Ordovas Montanes, Josede Sousa Casal, Joshua2026-07-0720262026-06-052026de Sousa Casal, Joshua. 2026. Mechanisms of structural cell adaptation and memory in intestinal health and disease. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32671544https://dash.harvard.edu/handle/1/42744109The gastrointestinal tract is a semipermeable barrier to the external environment, allowing the absorption of nutrients while restricting the entry of microbes. This is in part achieved through structural cells, including epithelial cells and fibroblasts, which coordinate amongst themselves and with the immune system to maintain dynamic barrier function. With the advent of single-cell technologies, we have gained unprecedented understanding of the diversity of the structural cells within the intestine, including the identification of previously undescribed enterocyte and fibroblast subsets. Recent work has demonstrated that not only do structural cell states shift and adapt to changes within our external environment, but these cells can encode long-lived memory of prior exposures. This has been shown to play a role in the persistence of allergic inflammation within the nasal mucosa and has implications in both intestinal nutrient adaptation and disease development. Despite these recent advancements, how individual cells within the intestine mechanistically remember and causatively contribute to healthy function and disease development is not well understood. Here we investigated how the intestine adapts to distinct lived environments and exposures with the hypothesis that structural cells can sense, adapt to and remember external exposures to direct intestinal physiology. Environmental adaptation within the duodenum is especially critical during the early years of childhood, when individuals are first introduced to diverse diets and microbes. Using single-cell RNA-sequencing (scRNA-seq), we evaluated the duodenal cellular composition of 88 healthy individuals between the ages of 0.5 – 12 years old from multiple sites within the US and Pakistan. Leveraging the diversity of our cohort, we define mucosal cellular composition and chart its developmental trajectory in early childhood. By performing comparative analyses of children residing in the United States and Pakistan, we identified a differentiated enterocyte subset expressing the aquaglyceroporin, AQP10, that is enriched in children from the US and correlated with dietary fat intake. Using organoid models, we demonstrate that emergence of this state depends on lipid exposure in intestinal stem cells. Furthermore, we identify a previously undescribed enteroendocrine cell positive for thyrotropin-releasing hormone with evidence for a local endocrine-epithelial immune circuit positively associated with age. Our work provides insight into pediatric duodenal mucosal development and illuminates how intestinal cellular dynamics are shaped by environmental adaptation. We next investigated the hypothesis that during disease, maladaptive memory states may accumulate in structural cells to establish an “inflammatory tipping point”, a cellular ecosystem that is unable to resolve inflammation. We demonstrate that in the DSS-induced murine colitis model, recovered mice are primed for accelerated disease development during subsequent rounds of colitis. Utilizing scRNA-seq, we find trophocytes, a subset of fibroblasts, expand during colitis and remains expanded following recovery. Using ex vivo cultures, we show that DSS-experienced fibroblasts intrinsically retain distinct transcriptional signatures and functionally support larger numbers of monocytes and macrophages through a mechanism not explained by elevated M-CSF but instead, by the production of the secreted WNT inhibitor, Sfrp1. As fibroblasts cannot be easily depleted, we optimized a chemogenetic system to selectively activate colonic fibroblasts. Using this approach, we demonstrate that fibroblast activation was sufficient to induce colon shortening, weight loss, and increased macrophage infiltration. When activated alongside DSS, this increased the severity of colitis. Taken together, our work demonstrates that defining structural cells through single-cell approaches has revealed novel adaptation and memory circuits in intestinal physiology. This memory can be cell intrinsic and influence subsequent immune cell interactions. We propose that the work presented here supports a new avenue of structural cell biology and its relation to immunology. By further understanding the mechanisms that govern structural cell memory, we can begin to reinforce or inhibit specific cell states that can restore healthy tissue function during chronic disease.application/pdfenImmunologyPhysiologyDevelopmental biologyThesis or Dissertation2026-07-070000-0003-2006-3992