Carriage of multiple genetic risk factors for MASH stimulates an altered inflammatory secretome in a human stem cell-based in vitro model

Background: Genetics play an important role in the development of metabolic dysfunction-associated steatohepatitis (MASH). Specifically, a combination of single nucleotide polymorphisms in known MASH-modifying genes - PNPLA3 (rs738409 C>G), TM6SF2 (rs58542926 C>T), GCKR (rs1260326 C>T), and MBOAT7 (rs641738 C>T) - has been integrated into a polygenic risk score for hepatic fat content (PRS-HFC). However, the impact of this polygenic risk on the hepatocyte-specific inflammatory secretome is unclear. Here, we aimed to identify inflammatory proteins related to an increased polygenic risk for MASH using a human stem cell-based in vitro model representing genetic variability in the MASH population.
Methods: 90 human skin-derived precursor (hSKP) cell lines were genotyped and classified according to their PRS-HFC. Five cell lines were selected from the low (RClow) and high (RChigh) risk categories and differentiated to hepatic cells (hSKP-HPCs) over 24 days. These cultures were then exposed for 24 hours to a mix consisting of fatty acids, fructose, lipopolysaccharide and TNF-alpha, mimicking a ‘MASH’ phenotype, or vehicle-matched control medium. Intracellular lipids were quantified using flow cytometry following BODIPY™ 493/503 staining for neutral lipids to validate the PRS-HFC in this in vitro system. Protein biomarkers were determined in the cell culture supernatant using the Olink® multiplex analysis platform.
Results: MASH-induced hSKP-HPC RChigh cultures exhibited a 3.5-fold increase in lipid load compared to RClow cultures (p = 0,0004). RChigh cultures secreted remarkably more interleukin 6 (IL6) than RClow cultures (5.2-fold, p < 0.0001) in response to the ‘MASH’ mix. While RClow and RChigh cultures secreted similar amounts of CC motif chemokine ligand (CCL) 2 (p = 0.053) and CXC motif chemokine ligand (CXCL) 10 (p = 0.56), an increased secretion in RChigh was also found for CCL7 (1.5-fold, p = 0.0058), CCL8 (7.1-fold, p < 0.0001), CCL19 (3.8-fold, p < 0.0001), CXCL8 (1.8-fold, p = 0.0007), and colony stimulating factor 3 (CSF3) (2.4-fold, p < 0.0001).
Conclusion: This in vitro study suggests that carriage of multiple genetic risk factors for MASH stimulates an altered inflammatory secretome involving at least IL6, CCL7, CCL8, CCL19, CXCL8, and CSF3. Employing pathway analysis and sequencing techniques will further uncover the underlying mechanisms responsible for the altered secretion of inflammatory proteins.