Environmental enteric dysfunction (EED), previously known as tropical or environmental enteropathy, is a significant pediatric health concern, particularly in low- and middle-income countries. It's characterized by chronic intestinal inflammation, leading to malnutrition, stunted growth, cognitive impairment, and reduced response to oral vaccines. EED patients typically exhibit villous atrophy, nutrient malabsorption, compromised barrier function, and inflammation. Current in vitro models are limited, hindering mechanistic understanding and the development of effective diagnostic biomarkers or therapeutics. While micronutrient deficiencies (like zinc and vitamin A) are associated with EED and abnormal intestinal permeability, nutritional interventions have yielded disappointing results, likely due to persistent nutrient absorption problems and inflammation. Other dietary interventions, such as omega-3 fatty acid supplementation, amino acid profile optimization, and multiple micronutrient supplementation, have also shown limited success. Dietary deficiencies increase susceptibility to environmental factors. For example, tryptophan deficiency decreases antimicrobial peptide secretion, increasing vulnerability to intestinal inflammation. Low serum tryptophan is linked to stunting in EED children. Tryptophan is crucial for protein synthesis and is a precursor for niacin, melatonin, and neurotransmitters. Amino acid supplementation, including tryptophan, has shown some promise in improving EED symptoms. Niacin deficiency is also implicated in EED, with niacin administration showing potential in ameliorating colitis. Niacin is essential for coenzymes NAD and NADP, vital for cell function. However, a direct mechanistic link between malnutrition and EED pathophysiology in humans remains unclear. The multifactorial nature of EED presents significant challenges for modeling. Limited murine models and the absence of human in vitro models necessitate a new approach. This research utilizes human organ-on-a-chip technology, specifically a human intestine-on-a-chip, to address this gap. The intestine-on-a-chip accurately replicates the structure and function of the human intestine, providing a suitable platform to study EED pathophysiology and develop new interventions.

Existing literature extensively documents the clinical manifestations and consequences of EED, highlighting its devastating impact on child health and development in resource-limited settings. Studies have consistently shown the correlation between EED and malnutrition, impaired growth, cognitive deficits, and diminished immune responses. Several studies have investigated the role of specific nutritional deficiencies, such as zinc, vitamin A, and more recently, tryptophan and niacin, in the pathogenesis of EED. While some evidence suggests that these deficiencies contribute to the disease process, the precise mechanisms and their interplay with other factors, such as the gut microbiome and host genetic predisposition, remain largely unclear. The existing animal models, primarily murine, have provided valuable insights, but their limitations in fully recapitulating the human intestinal environment and complex interactions restrict their translational potential. Therefore, a robust human-based in vitro model is crucial to advance our understanding of EED and develop targeted therapeutic strategies.

This study employed human intestine-on-a-chip technology, a microfluidic system lined with living human intestinal epithelium derived from patient-derived organoids. Intestine chips were created using intestinal epithelial cells from organoids derived from surgical biopsies of healthy children and pediatric EED patients. The chips facilitated the formation of differentiated three-dimensional villus-like epithelial structures and an overlying mucus layer, mimicking the in vivo environment. Gene expression profiling was conducted using Affymetrix Human Clariom D arrays, comparing healthy and EED intestine chips cultured in control medium and a nutrient-deficient medium lacking niacinamide and tryptophan (-N/-T). Differential gene expression analysis was performed using the limma R package. Pathway analysis utilized the COMPBIO natural language processing algorithm to identify functionally related gene clusters. Differential interference contrast (DIC) and immunofluorescence microscopy were used to assess villus structure. Apparent permeability (Papp) measurements using Cascade Blue tracer assessed intestinal barrier function. Fluorescently labeled dodecanoic acid measured fatty acid uptake, and untargeted metabolomic analysis using LC-MS/MS explored nutrient absorption and metabolism. A bead-based multiplexed ELISA quantified cytokine secretion.

The study's key findings demonstrate that the intestine-on-a-chip model successfully recapitulates several hallmarks of EED. Specifically: 1. **Gene Expression Changes:** EED chips cultured in -N/-T medium showed differential expression of 969 genes, with a significant overlap (60%) in the top 10 upregulated genes observed in a clinical EED signature from a separate study. This included upregulation of antimicrobial genes and downregulation of metallothioneins and genes involved in digestion and metabolism. 2. **Villus Blunting and Barrier Dysfunction:** Both healthy and EED chips cultured in -N/-T medium exhibited significant villus blunting and compromised barrier function, as evidenced by reduced epithelial height and increased Papp values. These changes were more pronounced in EED chips. 3. **Impaired Nutrient Absorption:** Nutritional deficiency led to downregulation of genes involved in fatty acid and amino acid uptake and metabolism. Immunofluorescence and fatty acid uptake assays confirmed reduced ApoB expression and impaired fatty acid uptake in both healthy and EED chips under -N/-T conditions. 4. **Altered Inflammatory Mediators:** EED chips exposed to -N/-T medium showed significantly higher levels of inflammatory cytokines (IL-6, ICAM-1, VCAM-1, IL-33, MCP-1, MIP-1 alpha, and IL-8) compared to healthy chips under the same conditions. These changes were observed both in the luminal and basal channels of the chips. 5. **Metabolomic Analysis:** Untargeted metabolomic analysis revealed that EED chips showed lower transport of several metabolites compared to healthy chips, particularly amino acid metabolites, nucleotides, cofactors, lipids, and carbohydrates. This supports the clinical observation of impaired nutrient metabolism in EED patients.

The findings of this study demonstrate the utility of the intestine-on-a-chip model in dissecting the complex interplay between nutritional deficiency and the pathogenesis of EED. The ability to recapitulate key transcriptomic, structural, functional, and inflammatory changes observed in EED patients in a controlled in vitro environment provides a powerful tool for mechanistic investigations. The results suggest that both genetic/epigenetic factors intrinsic to EED and nutritional deficiency contribute to the disease phenotype. While villus blunting and barrier dysfunction were observed in both healthy and EED chips under nutrient-deficient conditions, the greater extent of these changes and the amplified inflammatory response in EED chips highlight the synergistic effect of genetic predisposition and environmental factors in driving EED pathogenesis. Furthermore, the observed downregulation of nutrient transporters in EED chips exposed to nutritional deficiency suggests a direct impact on nutrient absorption independent of the reduced absorptive surface area due to villus blunting. This creates a potential positive feedback loop, where nutritional deficiency worsens the condition.

This study successfully established a human intestine-on-a-chip model that recapitulates key features of EED. The model reveals the combined effects of genetic predisposition and nutritional deficiency on intestinal morphology, barrier function, nutrient absorption, and inflammatory responses in EED. The intestine-on-a-chip technology offers a valuable platform for further investigation into EED pathophysiology, biomarker discovery, and personalized medicine approaches for optimizing nutritional interventions.

The study's limitations include the relatively small number of EED patient samples used. While the findings were consistent across the available samples and validated against large-scale clinical data, further studies with larger cohorts are needed to enhance generalizability and confirm the consistency of the observations. The study focused on a specific nutrient deficiency model (-N/-T) and future investigations should explore the effects of other nutritional deficiencies and their combinations to build a more complete picture of EED pathogenesis.