The increasing burden of Crohn's disease (CD) alongside globalization and urbanization has prompted research into the environmental factors driving this rise. Genetic drift is less implicated than environmental and dietary changes affecting the gut microbiome, immune system, and intestinal mucosa, ultimately contributing to chronic inflammation in CD. Current therapies often fail to provide complete disease control, highlighting a critical knowledge gap in CD's etiology and hindering the development of targeted treatments. While Western populations have experienced near-universal modernization, hindering the isolation of specific environmental and dietary factors, China's ongoing transition from a rural to an urban society presents a unique opportunity. This transition parallels a dramatic increase in CD incidence, offering a valuable setting to study CD evolution and compare it to Western patterns. A meta-analysis comparing Eastern and Western populations revealed that higher fat intake, along with increased monounsaturated fatty acids (MUFA), n-3 polyunsaturated fatty acids (PUFA), and n-6 PUFA, are associated with CD primarily in Eastern populations. This underscores the need for investigations focusing on specific environmental contexts and diverse populations.

Existing research on CD pathogenesis often relies on disease models that don't fully capture the complexity of the human condition. Human cohorts and biospecimens are essential for a more comprehensive understanding of the contributing factors. To address this, the Study Of Urban and Rural Crohn disease Evolution (SOURCE) cohort was established. This multi-center, multi-omics cross-sectional study encompassed 380 newly diagnosed CD patients and controls from both urban and rural settings in Guangdong province, China, and Israel. Data collected included demographics, clinical characteristics, dietary exposures (using a food frequency questionnaire – FFQ), environmental exposures (childhood factors, dietary and smoking habits, sanitation), fecal microbiome, ileal transcriptome, and fecal metabolome. Previous studies have highlighted the importance of the gut microbiome and its interaction with host genetics and environmental factors in the development of CD. However, the SOURCE study distinguishes itself by its unique cohort design and the comprehensive integration of diverse data types, aiming to provide a more holistic understanding of CD pathogenesis.

The SOURCE cohort included 380 participants: 40 newly diagnosed CD patients and 121 healthy urban residents of Guangzhou, and 162 healthy rural residents of Shaoguan district in China; and 25 newly diagnosed CD patients and 32 healthy controls in Israel. Data collected encompassed demographics, clinical characteristics (CRP, fecal calprotectin), FFQ data, environmental questionnaires (IOIBD), microbiome analysis (16S rRNA gene sequencing, metagenomics), ileal transcriptomics, and stool metabolomics. The Chinese participants were all Asian, while the Israeli participants were all White. Analysis included stratification of rural Chinese participants based on time spent in urban environments (<50% vs. ≥50%), allowing comparison of microbial and metabolic profiles across rural, rural-urban, and urban groups. Statistical methods involved unsupervised PCOA, alpha/beta diversity calculations, MaAsLin multivariate analyses (controlling for age and gender), HAllA analysis (to capture complex interactions in the rural Chinese group), PCA (for dietary patterns), PERMANOVA (quantifying factors affecting microbial composition), WGCNA (for ileal transcriptomics), and DIABLO (for multi-omics integration). Specific details of DNA extraction, 16S amplicon sequencing, shotgun metagenomics sequencing, metabolomics profiling (LC-MS and UPLC-MS/MS for Sheba and SYS samples respectively), RNA extraction, and RNA-seq analysis are provided in the methods section. The study was approved by the relevant ethics committees in both countries.

