Gut Microbiome Composition Is Associated With Future Onset of Crohn's Disease in Healthy First-Degree Relatives.

BACKGROUND & AIMS: The cause of Crohn's disease (CD) is unknown, but the current hypothesis is that microbial or environmental factors induce gut inflammation in genetically susceptible individuals, leading to chronic intestinal inflammation. Case-control studies of patients with CD have cataloged alterations in the gut microbiome composition; however, these studies fail to distinguish whether the altered gut microbiome composition is associated with initiation of CD or is the result of inflammation or drug treatment. METHODS: In this prospective cohort study, 3483 healthy first-degree relatives (FDRs) of patients with CD were recruited to identify the gut microbiome composition that precedes the onset of CD and to what extent this composition predicts the risk of developing CD. We applied a machine learning approach to the analysis of the gut microbiome composition (based on 16S ribosomal RNA sequencing) to define a microbial signature that associates with future development of CD. The performance of the model was assessed in an independent validation cohort. RESULTS: In the validation cohort, the microbiome risk score (MRS) model yielded a hazard ratio of 2.24 (95% confidence interval, 1.03-4.84; P = .04), using the median of the MRS from the discovery cohort as the threshold. The MRS demonstrated a temporal validity by capturing individuals that developed CD up to 5 years before disease onset (area under the curve > 0.65). The 5 most important taxa contributing to the MRS included Ruminococcus torques, Blautia, Colidextribacter, an uncultured genus-level group from Oscillospiraceae, and Roseburia. CONCLUSION: This study is the first to demonstrate that gut microbiome composition is associated with future onset of CD and suggests that gut microbiome is a contributor in the pathogenesis of CD.

Immune response and barrier dysfunction-related proteomic signatures in preclinical phase of Crohn's disease highlight earliest events of pathogenesis.

OBJECTIVE: The measure of serum proteome in the preclinical state of Crohn's disease (CD) may provide insight into biological pathways involved in CD pathogenesis. We aimed to assess associations of serum proteins with future CD onset and with other biomarkers predicting CD risk in a healthy at-risk cohort. DESIGN: In a nested case-control study within the Crohn's and Colitis Canada Genetics Environment Microbial Project (CCC-GEM) cohort, which prospectively follows healthy first-degree relatives (FDRs), subjects who developed CD (n=71) were matched with four FDRs remaining healthy (n=284). Using samples at recruitment, serum protein profiles using the Olink Proximity Extension Assay platform was assessed for association with future development of CD and with other baseline biomarkers as follows: serum antimicrobial antibodies (AS: positive antibody sum) (Prometheus); faecal calprotectin (FCP); gut barrier function using the fractional excretion of lactulose-to-mannitol ratio (LMR) assay. RESULTS: We identified 25 of 446 serum proteins significantly associated with future development of CD. C-X-C motif chemokine 9 (CXCL9) had the highest OR with future risk of CD (OR=2.07 per SD, 95% CI 1.58 to 2.73, q=7.9e-5), whereas matrix extracellular phosphoglycoprotein had the lowest OR (OR 0.44, 95% CI 0.29 to 0.66, q=0.02). Notably, CXCL9 was the only analyte significantly associated with all other CD-risk biomarkers with consistent direction of effect (FCP: OR=2.21; LMR: OR=1.67; AS: OR=1.59) (q<0.05 for all). CONCLUSION: We identified serum proteomic signatures associated with future CD development, reflecting potential early biological processes of immune and barrier dysfunction.

Altered Gut Microbiome Composition and Function Are Associated With Gut Barrier Dysfunction in Healthy Relatives of Patients With Crohn's Disease.

