P0210 ORMDL proteins regulate intestinal epithelial cell homeostasis by modulating ER architecture, autophagy, and DNA damage response
Authors: Raschdorf C , Tran F , Stengel S , Yang H , Pimenta Bernardes J , Lopez-Agudero V , Falk-Paulsen M , Jentzsch M , Bordoni D , Messner B , Aden K , Kaser A , Rosenstiel P 
Published: January 2025

Abstract

Background
ORM1-like protein 3 (ORMDL3) is a risk gene for asthma and inflammatory bowel disease (IBD), identified in genome-wide association studies (GWAS).1,2 It encodes an endoplasmic reticulum (ER)-resident transmembrane protein implicated in calcium homeostasis, sphingolipid biosynthesis, and regulation of the unfolded protein response (UPR) upon ER stress.3,4
However, the role of ORMDL3 and its homologous protein family members ORMDL1 and ORMDL2, which exhibit partly redundant functions, remains unclear in the context of intestinal inflammation.

Methods
Constitutive and conditional intestinal epithelial cell-specific knockout (KO) mice for Ormdl1, Ormdl2, and Ormdl3 were generated. Histological analysis of small intestinal (SI) Paneth cells (lysozyme immunohistochemistry), goblet cells (periodic acid-Schiff staining) and ER morphology in Paneth cells (transmission electron microscopy [TEM]) was conducted. Interactions between ORMDL proteins and autophagy-related proteins were examined by co-immunoprecipitation and immunoblot assays in irradiated mouse embryonic fibroblasts (iMEFs). Organoids derived from Ormdl1/2/3ΔIEC mice were used for bulk RNA sequencing.

Results
The Ormdl1/2/3 triple KO was embryonically lethal, while Ormdl3-KO and Ormdl1/3 double KO mice exhibited reduced body size, weight, and embryonic viability, along with fewer Paneth and goblet cells. To eliminate any compensatory effects of the multiple ORMDL proteins, the Ormdl1/2/3ΔIEC mice were analyzed. These mice showed no macroscopic phenotype but displayed microscopic inflammation, reduced Paneth and goblet cells, and increased SI tuft cells. Both constitutive and conditional Ormdl knockout mice showed massive dilation of the ER.
While autophagy is a known compensatory mechanism resolving ER stress, we demonstrated that ORMDL3 co-localized and co-precipitated with the autophagy-related protein LC3 (microtubule-associated protein 1A/1B-light chain 3). Consistent with this, loss of ORMDL proteins compromised autophagic flux.
Bulk RNA sequencing of SI organoids from Ormdl1/2/3ΔIEC mice revealed reduced expression of genes involved in lipid biosynthesis, tuft cell differentiation via BMP-signalling, and innate immunity, including antimicrobial peptides, likely due to loss of Paneth cells. In contrast, DNA damage response genes were upregulated, as reflected by an increase in γ-H2AX-positive epithelial cells.

Conclusion
The data suggest a critical regulatory role for ORMDL proteins in the ER-autophagy axis within epithelial cells, shaping mucosal homeostasis and expression of antimicrobial peptides, thereby conferring protection against IBD. Further studies are required to elucidate the molecular basis of the function of ORMDL proteins in intestinal inflammation.

References
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2.McGovern DPB, Gardet A, Törkvist L, et al. Genome-wide association identifies multiple ulcerative colitis susceptibility loci. Nat. Genet. 2010;42(4):332-337. doi:10.1038/ng.549
3.Hjelmqvist L, Tuson M, Marfany G, Herrero E, Balcells S, Gonzàlez-Duarte R. ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins. Genome Biol. 2002;3(6). doi:10.1186/gb-2002-3-6-research0027
4.Kiefer K, Carreras-Sureda A, García-López R, et al. Coordinated Regulation of the Orosomucoid-like Gene Family Expression Controls de Novo Ceramide Synthesis in Mammalian Cells. J. Biol. Chem. 2014;290(5):2822-2830. doi:10.1074/jbc.m114.595116