Single-Cell Analysis Reveals Regional Reprogramming during Adaptation to Massive Small Bowel Resection in Mice

Abstract

Synopsis:Here, single-cell RNA sequencing reveals interactions between the retinoid metabolism pathway and 'regional reprogramming' of distal small intestinal epithelium to a proximal identity following proximal small bowel resection. This provides novel insight into physiological adaptation to short gut syndrome. Abstract:Background & Aims: The small intestine (SI) displays regionality in nutrient and immunological function. Following SI tissue loss (as occurs in short gut syndrome, or SGS), remaining SI must compensate, or 'adapt'; the capacity of SI epithelium to reprogram its regional identity has not been described. Here, we apply single-cell resolution analyses to characterize molecular changes underpinning adaptation to SGS.Methods: Single-cell RNA-sequencing was performed on epithelial cells isolated from distal SI of mice following 50% proximal small bowel resection (SBR) vs. sham surgery. Single-cell profiles were clustered based on transcriptional similarity, reconstructing differentiation events from intestinal stem cells (ISCs) through to mature enterocytes. An unsupervised computational approach to score cell identity was used to quantify changes in regional (proximal vs distal) SI identity, validated using immunofluorescence, immunohistochemistry, qPCR, western blotting, and RNA-FISH.Results: Uniform Manifold Approximation and Projection-based clustering and visualization revealed differentiation trajectories from ISCs to mature enterocytes in sham and SBR. Cell identity scoring demonstrated segregation of enterocytes by regional SI identity: SBR enterocytes assumed more mature proximal identities. This was associated with significant upregulation of lipid metabolism and oxidative stress gene expression, which was validated via orthogonal analyses. Observed upstream transcriptional changes suggest retinoid metabolism and proximal transcription factor Creb3l3 drive proximalization of cell identity in response to SBR.Conclusions: Adaptation to proximal SBR involves regional reprogramming of ileal enterocytes toward a proximal identity. Interventions bolstering the endogenous reprogramming capacity of SI enterocytes-conceivably by engaging the retinoid metabolism pathway-merit further investigation, as they may increase enteral feeding tolerance, and obviate intestinal failure, in SGS.

Lipid absorption and overall intestinal lymphatic transport are impaired following partial small bowel resection in mice

“…In agreement with this reprogramming, we observed a progressive increase in expression of ApoB in the proximal intestine and MTTP in the remaining distal intestinal segment, which regulate the assembly of chylomicrons, in the resected mice (Figure 1A-B; ANOVA p<0.05 and p<0.01, respectively). As we have previously shown in single-cell analysis at early time points after resection, there was an increase in the intestinal lipid-sensing gene FABP2 in the distal intestine (Figure 1B; ANOVA p<0.005) (16). Simultaneously, in the distal intestine, there was a decrease in the long-chain fatty acid transporter gene, CD36, the cholesterol transporter gene, Abca1, and the bile acid binding gene, FABP6, expression in the distal intestine with increasing loss of bowel (Figure 1B; ANOVA p<0.005, p<0.005, and p<0.05, respectively).…”

“…At an early time point after resection, postoperative day 7, the distal intestine reprograms to a more proximal identity with an upregulation of genes associated with lipid handling and metabolism (16). In agreement with this reprogramming, we observed a progressive increase in expression of ApoB in the proximal intestine and MTTP in the remaining distal intestinal segment, which regulate the assembly of chylomicrons, in the resected mice (Figure 1A-B; ANOVA p<0.05 and p<0.01, respectively).…”

Teduglutide Promotes Epithelial Tight Junction Pore Function in Murine Short Bowel Syndrome to Alleviate Intestinal Insufficiency

“…Short bowel syndrome occurs after major intestinal resection and may cause intestinal insufficiency or intestinal failure (IF) [1]. Resection-associated intestinal adaptation compensates for the reduced absorptive mucosal surface area and may involve hyperphagia, polydipsia, prolonged gastrointestinal transit time, absorptive surface area expansion, increased epithelial transport, and increased mesenteric blood flow [2,3]. Besides macronutrient malabsorption, high intestinal salt and water losses are the major challenge to achieve oral autonomy.…”

Lipid absorption and overall intestinal lymphatic transport are impaired following partial small bowel resection in mice

“…Malabsorption of cholesterol and triglycerides after SBR. At an early time point after resection, postoperative day 7, the distal intestine reprograms to a more proximal identity with an upregulation of genes associated with lipid handling and metabolism 16 . In agreement with this reprogramming, we observed a progressive increase in expression of ApoB in the proximal intestine and MTTP in the remaining distal intestinal segment, which regulate the assembly of chylomicrons, in the resected mice (Fig.…”