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

Abstract

Objective: The epithelial layer of the GI tract is equipped with innate immune receptors to sense invading pathogens. Dysregulation in innate immune signalling pathways is associated with severe inflammatory diseases, but the responsiveness of GI epithelial cells to bacterial stimulation remains unclear.

Design: We generated 42 lines of human and murine organoids from gastric and intestinal segments of both adult and fetal tissues. Genome-wide RNA-seq of the organoids provides an expression atlas of the GI epithelium. The innate immune response in epithelial cells was assessed using several functional assays in organoids and two-dimensional monolayers of cells from organoids.

Results: Results demonstrate extensive spatial organisation of innate immune signalling components along the cephalocaudal axis. A large part of this organisation is determined before birth and independent of exposure to commensal gut microbiota. Spatially restricted expression of Toll-like receptor 4 (Tlr4) in stomach and colon, but not in small intestine, is matched by nuclear factor kappa B (NF-κB) responses to lipopolysaccharide (LPS) exposure. Gastric epithelial organoids can sense LPS from the basal as well as from the apical side.

Conclusion: We conclude that the epithelial innate immune barrier follows a specific pattern per GI segment. The majority of the expression patterns and the function of TLR4 is encoded in the tissue-resident stem cells and determined primarily during development.

Keywords: epithelial barrier; gastrointestinal immune response; mucosal immunity.

Figure 1

Transcriptome profiling of human and murine GI organoids reveals segment-specific expression of pattern recognition receptor signalling components. Organoids were initiated from three mice or three human donors each from the indicated segments of the GI tract, expanded to a maximum of passage 5, and RNA of each line was subjected to RNA sequencing (one dataset per line). (A) Scheme of setup. (B) Representative light microscope images of mouse and human organoids derived from the indicated segments of the GI tract. scale bar, 200 µm. (C) Hierarchical clustering of total mRNA sequencing. (D) Multidimensional scaling analysis of RNA sequencing, highlighting the closeness of the six gastric organoids and the six proximal intestinal organoids in each species. (E) Heatmaps displaying normalised and scaled gene counts of selected gastric and intestinal cell marker genes through the GI tract for mouse and human organoids.

Figure 2

Expression of innate immune signalling components is GI segment specific and species- pecific. (A) Lists of differentially expressed genes were generated, comparing six gastric organoids (combined corpus and pylorus) and six proximal intestinal organoids (combined duodenum and jejunum) or six proximal intestinal and three colonic organoids. Venn diagrams display number of differentially expressed genes (p<0.05 and log₂ fold change >±1 (twofold change)). Selected genes chosen for biological interest are listed. Black: genes known for tissue identity. Blue: genes known for immune function. (B) GO enrichment analysis (biological process) comparing six gastric, six proximal intestinal and three colonic organoids. Scores of the top GO terms, indicating the enrichment p value. (C) Heatmap of all genes in GO:0009605 – response to external stimulus (1939 mouse genes, 2012 human genes). (D) Heatmap displaying normalised and scaled gene counts of selected genes from GO:0009605 for mouse and human organoids. Genes identified by the analysis as differentially expressed are marked in red. GO, Gene Ontology.

Figure 3

Expression and function of TLR2, 4 and 5 signalling pathways is GI segment specific and species specific. (A and B) Normalised gene counts of Tlr4, Cd14, Lbp and Md2 in murine and human organoids. Right: graphical summary of resulting hypothesis. (C and D) Separate lines of organoids from the indicated gut segments of three individual mice (C) or human patients (D) were exposed to LPS 100 ng/mL in the supernatant. After 2 hours, cells were harvested, RNA was prepared and expression of Cxcl2 (C) or human analogue IL-8 (D) was determined by qPCR. (E–H) Normalised gene counts of Tlr2 and Tlr5 in murine (E and G) or human (F and H) organoids (left panels) and functional test of the respective TLRs using Pam3CSK4 1 µg/mL or flagellin 100 ng/mL (right panels). Organoid seeding, cell processing and qPCR was performed as in C and D. Bars in A and B and E–H (left panels) represent means with SD of normalised gene counts from RNA sequencing of three organoid lines per segment (see figure 1). Normalisation of sequencing data is explained in the online supplementary methods. qPCR results in C–H (right panels), were normalised to GAPDH and then to the average of the mock controls. Bars in A–H represent mean with SD of cells from three biological samples (n=3). All results are representative of at least three independent experiments. LPS, lipopolysaccharide.

