Abstract
Emergent advancements on the role of the intestinal microbiome for human health and disease necessitate well-defined intestinal cellular models to study and rapidly assess host, microbiome, and drug interactions. Differentiated Caco-2 cell line is commonly utilized as an epithelial model for drug permeability studies and has more recently been utilized for investigating host-microbiome interactions. However, its suitability to study such interactions remains to be characterized. Here, we employed multilevel proteomics to demonstrate that both spontaneous and butyrate-induced Caco-2 differentiations displayed similar protein and pathway changes, including the downregulation of proteins related to translation and proliferation and upregulation of functions implicated in host-microbiome interactions, such as cell adhesion, tight junction, extracellular vesicles, and responses to stimuli. Lysine acetylomics revealed that histone protein acetylation levels were decreased along with cell differentiation, while the acetylation in proteins associated with mitochondrial functions was increased. This study also demonstrates that, compared to spontaneous differentiation methods, butyrate-containing medium accelerates Caco-2 differentiation, with earlier upregulation of proteins related to host-microbiome interactions, suggesting its superiority for assay development using this intestinal model. Altogether, this multiomics study emphasizes the controlled progression of Caco-2 differentiation toward a specialized intestinal epithelial-like cell and establishes its suitability for investigating the host-microbiome interactions.