BACKGROUND & AIMS: The gut microbiome has been suggested to play a role in gut barrier hemostasis, but data are scarce and limited to animal studies. We therefore aimed to assess whether alterations in gut microbial composition and functional pathways are associated with gut barrier function in a cohort of healthy first-degree relatives of patients with Crohn's disease. METHODS: We used the Crohn's and Colitis Canada Genetic Environmental Microbial (CCC-GEM) cohort of healthy first-degree relatives of patients with Crohn's disease. Gut barrier function was assessed using the urinary fractional excretion of lactulose-to-mannitol ratio (LMR). Microbiome composition was assessed by sequencing fecal 16S ribosomal RNA. The cohort was divided into a discovery cohort (n = 2472) and a validation cohort (n = 655). A regression model was used to assess microbial associations with the LMR. A random forest classifier algorithm was performed to assess microbial community contribution to barrier function. RESULTS: Individuals with impaired barrier function (LMR >0.025) had reduced alpha-diversity (Chao1 index, P = 4.0e-4) and altered beta-diversity (Bray-Curtis dissimilarity index, R2 = 0.001, P = 1.0e-3) compared with individuals with an LMR ≤0.025. When taxa were assessed individually, we identified 8 genera and 52 microbial pathways associated with an LMR >0.025 (q < 0.05). Four genera (decreased prevalence of Adlercreutzia, Clostridia UCG 014, and Clostridium sensu stricto 1 and increased abundance of Colidextribacter) and 8 pathways (including decreased biosynthesis of glutamate, tryptophan, and threonine) were replicated in the validation cohort. The random forest approach revealed that the bacterial community is associated with gut barrier function (area under the curve, 0.63; P = 1.4e-6). CONCLUSIONS: The gut microbiome community and pathways are associated with changes in gut barrier function. These findings may identify potential microbial targets to modulate gut barrier.

Mediterranean-Like Dietary Pattern Associations With Gut Microbiome Composition and Subclinical Gastrointestinal Inflammation.

BACKGROUND & AIMS: Case-control studies have shown that patients with Crohn's disease (CD) have a microbial composition different from healthy individuals. Although the causes of CD are unknown, epidemiologic studies suggest that diet is an important contributor to CD risk, potentially via modulation of bacterial composition and gut inflammation. We hypothesized that long-term dietary clusters (DCs) are associated with gut microbiome compositions and gut inflammation. Our objectives were to identify dietary patterns and assess whether they are associated with alterations in specific gut microbial compositions and subclinical levels of gut inflammation in a cohort of healthy first-degree relatives (FDRs) of patients with CD. METHODS: As part of the Genetic, Environmental, Microbial (GEM) Project, we recruited a cohort of 2289 healthy FDRs of patients with CD. Individuals provided stool samples and answered a validated food frequency questionnaire reflecting their habitual diet during the year before sample collection. Unsupervised analysis identified 3 dietary and 3 microbial composition clusters. RESULTS: DC3, resembling the Mediterranean diet, was strongly associated with a defined microbial composition, with an increased abundance of fiber-degrading bacteria, such as Ruminococcus, as well as taxa such as Faecalibacterium. The DC3 diet was also significantly associated with lower levels of subclinical gut inflammation, defined by fecal calprotectin, compared with other dietary patterns. No significant associations were found between individual food items and fecal calprotectin, suggesting that long-term dietary patterns rather than individual food items contribute to subclinical gut inflammation. Additionally, mediation analysis demonstrated that DC3 had a direct effect on subclinical inflammation that was partially mediated by the microbiota. CONCLUSIONS: Overall, these results indicated that Mediterranean-like dietary patterns are associated with microbiome and lower intestinal inflammation. This study will help guide future dietary strategies that affect microbial composition and host gut inflammation to prevent diseases.

Anti-Microbial Antibody Response is Associated With Future Onset of Crohn's Disease Independent of Biomarkers of Altered Gut Barrier Function, Subclinical Inflammation, and Genetic Risk.

