Introduction
Ulcerative colitis (UC) is an inflammatory disorder of the colon of unknown aetiology characterised by intestinal barrier dysfunction associated with colonic epithelial goblet cell depletion and a shift in intestinal stem cell retention and function1,2. Pancreastatin (PST), an intestinal epithelial cells-derived peptide highly expressed in colonic tissues of patients with active UC, positively correlates with UC early onset, disease severity, and inflammatory markers3. However, the effect of PST inhibition on colonic epithelial barrier function remains undetermined.
Aims & Methods
This study aimed to investigate the impact of pancreastatin inhibitor 8 (PSTI8) on colonic epithelial barrier function in a preclinical mouse model of UC. Male and female C57BL/6 mice were treated intrarectally with PSTi8 (2.5mg/mL/kg) or PBS for six days and received 5% dextran sulfate sodium (DSS) to induce colitis or water (control) for five days. The disease activity index (DAI) was assessed daily. ELISA quantified inflammatory cytokines, interleukin (IL)-6, IL-18 and IL-22. Markers to characterise differentiated goblet cells (Mucin, MUC2), transcription factors for goblets’ lineage commitment (growth factor independent, GFI1 and Kruppel-like factor, KLF1), and colonic stem cells plasticity (fast-cycling Lgr5, fetal-like Ly6a, and reserve Hopx cells) were quantified via RT-PCR and immunofluorescence.
Results
PSTi8 treatment increased stool consistency (p<0.01) and delayed the onset of bleeding in colitic male mice compared to PBS-treated mice (2-4 days). Conversely, no differences in the DAI were noted between colitic PSTi8 and PBS-treated female mice. In non-colitic conditions, PSTi8 treatment increased (p<0.03) IL-22 in males and decreased (p<0.01) its level in female mice with no effect of IL-6. In colitic conditions, although IL-22 levels were decreased (p<0.01) in female mice and IL-6 levels were increased (p<0.001) in male mice, PSTi8 treatment did not show any effect. Conversely, in colitic females, IL-18 was significantly increased (p<0.02), and PSTi8 abolished that increase. The mRNA expression level of MUC2 remains unaltered in male and female non-colitic and colitic mice treated or not with PSTi8. Yet, immunofluorescence staining in non-colitic and colitic conditions demonstrated a decrease of MUC2 in PSTi8-treated female mice. In concordance, transcription factors KLF1 and GFI1 were significantly increased (p<0.001 and p<0.02, respectively) in colitic female mice, and PSTi8 treatment abolished that increase. No differences were seen in male mice except for a significant increase of KLF1 in PSTi8-treated colitic mice. In colitic conditions, fast-cycling cells marker Lgr5 expression in both sexes was significantly decreased (p<0.0001), and treatment with PSTi8 did not show any effect. However, in colitic male mice, fetal-like stem cells makers Ly6a expression was significantly increased (p<0.007), and PSTi8 treatment significantly decreased (p<0.02) that expression. Although a significant decrease (p<0.0001) of reserve cells marker Hopx was only seen in colitic female mice, PSTi8 treatment did not regulate that marker in male and female mice.
Conclusion
PST inhibition protects male mice from DSS-mediated colitis while exacerbating susceptibility to colitis in female mice. PST inhibition regulates the expansion of goblet and intestinal stem cells in a sex-dependent manner. Further investigation is needed to delineate the signalling pathways involved in PST action on the mucosa in both sexes. Targeting PST may lead to novel therapeutic strategies in UC.
References
1. Singh V, Johnson K, Yin J, et al. Chronic Inflammation in Ulcerative Colitis Causes Long-Term Changes in Goblet Cell Function. Cmgh. 2022;13(1):219-232.
2. Yui S, Azzolin L, Maimets M, et al. YAP/TAZ-dependent reprogramming of colonic epithelium links ECM remodeling to tissue regeneration. Cell Stem Cell. 2018;22(1):35-49.e7.
3. Eissa N, Elgazzar O, Hussein H, Hendy GN, Bernstein CN, Ghia JE. Pancreastatin reduces alternatively activated macrophages, disrupts the epithelial homeostasis and aggravates colonic inflammation. A descriptive analysis. Biomedicines. 2021;9(2):1-18.
