptom-related words compared with neutral words when they are presented subliminally but not when they are presented supraliminally. In contrast, healthy controls demonstrate the opposite pattern. Implications for the cognitive mechanisms in IBS, and future research directions, are discussed.

Effects of microflora on the neonatal development of gut mucosal T cells and myeloid cells in the mouse.

Colonization with commensal flora in very early life may profoundly influence intestinal lymphoid development and bias later immune responses. We defined gut-homing T cell phenotypes and the influence of flora on intestinal immune development in mice. Intestinal T cells were phenotyped and quantified in conventional (CV), germfree (GF) and conventionalized germfree (GF/CV) neonatal mice by immunohistochemistry. Mucosal adressin cell adhesion molecule 1 (MAdCAM-1) was expressed by mucosal vessels at birth in CV and GF mice and was more prevalent in CV than GF small intestine, but was distributed similarly and did not change with age. Less MAdCAM-1 was expressed in the colon; its distribution became restricted after weaning, with no difference between CV and GF mice. CD3(+)beta(7) (+) cells were present in similar numbers in CV and GF intestine at birth. They were CD62L(-) in CV mice and were accompanied by further CD3(+)beta(7) (+)CD62L(-) T cells as development progressed, but in GF and GF/CV intestine they expressed CD62L and numbers did not change. IEL numbers increased at weaning in CV mice in both small and large intestine, but showed delayed development in GF intestine. Macrophages were present at high levels from birth in GF intestine, but dendritic cells did not develop until day 16. Thus, fetus-derived T cells seed the intestinal lamina propria before birth via beta-MadCAM interactions. Their activation status depends on the microbiological status of the dam, and without a commensal flora they remain naive. We propose that these cells regulate antigen responsiveness of the developing mucosal T cell pool.

Intestinal alpha beta T cells differentiate and rearrange antigen receptor genes in situ in the human infant.

Intestinal Ag exposure during neonatal life influences appropriate adult immune responses. To define the mechanisms shaping the T cell repertoire during this period, we examined T cell differentiation and receptor diversity in the intestine of human infants. Developmental phenotypes of intraepithelial and lamina propria intestinal T cells from infants aged 1 day to 2 years were assessed ex vivo by flow cytometry and in situ by triple-fluorescent immunohistochemistry. Gene recombination-specific enzymes were assessed by PCR. TCR beta-chain V region gene diversity was determined by sequencing. Several different early lineage T cell populations were present neonatally: CD3(+)4(-)8(-) T cells were present at birth and numbers decreased during the neonatal period; CD3(+)4(+)8(+) T cells were present in low numbers throughout infancy; and CD3(+)4(+)8(-) or CD3(+)4(-)8(+) T cells increased with age. Very early lineage T cells, CD3(-)2(-)7(+) and CD3(-)2(+)7(+), were present neonatally, but were essentially absent at 1 year. Most lamina propria T cells differentiated rapidly after birth, but maturation of intraepithelial T cells took place over 1 year. Intestinal samples from infants less than 6 mo old contained transcripts of T early alpha and TdT, and 15 of 19 infant samples contained mRNA for RAG-1, some coexpressing RAG-2. TCR beta-chain repertoires were polyclonal in infants. Immature T cells, pre-T cells, and genes involved in T cell recombination were found in the intestine during infancy. T cell differentiation occurs within the neonatal human intestine, and the TCR repertoire of these developing immature T cells is likely to be influenced by luminal Ags. Thus, mucosal T cell responsiveness to environmental Ag is shaped in situ during early life.

Betel nut and smoking: are they both protective in ulcerative colitis? A pilot study

Two-hundred and twenty-three Asian patients with inflammatory bowel disease were sent a questionnaire about smoking and betel nut chewing habits. The 116 responses from patients with ulcerative colitis were compared with answers from 79 healthy members of the community. Twelve per cent of male patients were currently smokers compared with 31 per cent of healthy controls (X(2) = 20.9 P < O.05). No such differences existed between female patients and controls. Thirteen per cent of male patients regularly used betel nut compared with 20 per cent (X(2) = 3.9 P < O.05). Both smoking and betel nut chewing appear to reduce the risk of developing ulcerative colitis, although these effects may be linked.