BACKGROUND AND AIMS: Altered host immune reactivity to microbial antigens is hypothesized to trigger the onset of Crohn's disease (CD). We aimed to assess whether increased serum anti-microbial antibody response in asymptomatic first-degree relatives (FDRs) of CD patients is an independent risk factor for future CD development. METHODS: We measured host serum antibody response to 6 microbial antigens at enrollment (Prometheus enzyme-linked immunosorbent assay test: anti-Saccharomyces cerevisiae antibodies immunoglobulin A/immunoglobulin G, anti-OmpC, anti-A4-Fla2, anti-FlaX, anti-CBir1) and derived the sum of positive antibodies (AS). We used samples at enrollment of prospectively followed healthy FDRs from a nested case-control cohort of the Crohn's and Colitis Canada Genetics Environment Microbial Project. Those who later developed CD (n = 77) were matched 1:4 by age, sex, follow-up duration, and geographic location with control FDRs remaining healthy (n = 307). To address our research aims, we fitted a multivariable conditional logistic regression model and performed causal mediation analysis. RESULTS: High baseline AS (≥2) (43% of cases, 11% of controls) was associated with higher risk of developing CD (adjusted odds ratio, 6.5; 95% confidence interval, 3.4-12.7; P < .001). Importantly, this association remained significant when adjusted for markers of gut barrier function, fecal calprotectin, C-reactive protein, and CD-polygenic risk score, and in subjects recruited more than 3 years before diagnosis. Causal mediation analysis showed that the effect of high AS on future CD development is partially mediated (42%) via preclinical gut inflammation. CONCLUSIONS: Our results suggest that increased anti-microbial antibody responses are associated with risk of future development of CD, independent of biomarkers of abnormal gut barrier function, subclinical inflammation, and CD-related genetic risks. This suggests that anti-microbial antibody responses are an early predisease event in the development of CD.

Comparison of Co-housing and Littermate Methods for Microbiota Standardization in Mouse Models.

The intestinal microbiota is a fundamental factor that broadly influences physiology. Thus, studies using transgenic animals should be designed to limit the confounding effects of microbiota variation between strains. Here, we report the impact on intestinal microbiota of co-housed versus F2-generation littermates, two commonly used techniques to standardize microbiota in animal models. Our results establish that while fecal microbiota is partially normalized by extended co-housing, mucosal communities associated with the proximal colon and terminal ileum remain stable and distinct. In contrast, strain inter-crossing to generate F2 littermates allows robust microbiota standardization in fecal, colon, and ileum sampling locations. Using reciprocal inter-crosses of P1 parents, we identify dissymmetry in F2 community structures caused by maternal transmission, in particular of the Verrucomicrobiaceae. Thus, F2 littermate animals from a unidirectional P1 cross should be used as a standard method to minimize the influence of the microbiota in genotype-phenotype studies.

Innate Immune Influences on the Gut Microbiome: Lessons from Mouse Models.

The gut microbiota is important in health and disease. Whereas the intestinal immune system has evolved to protect the mucosal barrier against pathogens, there is much interest in understanding how it influences the composition and functions of resident microbial communities. Overall, host innate immunity exerts little influence on the microbiota at homeostasis, but increases upon immune activation and the onset of inflammation, as well as in the presence of certain members of the microbiota. However, many experiments have not adequately incorporated study design to detect such immune influences, including using proper control groups, precise sampling and timing, and measures beyond broad-scale descriptions of dysbiosis for microbial analysis. We discuss these and other challenges in the context of current understanding of chronic inflammatory disease.

The interplay between microbes and the immune response in inflammatory bowel disease.

The aetiology and pathogenesis of inflammatory bowel disease (IBD) remains unclear but involves a complex interplay between genetic risk, environmental exposures, the immune system and the gut microbiota. Nearly two decades ago, the first susceptibility gene for Crohn's disease, NOD2, was identified within the IBD 1 locus. Since then, over 230 genetic risk loci have been associated with IBD and yet NOD2 remains the strongest association to date. As an intracellular innate immune sensor of bacteria, investigations into host-microbe interactions, involving both innate and adaptive immune responses, have become of particular interest in understanding the pathogenesis of IBD. Advancements in sequencing technology have lead to the groundbreaking characterization of the gut microbiota and its role in health and disease. While an altered microbiome has been described for IBD, whether it is a cause or an effect of the intestinal inflammation has yet to be determined. Moreover, the bidirectional relationship between the gut microbiota and the mucosal immune system adds to the multifaceted complexity of intestinal homeostasis. A better understanding of how host genetics, including NOD2, influence immune-microbe interactions and alter susceptibility to IBD is necessary in order to develop therapeutic and preventative treatments.

Determinants of IBD Heritability: Genes, Bugs, and More.

