LCN2 contributions to an altered secretory environment in the intestinal epithelium in Crohn’s disease

Background: The intestinal epithelium is a single but heterogeneous layer of cells that acts as a barrier between the luminal contents of the gastrointestinal tract and underlying mucosa. In patients with inflammatory bowel disease (IBD), dysregulated immune response and impaired mucosal healing lead to chronic inflammation. Our lab has identified a unique cell state, termed inflammatory secretory progenitor (ISP) cell state, in the intestinal epithelium of pediatric patients with Crohn’s disease. ISP gene expression is found in OLFM4-/REG1A+ secretory progenitors that maintain an inflammatory profile with the expression of immune-related and antimicrobial peptides, and antigen presentation proteins. We posit that the presence of cells in an ISP state may modulate epithelial response to injury and sustained inflammation, such as impaired repair that poises the epithelium for exacerbated disease pathogenesis or oncogenesis. Consistent with previous findings that show it is upregulated in Crohn’s, one of the ISP markers is lipocalin-2 (LCN2). First identified for its role in innate immunity, LCN2 is a secreted molecule expressed by multiple cell types. LCN2 can facilitate cellular uptake of different small weight molecules, including iron-binding compounds. Epithelial cell expression of LCN2 could therefore modify local iron stores during intestinal inflammation and repair. Mice with whole body deletion of Lcn2 are more sensitive to bacterial infection, but also appear to be less susceptible to acute colitis. No established role for epithelial expression of LCN2 in patients with IBD exists. The functional consequence of ISPs has yet to be determined, and thus the contribution of individual markers like LCN2 to disease development is not clear. We hypothesize: that ISPs give rise to an inflamed secretory environment in part via the upregulation of LCN2 which contributes to epithelial barrier damage. Methods: Stem cell derived 3D organoids allow us to examine epithelial-intrinsic defects that may contribute to disease. Given LCN2’s role in iron modulation, we generated Lcn2 knockdown colonoids—organoids derived from colonic epithelium— to probe sensitivity to iron-mediated cell death (ferroptosis) and inflammatory cytokines using organoid morphology, western blot, and immunostaining. We also evaluated single cell RNA-sequencing data for LCN2 expression in ISP and other cell types and confirmed findings using immunostaining in control and Crohn’s colonoids with or without cytokine treatment. Results & conclusions: Preliminary data show that Lcn2 KD murine colonoids show no morphological differences to control colonoids but exhibited reduced expression of ferroptosis marker TFRC at homeostasis and under cytokine stimulation via western blot. Cytokine-stimulated control colonoids exhibited increased membrane localization of TFRC compared to Lcn2 KD colonoids via immunostaining. Given goblet cells are the primary secretory cell type in the colon, our human data at the single cell and protein level (immunostaining) also support that goblet cell expression of LCN2 is altered in Crohn’s patients and under cytokine stimulation in Crohn’s disease colonoids. Taken together, our data suggest that LCN2 expression in ISPs may promote enhanced susceptibility of secretory progenitors and goblet cells to ferroptosis. Thus, the presence of ISPs may promote an aberrant secretory phenotype in the intestinal epithelium by altering the antimicrobial peptide content and susceptibility to cell death. Ongoing and future studies will evaluate whether this altered secretory phenotype contributes directly to alterations in mucus secretion and composition, and immune-epithelial crosstalk. University of Pennsylvania School of Veterinary Medicine; Institute for Infectious & Zoonotic Disease Martin and Pamela Winter Infectious Disease Pre-Doctoral Fellowship This abstract was presented at the American Physiology Summit 2025 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.