Longitudinal Profiling Reveals Coupled Immune and Structural Defects in Developing Cystic Fibrosis Ileum

Background: In humans, Cystic Fibrosis (CF) gastrointestinal (GI) pathology begins early in fetal development. However, in CF mice, similar changes occur postnatally during weaning (P14 to P21), mimicking GI pathologies seen in the human CF intestine, including ileal obstruction, goblet cell hyperplasia, gut inflammation, and dysbiosis. This study hypothesizes that significant ileal structural changes during maturation correlate with early immune cell population establishment and subsequent inflammation in the small intestine (SI) of CF mice. Methods: Ileal samples were collected from B6.129P2-Cftr-/-tm1Unc (CF) and littermate heterozygous (HET) mice on P1, 3, 14, 21, and 28 (n=4/group). Tissues were analyzed for goblet cell counts (GC) (PAS), muscle thickness, and crypt-villus axis (CVA) length (H&E). Data are expressed as fold change: CF relative to HET. CFTR distribution was assessed by immunofluorescence (IF). Immune cells from the SI (P1, 14, 21; n=4-8/group) were analyzed by CyTOF using a 28-antibody panel. Data were bead-normalized, gated on CD45+ cells, and clustered using FlowJo/FlowSom after down-sampling. Pro-inflammatory cytokines were assessed by CyTOF. Results: IF analysis showed CFTR apical staining in HET enterocytes that was absent in CF mice. CF mice exhibited higher goblet cell numbers after P1, and muscle thickness and CVA length at all time points compared to HET mice. GC were initially lower in CF mice at P1 (0.83× HET levels), but surpassed HET by P3 (1.3×) and continued to rise, reaching a fold difference of 1.57× at P28. Muscle layer thickness was significantly higher in CF mice at all stages, increasing from 1.9× (P1) and 1.76× (P3) to 8.01× (P21) and 8.49× (P28). CVA length showed a similar trend, with modest increases at P1–P3 (1.13× to 1.15× HET values) and stronger divergence at later stages (1.96× at P14, 2.54× at P21, and 1.72× at P28), indicating accelerated crypt-villus maturation and remodeling in the intestine. Immune cell profiling identified seven lymphoid and twelve myeloid clusters. Regulatory T cells (Treg), increased over time in healthy tissue but showed a 3-fold decline in CF gut compared to HET mice at P21. Opposite trends were observed for NK cells, which were significantly higher in CF gut at P21 (3-fold increase). Notably, CF ileal tissues had increasing proportions of MHCIIhiCD24+CD8+ T cells from P1 to P21 (7% of lymphoid cells by P21), which also expressed increasing levels of IFN-γ, IL-17A, and TNF-α. Additionally, populations of monocytes, CD68 + macrophages, and neutrophils emerged at P14 and P21 in the CF SI, but were absent in HET SI. Conclusions: Postnatal CF mouse ileum showed progressive goblet cell hyperplasia, muscle hypertrophy, and CVA elongation. These structural changes parallel early immune shifts, beginning with a distinct lymphoid population at birth and followed by inflammatory myeloid and cytokine-producing lymphoid cells emerging between P14 and P21. Together, these coordinated structural and immune deviations indicate a transition to active inflammation and disrupted tolerance at weaning, implicating early immune imbalance in CF ileal pathology. Supported by CFF This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.