The study revealed several key findings: 1. **Rural-to-urban transition mirrored CD-associated changes:** Unsupervised analysis demonstrated that time spent in urban environments by rural Chinese residents significantly altered their gut microbiome and metabolome, mirroring changes observed in CD patients. Specifically, lower microbial diversity and shifts in ASV abundance towards taxa (like *Bacteroides*, *R. gnavus*, *Fusobacteriaceae*) enriched in CD were observed in the rural-urban group. Metabolite changes in this group also showed significant overlap with CD-associated metabolic shifts. 2. **Diet linked with microbial variations:** PCA and PERMANOVA analyses indicated that dietary factors (total and saturated fat, fruits, iron, dairy, added sugar) were significantly associated with gut microbial composition in both cohorts, with fat consumption showing a stronger link in China. MaAsLin2 analyses identified ASVs whose abundance decreased with higher iron and fat consumption; these were enriched for taxa previously observed to be decreased in CD. In contrast, ASVs increasing with higher fat and iron consumption were enriched for bacteria typically found in saliva. 3. **CD-increased taxa enriched in mucosal biopsies:** Analysis of the microbiome in both stool and ileal biopsies revealed that CD patients had reduced alpha diversity, decreased microbial health index and an increased abundance of CD-associated taxa in their biopsies, notably *Enterobacteriaceae*, *Actinomyces*, and *Fusobacteriaceae*. 4. **Correlation between mucosal transcriptomics, diet, and metabolites:** WGCNA on ileal transcriptomics data identified gene co-expression modules associated with CD in both cohorts. These modules were linked with clinical factors, biomarkers, and dietary, bacterial, and metabolomic features. Diet-linked metabolites were primarily associated with host epithelial metabolic functions, while microbe-linked metabolites were linked to host immune responses. Specific dietary factors such as manganese, vitamin D, and coffee showed negative correlations with CD-associated modules, while sugar and saturated fat showed positive correlations. Several metabolites showed promise for therapeutic intervention; azelate (used to suppress skin inflammation), and compounds related to the dopaminergic system (phenethanolamine & L-dopa). Notably, mitochondrial dysfunction was highlighted, with metabolites suppressing mitochondrial respiration linked to CD-associated bacteria. 5. **Multi-omics integration:** sPLS and DIABLO analyses revealed a high degree of shared variation across different omics layers (transcriptomics, metabolomics, microbiome, dietary data), further solidifying the interconnectedness of these factors in CD pathogenesis. Key bacterial taxa (*R. gnavus*, *E. Ramosums*) associated with immune-related modules clustered with CD, while those like fibers and *A. putredinis* clustered with controls.

The findings from the SOURCE study provide crucial insights into the interplay between diet, environment, microbiome, and host transcriptome in CD pathogenesis. The study's unique design, combining data from both urban and rural settings in China and Israel, and integrating multi-omics data, allows for a comprehensive understanding of CD's etiology beyond a simple urban/rural dichotomy. The observation that changes mimicking those in CD patients appear in rural individuals with significant urban exposure highlights the continuous impact of environmental factors on gut health. The identification of specific dietary factors (manganese, vitamin D, coffee) positively correlated with healthier gut states offers potential avenues for dietary interventions and preventive strategies. Similarly, the correlation of several metabolites with healthier states, some already used in other inflammatory conditions, proposes promising therapeutic targets. The integration of the different omics data layers reinforces the complex interactions and interdependence of these factors in CD development. The integration of multi-omics data, including transcriptomics, metabolomics, and microbiome profiling, contributes significantly to understanding the underlying mechanisms of the disease. This comprehensive approach goes beyond traditional approaches that focus on single factors.

The SOURCE study provides a comprehensive understanding of the interplay between diet, environment, microbiome, and host in Crohn’s disease. The study’s unique design and multi-omics approach offer valuable insights, including the identification of potential dietary interventions and therapeutic targets. Future research could focus on interventional studies to validate the findings, expand the cohort size for increased statistical power, and explore the use of identified metabolites and dietary factors as potential therapeutic strategies.

The study's relatively small sample size, despite the comprehensive data collection, limits the generalizability of findings to other populations. The reliance on FFQs for dietary information may introduce some inaccuracies, although the comprehensive questionnaire likely captured most dietary variance. Additionally, the two cohorts originated from specific geographic locations, potentially influencing the generalizability of certain results. Finally, the more limited transcriptomics and metabolomics data available for only a subset of participants is a limitation, although the findings in this subgroup were independently validated and still produced important and substantial results.