Germ‐free and microbiota‐associated mice yield small intestinal epithelial organoids with equivalent and robust transcriptome/proteome expression phenotypes

Abstract

Intestinal epithelial organoids established from gut tissue have become a widely used research tool. However, it remains unclear how environmental cues, divergent microbiota composition and other sources of variation before, during and after establishment confound organoid properties, and how these properties relate to the original tissue. While environmental influences cannot be easily addressed in human organoids, mice offer a controlled assay-system. Here, we probed the effect of donor microbiota differences, previously identified as a confounding factor in murine in vivo studies, on organoids. We analysed the proteomes and transcriptomes of primary organoid cultures established from two colonised and one germ-free mouse colony of C57BL/6J genetic background, and compared them to their tissue of origin and commonly used cell lines. While an imprint of microbiota-exposure was observed on the proteome of epithelial samples, the long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead, stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts and closely mimicked expression patterns in the gut epithelium. This included the inflammasome components ASC, Naip1-6, Nlrc4 and Caspase-1, which were highly expressed in all organoids compared to the reference cell line m-IC c12 or mouse embryonic fibroblasts. Taken together, these results reveal that the donor microbiota has little effect on the organoid phenotype and suggest that organoids represent a more suitable culture model than immortalised cell lines, in particular for studies of intestinal epithelial inflammasomes.

Intestinal restriction of Salmonella Typhimurium requires caspase-1 and caspase-11 epithelial intrinsic inflammasomes

“…This expression profile is corroborated by our finding that levels of pro-caspase-11, but not pro-caspase-1, increased in IECs in response to IFN-γ treatment. In a recent publication by Hausmann et al, it was reported that TNF-α treatment of murine small intestine-derived enteroids also increased Casp11 expression [17]. Together with our findings, this suggests that although caspase-1 is sufficient to protect naïve IECs, as the infection proceeds, host inflammatory responses upregulate caspase-11 expression in IECs, leading to increased caspase-11 activity; the combined efforts of these two caspases form a multilayered innate defense that controls intracellular Salmonella burdens and protects the host from pathogen attack.…”

Bacterial detection by NAIP/NLRC4 elicits prompt contractions of intestinal epithelial cell layers

“…Our results highlight a previously unappreciated dynamic behavior of intestinal epithelia. epithelium (6,9). Organoid models have for these reasons become attractive tools for physiological studies of gut infection (25,(29)(30)(31)(32).…”

“…As a system to sense mucosal intrusions, IECs express pattern recognition receptors (PRRs) [e.g., Toll-like receptors associated with cell membranes (6), and Nod-like receptors (NLRs) that form inflammasomes in the cytosol] (7)(8)(9). Epithelial recognition of pathogens through PRRs elicits a panel of countermeasures, including production of proinflammatory cytokines, chemokines, lipids (10)(11)(12)(13), secretion of antimicrobial peptides (11,14), and the death and expulsion of infected IECs into the lumen (12,15,16).…”

Regulation of Enteroendocrine Cell Networks by the Major Human Gut Symbiont Bacteroides thetaiotaomicron

“…However, the finding that the transcriptome and proteome of small intestinal stem cell-derived organoids from SPF and GF mice co-cluster (Hausmann et al, 2020), would argue against this possibility although in this study no account was made for any possible differences in EEC distribution or number. Indeed, whereas our analysis relied on cellular comparisons of EEC in epithelial cell monolayer cultures versus in intact tissue, other studies have used single or multi-omicsbased approaches in comparative studies (Lindeboom et al, 2018;Beumer et al, 2020;Hausmann et al, 2020;Ohki et al, 2020). Other possible confounding factors include comparing EECs in two-dimensional epithelial cell monolayers versus stem cell-derived three-dimensional organoids, other methodological differences including the age of the mice used, where in the small intestine crypts are obtained from Fuller et al (2012), the duration of culture, and the type and concentrations of growth and differentiation factors used.…”

“…Our contradictory findings may relate to our use of GF mice, as SPF mice are the usual source of intestinal crypts (Petersen et al, 2014;Roberts et al, 2019). However, the finding that the transcriptome and proteome of small intestinal stem cell-derived organoids from SPF and GF mice co-cluster (Hausmann et al, 2020), would argue against this possibility although in this study no account was made for any possible differences in EEC distribution or number. Indeed, whereas our analysis relied on cellular comparisons of EEC in epithelial cell monolayer cultures versus in intact tissue, other studies have used single or multi-omicsbased approaches in comparative studies (Lindeboom et al, 2018;Beumer et al, 2020;Hausmann et al, 2020;Ohki et al, 2020).…”