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Location-specific cell identity rather than exposure to GI microbiota defines many innate immune signalling cascades in the gut epithelium

The epithelial lining of the GI tract expresses pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) that enables it to detect pathogens whilst keeping the gut microbiota physically separate from the body's active immune cells. Aberrant PRR signalling such as SNPs disrupting the nucleotide-binding oligomerisation domain (NOD2) and TLR4 can cause inflammatory diseases such as IBD and necrotising enterocolitis. The role of PRR sensing in GI epithelial cells and their contribution to the pathologies of IBD and other diseases remains unclear. The epithelium must be able to attenuate inflammation by commensals, which may lead to some tolerance to some microbiota. PRR activation is also important for barrier function so may play a vital part in pathogen sensing.

Recently, advances in organoid development have enabled further studies of pattern recognition as they are primary non-transformed cells that can differentiate into cell layers of the epithelium that they are generated from. Organoids retain many of the characteristics of their original epithelium, but do not harbour immune cells, making analysis of just the epithelial response and PRR signalling possible.

In this study, the authors established a biobank of human and murine GI organoids from tissue-resident stem cells and analysed gene expression across the different segments of the GI tract. Organoids were developed form gastric corpus, gastric pylorus, intestinal duodenum, jejunum, ileum and colon from three patient samples.

Cluster analysis of the RNA‑seq data demonstrated that organoids clustered according to the segment tissue they originated from. Tissue markers such as Muc5ac, Muc6, Cdx1 and Cdx2 were expressed in the relevant organoids confirming that tissue identity is maintained.

The dataset was combined to make three regions, stomach, proximal small intestine and colon. Differential expression identified the genes Muc5ac, Muc6, Cdx1, Cdx2 and villin (Vil1), known markers of the GI border.

Tlr1, Tlr2, Tlr4, Nod2, NLR family pyrin domain containing 6 (Nlrp6) and many other genes involved in innate immunity were also differentially expressed and GO terms identified a significant enrichment of “Response to external stimulus”.

Heatmaps of the genes in this particular ontology demonstrated expression of specific groups of genes along the cephalocaudal axis in both human and mouse organoids suggesting that each segment expresses a specific set of innate immunity genes.

Human and mouse organoids expressed some similar genes such as Nod2, Nlrp6 and Tlr4, but others, such as Tlr2, Tlr5 or Tlr6 were different.

In both human and mouse, tissue and organoids, Tlr4 had high expression in the stomach and colon, but very low expression in the small intestine. However, Tlr4 has 3 co-factors, CD14, MD2 and LBP, all three were expressed in mice but only CD14 in humans suggesting that despite similar patterns Tlr4 is active in mouse gastric, ileal and colonic epithelium but not in human GI epithelium.

Consistent with this hypothesis, qPCR analysis demonstrated upregulation of Cxcl2 in response to LPS in mouse corpus and colon but not the jejunum. In human organoids, IL‑8, the human analog of Cxcl2, was not induced.

Specific patterning of PRRs Tlrs 2, 4 and 5 were detected at the level of expression of RNA and functional assays and whilst expression and function mostly correlated in some cases this was not consistent.

In transwell assays of LPS stimulation of Tlr4 in human organoids, LPS was unable to stimulate IL‑8 expression from both the apical or basal sides of the organoids whereas TNF-α induced IL‑8 from the basal side.

In mouse organoids basal LPS stimulation induced Cxcl2 in a concentration dependent manner and further analysis in organoids from Tlr2/4 KO mice demonstrated lack of basal stimulus.

In mouse organoids a variety of methods including microinjection of LPS demonstrated that LPS canstimulate both the apical and basal sides of the organoid and that this occurs via Tlr4.

Organoids generated from mouse embryos that had not been exposed to any GI microbiota had similar structures and characteristics to those of adult organoids. Gene expression analysis demonstrated clustering with the tissue of origin and analysis of tissue markers revealed a similar expression to adult organoids, confirming regional identity.

Differential expression revealed that markers of the GI border were expressed in the mouse embryo organoids. Cdx1, Muc5ac and Lyz1, well characterised GI border markers were detected as well as Tlr4 and other PRRs.

Similar GO terms and heatmap clustering to adult organoids was observed from the expression data demonstrating that patterning of most but not all PRRs was already encoded in the tissue-resident embryonic stem cells.

Expression of Tlr4 was observed in the stomach but not intestine and functional assays demonstrated no LPS mediated induction of Cxcl2 in proximal intestinal organoids but, like the adult organoids, the embryo-derived gastric organoids expressed Cxcl2.

Innate immune signalling is critical for the inflammatory response and tissue homeostasis and pattern recognition is a crucial part of this regulation. Pattern regulation receptors such as toll-like receptors have been characterised and studies suggest that some PRRs may be expressed due to stimulation of the GI tissue by microbiota at birth. The authors used RNA‑seq from organoids generated from different segments of the GI tract and demonstrated specific patterns of expression of PRRs across the GI tract. They also used sterile, embryo-derived organoids to demonstrate that expression of many PRRs throughout the GI tract, including Tlr4 was already encoded in the embryonic stem cells and independent of microbiota stimuli.

Reference

Kayisoglu O, Weiss F, Niklas C, et al. Location-specific cell identity rather than exposure to GI microbiota defines many innate immune signalling cascades in the gut epithelium. Gut 2020; 0:1–11
doi: 10.1136/gutjnl-2019-319919

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