Abstract

Background: osteopontin (OPN, encoded by SPP1) is involved in chronic liver disease. Previous studies reported that intestinal OPN regulates the gut microbiota, however the role of intestinal epithelial cell (IEC)-derived OPN in metabolic dysfunction-associated steatohepatitis (MASH) is unknown.

Methods: we generated Spp1 knock-in (Spp1^{KI IEC}) and knock-out (Spp1^ΔIEC) mice in IECs and fed both genders with high fat, fructose, and cholesterol diet to induce MASH, or with isocaloric control diet, for 6 months.

Results: immunohistochemistry revealed decreased OPN expression in IECs in WT mice with MASH compared to control diet. Spp1^ΔIEC showed worse hepatic inflammation (increased CD11b⁺ cell infiltration and up-regulation of pro-inflammatory cytokines) than WT mice regardless of diet but developed more liver fibrosis and increased serum ALT than WT mice, when fed MASH-inducing diet. Spp1^ΔIEC mice displayed clutched IECs with condensed cytoplasm and pyknotic nuclei, increased TUNEL⁺ IECs, downregulation of tight junction proteins, increased serum LPS and hepatic bacterial load, compared to WT mice, regardless of diet. Metagenomic analysis of gut microbiota revealed that Spp1^ΔIEC mice had enhanced β-diversity due to increased Deferribacteres and Verrucomicrobiota phyla. Multi-factor analysis revealed that 11 genera and 27 species were differentially upregulated in Spp1^ΔIEC mice, regardless of diet. Among them, Clostridium_XlVa, Parabacteroides and Dora regulate bile acid (BA) deconjugation and 7-dehydroxylation. Total BAs increased in portal serum and decreased in feces from Spp1^ΔIEC compared to WT mice with MASH, indicating less BA excretion. Both the MASH diet and the Spp1^ΔIEC genotype independently increased conjugated primary BAs, particularly taurocholic acid (TCA), in both genders. Unconjugated primary BAs decreased in MASH but were less affected in Spp1^ΔIEC mice. Secondary BAs were lower in serum, however taurodeoxycholic acid (TDCA) significantly increased in Spp1^ΔIEC mice, regardless of diet. The hepato-protective BA tauroursodeoxycholic acid (TUDCA), mildly increased in both genders, regardless of diet, but was less abundant than TCA and TDCA. Compared to WT mice, Spp1^{KI IEC} mice were protected from MASH shown by reduced gut permeability, liver inflammation, and fibrosis.

Conclusion: IEC-derived OPN protects against MASH by preserving gut permeability, changing the composition of the gut microbiome, and bile acids.