The gastrointestinal (GI) epithelium is composed of a single layer of cells with a turnover time of only a few days. Due to its location at the barrier between GI tract contents and the underlaying mucosa, the epithelium is constantly exposed to stress such as toxic agents and a variety of pathogens and susceptible to injury. Accordingly, the homeostatic growth as well as repair of injury in epithelium must be efficient and strictly regulated. Misregulated repair of the injured epithelium can lead to pathologies such as chronic inflammation or cancer. Underlying stromal cells such as fibroblasts provide growth factors and other signaling molecules regulating the epithelial cell stemness, differentiation and repair, but the stromal regulatory pathways during regeneration are poorly understood.

The aim of this study was to establish a consensus view on the heterogeneity of GI fibroblasts, as well as to map potential epithelium derived signals affecting fibroblast function in homeostatic and injury situations using literature review, in silico approaches, and murine primary intestinal fibroblast culture. Seurat and CellChat R packages were used to perform integration and interaction analyses of six previously published mouse and three human single- cell RNA-sequencing datasets of colonic epithelial and mesenchymal cells isolated in homeostatic and/or inflammatory conditions. Murine primary intestinal fibroblasts were treated with identified potential signaling factors ex vivo and 3’RNAseq was performed to identify transcriptional responses.

Both mesenchymal and epithelial cell clusters were identified in the scRNAseq data. Interestingly, similar fibroblast populations could be found in the murine and human data. I identified several epithelium-derived signaling molecules potentially targeting GI fibroblasts and focused on Gas6-Axl pathway and lactate. I confirmed high and specific expression of the Gas6 receptor Axl in intestinal fibroblasts, but recombinant Gas6 failed to induce significant changes in cultured primary fibroblasts. Lactate-treated primary intestinal fibroblasts reprogrammed their transcriptome with main alterations in metabolic pathways and induction of neutrophil-attracting chemokines.

In this work I suggest a consensus model for GI fibroblast subpopulations and suggest epithelium derived lactate as a powerful means to reprogram fibroblasts.