Defining the etiology of inflammatory bowel disease (IBD) continues to elude researchers, in part due to the possibility that there may be different triggers for a spectrum of disease phenotypes that are currently classified as either Crohn's disease (CD) or ulcerative colitis (UC). What is clear is that genetic susceptibility plays an important role in the development of IBD, and large genome-wide association studies using case-control approaches have identified more than 230 risk alleles. Many of these identified risk alleles are located in a variety of genes important in host-microbiome interactions. In spite of these major advances, the mechanisms behind the genetic influence on disease development remain unknown. In addition, the identified genetic risks have thus far failed to fully define the hereditability of IBD. Host genetics influence host interactions with the gut microbiota in maintaining health through a balance of regulated immune responses and coordinated microbial composition and function. What remains to be defined is how alterations in these interactions can lead to disease. The nature and cause of changes in the microbiota in patients with IBD are poorly understood. In spite of the large catalog of alterations in the microbiota of IBD patients, inflammation itself can alter the microbiota, leaving open the question of which is cause or effect. The composition and function of the gut microbiota are influenced by many factors, including environmental factors, dietary factors, and, as recent studies have shown, host genetic makeup. More than 200 loci have shown potential to influence the microbiota, but replication and larger studies are still required to validate these findings. It would seem reasonable to consider the combination of both host genetic makeup and the inheritance of the microbiota as interdependent heritable forces that could explain the nature of an individual's susceptibility to IBD or indeed the actual cause of IBD. In this review, we will consider the contribution of the host genetics, the microbiome, and the influence of host genetics on the microbiota to the heritability of IBD.

The Cytosolic Microbial Receptor Nod2 Regulates Small Intestinal Crypt Damage and Epithelial Regeneration following T Cell-Induced Enteropathy.

Loss of function in the NOD2 gene is associated with a higher risk of developing Crohn's disease (CD). CD is characterized by activation of T cells and activated T cells are involved in mucosal inflammation and mucosal damage. We found that acute T cell activation with anti-CD3 mAb induced stronger small intestinal mucosal damage in NOD2(-/-) mice compared with wild-type mice. This enhanced mucosal damage was characterized by loss of crypt architecture, increased epithelial cell apoptosis, delayed epithelial regeneration and an accumulation of inflammatory cytokines and Th17 cells in the small intestine. Partial microbiota depletion with antibiotics did not decrease mucosal damage 1 d after anti-CD3 mAb injection, but it significantly reduced crypt damage and inflammatory cytokine secretion in NOD2(-/-) mice 3 d after anti-CD3 mAb injection, indicating that microbial sensing by Nod2 was important to control mucosal damage and epithelial regeneration after anti-CD3 mAb injection. To determine which cells play a key role in microbial sensing and regulation of mucosal damage, we engineered mice carrying a cell-specific deletion of Nod2 in villin and Lyz2-expressing cells. T cell activation did not worsen crypt damage in mice carrying either cell-specific deletion of Nod2 compared with wild-type mice. However, increased numbers of apoptotic epithelial cells and higher expression of TNF-α and IL-22 were observed in mice carrying a deletion of Nod2 in Lyz2-expressing cells. Taken together, our results demonstrate that microbial sensing by Nod2 is an important mechanism to regulate small intestinal mucosal damage following acute T cell activation.

Genetics and innate and adaptive immunity in IBD.

Inflammatory bowel disease (IBD) is an abnormal inflammatory response within the gut to a trigger that has yet to be identified. The family history in many patients, especially those with Crohn's disease, suggests a genetic predisposition. It has been hypothesized that the abnormal inflammatory response is due in part to genetic alterations in the normal homeostatic processes that regulate host interactions with the normal gut microbes. Genetic studies have identified increasing numbers of genetic risk factors that involve a diverse series of pathways such as receptors of innate immune response, defects in epithelial barrier function, immune- and cytokine-related genes and genes involved in autophagy. Studies further suggest that abnormal immune responses in IBD patients are directed against the intestinal microbiota, with activation of both innate and adaptive immune responses. Indeed, studies have shown bacterial-derived antigen as drivers of T cell immune responses. More recently, Th17, regulatory T cells and unconventional innate-like T cells have been implicated in the induction and regulation of intestinal inflammation. The seminal discoveries of pathogen recognition receptors including Toll-like receptors and nucleotide-binding oligomerization domain receptors have changed our understanding of how immune cells respond to microbes and the role this may play in IBD pathogenesis. Understanding these mechanism will lead to new strategies in the treatment and prevention of IBD.

