TNFAIP8 controls murine intestinal stem cell homeostasis and regeneration by regulating microbiome-induced Akt signaling

The intestine is a highly dynamic environment that requires tight control of the various inputs to maintain homeostasis and allow for proper responses to injury. It was recently found that the stem cell niche and epithelium is regenerated after injury by de-differentiated adult cells, through a process that gives rise to Sca1+ fetal-like cells and is driven by a transient population of Clu+ revival stem cells (revSCs). However, the molecular mechanisms that regulate this dynamic process have not been fully defined. Here we show that TNFAIP8 (also known as TIPE0) is a regulator of intestinal homeostasis that is vital for proper regeneration. TIPE0 functions through inhibiting basal Akt activation by the commensal microbiota via modulating membrane phospholipid abundance. Loss of TIPE0 in mice results in injury-resistant enterocytes, that are hyperproliferative, yet have regenerative deficits and are shifted towards a de-differentiated state. Tipe0−/− enterocytes show basal induction of the Clu+ regenerative program and a fetal gene expression signature marked by Sca1, but upon injury are unable to generate Sca-1+/Clu+ revSCs and could not regenerate the epithelium. This work demonstrates the role of TIPE0 in regulating the dynamic signaling that determines the injury response and enables intestinal epithelial cell regenerative plasticity.

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Jason R Goldsmith Youhai Chen no software used Open source code for the R-suite was used for RNA-Seq analysis, as described within the methods section. For sc-RNASeq, analysis, initial data processing of samples was performed using Cellranger (v.2.1.0, 10x Genomics). Cellranger mkfastq was used to generate demultiplexed FASTQ files from the raw sequencing data. Next, cellranger count was used to align sequencing reads to the mouse reference (mm10), and generate single cell gene barcode matrices. Cellranger aggr was used to aggregate the matrices and normalize by mapped read depth to account for sequencing depth. Post processing and secondary analysis of the aggregated dataset was performed using the Seurat package (v.3.0) within R (v.3.5.1). Ingenuity Pathway Analysis (Qiagen) was also used. ImageJ with FIJI (NIH) was used for image analysis.

April 2018
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An N of 6 per biological experiment was chosen based on prior work with ischemia/reperfusion, where this sample size was determined based on power calculations to detect a histological score change of 1 with a variance of 0.5 within a group. This was extended to all animal models here for consitency.
Ischemia data was excluded when a mouse showed zero signs histologically and by gross observation of ischemic injury. This rarely happens when a clamp fails to hold and is obvious by gross and histological pathology. Other data were excluded if they fell outside of 3 SD of the mean of their group, with them included in that group for that analysis.
All experiments were repeated on different days. At least two different biological replicates were performed for experiments derived from primary cells, and often many more.
All mice used were age and gender matched, and were otherwise randomly selected.
Investigators were blinded during all histological analyses. Blinding in other samples was not possible, as all primary tissues from which subsequent samples were derived were labeled by genotype. Anti-B-catenin was validated by prior personal use and manufacturer provided citations. Anti-YAP was validated by Western and by manufacturer provided citations. The anti-CD45.1 antibodies were previously validated in the lab by flow cytometry of immune cells gathered from mice of each CD45-isotype; anti-pGSK3!S9 and anti-pAKTS473 antibodies were validated by prior use in the lab and manufacturer provided citations. Anti-Ki67 was validated by literature review and comparison to known ki-67 staining patterns. Anti-activated-Caspase-3 was validated by manufacturer provided citations. Anti-human lysozyme and antikeratin-20 were validated by an enteroid-specific IF protocol publication to cross-react in mice and this was confirmed by the staining pattern observed. Phalloidin-555 was validated based on manufacturer provided citations and prior use in the lab. !actin was validated based on numerous prior references using this antibody. anti-Rictor was validated based on manufacturer provided citations; anti-TNFAIP8 was validated using purified protein and knockout mouse tissue in our lab. Fluorescent secondary antibodies from Cell Signal and Jackson ImmunoResearch were validated by prior use in our lab, as well as manufacturer data sheets with listed publications. VectaKit Elite ABC kits were validated by prior lab use and manufacturer data sheets with listed publications. Streptavidin conjugate was recommended by Dr. Jerry Turner who previously validated the antibody.
CMT-93, ATCC® CCL-223™ Cell lines were authenticated based on gross morphology, which is distinctive, and verified by sequencing to be colonic and polyploid.
Cells were certified free of mycoplasma contamination when they were received.