Figure 4

TLR4 senses apical and basal LPS stimulation in murine gastric organoids. (A–C) Organoids from the indicated gut segments of wildtype mice (B), or the stomach of wildtype, Myd88 ^−/− or Tlr2/4 ^−/− mice (C) were grown in Matrigel. Two hours after exposure to LPS 100 ng/mL in the supernatant, cells were harvested, RNA was prepared and expression of Cxcl2 was determined by qPCR. (D) Gastric organoids generated from GFP-p65 knockin mice were stimulated with LPS and GFP-p65 translocation monitored by live cell confocal microscopy. Stills of the movie are shown. White arrow points to a single nucleus (see also online supplementary movie and online supplementary figure S5). Scale bar: 100 µm. (E–G) Cells from murine gastric organoids were seeded on conventional cell culture plates to form a two dimentional monolayer of cells, stimulated with LPS at the indicated concentrations for 2 hours, fixed and stained for p65 and nuclei (F). Cells with nuclear p65 were counted and are presented as percent of total cells (G). Scale bar, 10 µm. (H and I) Cells from murine gastric organoids were seeded onto transwells, and 100 ng/mL LPS was added either to the lower or the upper compartment. After 2 hours, mRNA levels of Cxcl2 were determined by qPCR. (J–K) organoids were microinjected with LPS 100 ng/mL starting concentration either inside the organoid or outside. After 2 hours, cells were harvested, RNA prepared and Cxcl2 levels quantified by qPCR. Results were normalised to Gapdh, and each outside injection was normalised to the paired inside injection on the same plate. Bars in B and G represent means with SD of cells from three mice (n=3). Bars in C and I represent means with SD of technical triplicates. Bars in K represent means with SD of 20 paired samples (apical/basal) in six experiments. Statistical significance was tested with paired two-sided t-test of the underlying ΔΔCq data. All results are representative of at least three independent experiments. LPS, lipopolysaccharide.

Figure 5

Transcriptome profiling of adult and embryonic mouse-derived GI organoids. Organoids were generated from E16 stomach or proximal intestine and expanded to a maximum of passage 5; RNA of each line was subjected to RNA sequencing (one dataset per line). (A) Representative images of organoids. Scale bar, 200 µm. (B) RNA was isolated from two organoid lines generated from embryonic stomach and three lines generated from proximal intestine (all E16). Transcriptomes were sequenced and compared with transcriptomes of adult-derived organoids. Multidimensional scaling analysis of total mRNA sequencing, highlighting the closeness of the embryo-derived organoids to the adult-derived organoids (data from adult mice also shown in figure 1). (C) Heatmap displaying normalised and scaled gene counts of selected gastric and intestinal cell markers through the GI tract of adult mouse and embryo-derived gastric and proximal intestinal organoids.

Figure 6

Expression of innate immune signalling components is segment specific in murine embryo-derived organoids. (A) Lists of differentially expressed genes were generated, comparing six gastric organoids (combined corpus and pylorus) and six proximal intestinal organoids (combined duodenum and jejunum) or two embryonic gastric organoids and three embryonic proximal intestinal organoids. Venn diagrams display number of differentially expressed genes (p<0.05 and log₂fold change >±1 (twofold change)). Selected genes chosen for biological interest are listed. Black: genes known for tissue identity. Blue: genes known for immune function. (B) GO term enrichment analysis (biological process) of 1626 overlapping adult versus embryonic differentially expressed genes. Scores of the top GO terms, indicating the enrichment p value. (C) Heatmap of all genes in GO:0009605 – response to external stimulus (1974 genes). (D) Heatmap displaying normalised and scaled gene counts of selected genes from GO:0009605 for adult and embryonic mouse-derived organoids. GO, Gene Ontology.

Figure 7

Tlr4 expression and function are mainly developmentally rather than environmentally programmed. (A) Normalised gene counts of Tlr4 in murine organoids. Data from RNA-Seq are shown in figures 1 and 4. Bars represent mean with SD of three embryonic proximal intestinal organoids or two lines for embryo-derived gastric organoids. (B) RNA was prepared from embryo-derived organoids. Expression of indicated genes was assessed by conventional PCR. Mucus genes are marker genes for stomach or proximal intestine. Each lane shows PCR results from the indicated organoid lines, n=3 stomach and n=3 proximal intestine. (C) Organoids in culture were exposed to 100 ng/mL LPS for 2 hours, cells were harvested, RNA was prepared and expression of Cxcl2 was quantified by qPCR. Results were normalised to Gapdh and then to the paired non-stimulated control. Bars represent means with SD of three organoid lines for adult murine corpus organoids and three technical replicates for embryo-derived organoids. Results are representative of at least three independent experiments. LPS, lipopolysaccharide.