Nod2 activates NF-kB in CD4+ T cells but its expression is dispensable for T cell-induced colitis.

Although the etiology of Crohn's disease (CD) remains elusive this disease is characterized by T cell activation that leads to chronic inflammation and mucosal damage. A potential role for maladaptation between the intestinal microbiota and the mucosal immune response is suggested by the fact that mutations in the pattern recognition receptor Nod2 are associated with higher risks for developing CD. Although Nod2 deletion in CD4(+) T cells has been shown to impair the induction of colitis in the murine T cell transfer model, the analysis of T cell intrinsic Nod2 function in T cell differentiation and T cell-mediated immunity is inconsistent between several studies. In addition, the role of T cell intrinsic Nod2 in regulatory T cell (Treg) development and function during colitis remain to be analyzed. In this study, we show that Nod2 expression is higher in activated/memory CD4(+) T cells and its expression was inducible after T cell receptor (TCR) ligation. Nod2 stimulation with muramyl dipeptide (MDP) led to a nuclear accumulation of c-Rel NF-kB subunit. Although functionally active in CD4(+) T cells, the deletion of Nod2 did not impair the induction and the prevention of colitis in the T cell transfer model. Moreover, Nod2 deletion did not affect the development of Foxp3(+) Treg cells in the spleen of recipient mice and Nod2 deficient CD4 T cells expressing the OVA specific transgenic TCR were able to differentiate in Foxp3(+) Treg cells after OVA feeding. In vitro, CD25(+) Nod2 deficient T cells suppressed T cell proliferation as well as wild type counter parts and T cell stimulation with MDP did not affect the proliferation and the cytokine secretion of T cells. In conclusion, our data indicate that Nod2 is functional in murine CD4(+) T cells but its expression is dispensable for the T cell regulation of colitis.

An oral CD3-specific antibody suppresses T-cell-induced colitis and alters cytokine responses to T-cell activation in mice.

BACKGROUND & AIMS: New therapeutic approaches are needed for inflammatory bowel diseases. A monoclonal antibody against CD3 (anti-CD3) suppresses T-cell-mediated autoimmune diseases such as experimental allergic encephalomyelitis. We explored the effects of anti-CD3 in mice with colitis. METHODS: Severe combined immunodeficient mice were given injections of CD4(+)CD45RB(high) T cells to induce colitis. Four weeks later, the mice were given 2 or 5 µg/day of anti-CD3 or hamster immunoglobulin (Ig)G (control), via gavage, for 5 or 10 days. The effect of oral anti-CD3 on cytokine responses was studied by activating T cells using intraperitoneal injections of anti-CD3 monoclonal antibody 2 days after oral administration of the antibody. We collected intestine samples for histology analysis and cells were analyzed by flow cytometry. Cytokines in sera were analyzed by cytometric bead array. RESULTS: Oral administration of anti-CD3 protected the mice from wasting disease and intestinal inflammation. Analyses of spleen and mesenteric lymph node cells showed no differences in total cell counts, or percentages of CD4(+) and forkhead box P3(+) regulatory T cells, between mice given anti-CD3 or the control immunoglobulin. Colitis therefore was not suppressed by induction of forkhead box P3(+) regulatory T cells, or depletion or limited expansion of T cells. Oral administration of anti-CD3 ameliorated the enteropathy induced by intraperitoneal injection of the antibody. In mice with enteropathy, oral anti-CD3 reduced levels of inflammatory cytokines such as interferon-γ, tumor necrosis factor-α, and interleukin (IL)-6; it also increased levels of the anti-inflammatory cytokines IL-10 and transforming growth factor-ß. The effects of oral anti-CD3 required IL-10. CONCLUSIONS: Oral administration of anti-CD3 to mice induces changes in the mucosal immune response that prevent colitis, independent of specific antigen, and reduce T-cell activation in an IL-10-dependent manner. Oral anti-CD3 therefore might be developed for the treatment of patients with inflammatory bowel disease.