Development of an Inflamed High Throughput Stem-cell-based Gut Epithelium Model to Assess the Impact of Annexin A1

Stem Cell Rev Rep. 2024 Jul;20(5):1299-1310. doi: 10.1007/s12015-024-10708-4. Epub 2024 Mar 18.

Abstract

Objective and design: Annexin A1 (ANXA1) plays a role in maintaining intestinal hemostasis, especially following mucosal inflammation. The published data about ANXA1 was derived from experimental animal models where there is an overlapping between epithelial and immune cells. There is no in vitro gut epithelial model that can assess the direct effect of ANXA1 on the gut epithelium.

Methods: We developed high-throughput stem-cell-based murine epithelial cells and bacterial lipopolysaccharides (LPS) were used to induce inflammation. The impact of ANXA1 and its functional part (Ac2-26) was evaluated in the inflamed model. Intestinal integrity was assessed by the transepithelial electrical resistance (TEER), and FITC-Dextran permeability. Epithelial junction proteins were assessed using confocal microscopy and RT-qPCR. Inflammatory cytokines were evaluated by RT-qPCR and ELISA.

Results: LPS challenge mediated a damage in the epithelial cells as shown by a drop in the TEER and an increase in FITC-dextran permeability; reduced the expression of epithelial junctional proteins (Occludin, ZO-1, and Cadherin) and increased the expression of the gut leaky protein, Claudin - 2. ANXA1 and Ac2-26 treatment reduced the previous damaging effects. In addition, ANXA1 and Ac2-26 inhibited the inflammatory responses mediated by the LPS and increased the transcription of the anti-inflammatory cytokine, IL-10.

Conclusion: ANXA1 and Ac2-26 directly protect the epithelial integrity by affecting the expression of epithelial junction and inflammatory markers. The inflamed gut model is a reliable tool to study intestinal inflammatory diseases, and to evaluate the efficacy of potential anti-inflammatory drugs and the screening of new drugs that could be candidates for inflammatory bowel disease.

Keywords: 3D Organoids; Annexin1; High-throughput (HTP) cell Model; Inflamed Model; Polarized Epithelial Cells.

MeSH terms

  • Animals
  • Annexin A1* / genetics
  • Annexin A1* / metabolism
  • Cytokines / metabolism
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism
  • Epithelial Cells / pathology
  • Inflammation* / metabolism
  • Inflammation* / pathology
  • Intestinal Mucosa* / drug effects
  • Intestinal Mucosa* / metabolism
  • Intestinal Mucosa* / pathology
  • Lipopolysaccharides* / pharmacology
  • Mice
  • Peptides
  • Permeability
  • Stem Cells / cytology
  • Stem Cells / drug effects
  • Stem Cells / metabolism

Substances

  • Annexin A1
  • Lipopolysaccharides
  • Cytokines
  • annexin A1 peptide (2-26)
  • Peptides