Bowel resection accelerates enterocyte proliferation in the remaining gut that may have suboptimal absorptive and digestive capacity because of proliferation-associated decrease in functional differentiation markers. We hypothesized that although Slfn3 is an important regulator of murine enterocytic differentiation, Slfn3 would have less impact on the bowel resection adaptation where accelerated proliferation takes priority over differentiation. We assessed proliferation, cell shedding, and enterocyte differentiation markers of mucosa from resected and postoperative bowel of wild type (WT) and Slfn3 knockout (Slfn3KO) mice. Villus length and crypt depth were increased in WT mice and was even longer in Slfn3KO mice, while female Slfn3KO mice displayed even deeper crypts than both WT sexes after resection. Mitotic marker, Phh3+, and proliferation marker expression of Lgr5, FoxL1, and PDGFRα were increased after resection in male WT but this effect was blunted in male Slfn3KO mice. Cell shedding regulators Villin1 and TNFα were downregulated in female mice and male WT mice only, while Gelsolin and EGFR increased expression in all mice. Slfn3 expression increased after resection in WT mice but differentiation markers sucrase isomaltase, Dpp4, Glut2, and SGLT1 were all decreased. This suggests that enterocytic differentiation effort is incompatible with a rapid proliferation shift in intestinal adaptation. Slfn3 absence potentiates villus length and crypt depth, suggesting that the differentiating stimulus of Slfn3 signaling may restrain mucosal mass increase through regulating Villin1, Gelsolin, EGFR, TNFα, and proliferation markers. Slfn3 may therefore be an important regulator not only of "normal" enterocytic differentiation but also the response to bowel resection.

Background Anastomotic failure causes morbidity and mortality even in technically correct anastomoses. Initial leaks must be prevented by mucosal reapproximation across the anastomosis. Healing is a concerted effort between intestinal epithelial cells (IECs), immune cells, and commensal bacteria. IEC TLR4 activation and signaling is required for mucosal healing, leading to inflammatory factor release that recruits immune cells to limit bacteria invasion. TLR4 absence leads to mucosal damage from loss in epithelial proliferation, attenuated inflammatory response, and bacteria translocation. We hypothesize after anastomosis, an imbalance in microbiota will occur due to a decrease in TLR4 expression and will lead to changes in the immune milieu. Results We isolated fecal content and small intestinal leukocytes from murine, Roux-en-Y and end-to-end anastomoses, to identify microbiome changes and subsequent alterations in the regulatory and pro-inflammatory immune cells 3 days post-operative. TLR4+ IECs were impaired after anastomosis. Microbiome diversity was reduced, with Firmicutes, Bacteroidetes, and Saccharibacteria decreased and Proteobacteria increased. A distinct TCRβhi CD4+ T cells subset after anastomosis was 10–20-fold greater than in control mice. 84% were Th17 IL-17A/F+ IL-22+ and/or TNFα+. iNKT cells were increased and TCRβhi. 75% were iNKT IL-10+ and 13% iNKTh17 IL-22+. Additionally, Treg IL-10+ and IL-22+ cells were increased. A novel dendritic cell subset was identified in anastomotic regions that was CD11bhi CD103mid and was 93% IL-10+. Conclusions This anastomotic study demonstrated a decrease in IEC TLR4 expression and microbiome diversity which then coincided with increased expansion of regulatory and pro-inflammatory immune cells and cytokines. Defining the anastomotic mucosal environment could help inform innovative therapeutics to target excessive pro-inflammatory invasion and microbiome imbalance.

The intestinal epithelium requires self-renewal and differentiation in order to function and adapt to pathological diseases such as inflammatory bowel disease, short gut syndrome, and ulcers. The rodent Slfn3 protein and the human Slfn12 analog are known to regulate intestinal epithelial differentiation. Previous work utilizing a pan-Slfn3 knockout (KO) mouse model revealed sex-dependent gene expression disturbances in intestinal differentiation markers, metabolic pathways, Slfn family member mRNA expression, adaptive immune cell proliferation/functioning genes, and phenotypically less weight gain and sex-dependent changes in villus length and crypt depth. We have now created a Vil-Cre specific Slfn3KO (VC-Slfn3KO) mouse to further evaluate its role in intestinal differentiation. There were increases in Slfn1, Slfn2, Slfn4, and Slfn8 and decreases in Slfn5 and Slfn9 mRNA expression that were intestinal region and sex-specific. Differentiation markers, sucrase isomaltase (SI), villin 1, and dipeptidyl peptidase 4 and glucose transporters, glucose transporter 1 (Glut1), Glut2, and sodium glucose transporter 1 (SGLT1), were increased in expression in VC-Slfn3KO mice based on intestinal region and were also highly female sex-biased, except for SI in the ileum was also increased for male VC-Slfn3KO mice and SGLT1 was decreased for both sexes. Overall, the variations that we observed in these VC-Slfn3KO mice indicate a complex regulation of intestinal gene expression that is sex-dependent.

Background: The intestinal lining renews itself in a programmed fashion that can be affected by adaptation to surgical procedures such as gastric bypass. Methods: To assess adaptive mechanisms in the human intestine after Roux-en-Y gastric bypass (RYGB), we biopsied proximal jejunum at the anastomotic site during surgery to establish a baseline and endoscopically re-biopsied the same area 6–9 months after bypass for comparison. Laser microdissection was performed on pre- and post-RYGB biopsies to isolate enterocytes for RNA sequencing. Results: RNA sequencing suggested significant decreases in gene expression associated with G2/M DNA damage checkpoint regulation of the cell cycle pathway, and significant increases in gene expression associated with the CDP-diacylglycerol biosynthesis pathway TCA cycle II pathway, and pyrimidine ribonucleotide salvage pathway after RYGB. Since Schlafen 12 (SLFN12) is reported to influence enterocytic differentiation, we stained mucosa for SLFN12 and observed increased SLFN12 immunoreactivity. We investigated SLFN12 overexpression in HIEC-6 and FHs 74 Int intestinal epithelial cells and observed similar increased expression of the following genes that were also increased after RYGB: HES2, CARD9, SLC19A2, FBXW7, STXBP4, SPARCL1, and UTS. Conclusions: Our data suggest that RYGB promotes SLFN12 protein expression, cellular mechanism and replication pathways, and genes associated with differentiation and restitution (HES2, CARD9, SLC19A2), as well as obesity-related genes (FBXW7, STXBP4, SPARCL1, UTS).