Loss of Slfn3 induces a sex-dependent repair vulnerability after 50% bowel resection

Abstract

Bowel resection accelerates enterocyte proliferation in the remaining gut with suboptimal absorptive and digestive capacity because of a proliferation-associated decrease in functional differentiation markers. We hypothesized that although schlafen 3 (Slfn3) is an important regulator of enterocytic differentiation, Slfn3 would have less impact on bowel resection adaptation, where accelerated proliferation takes priority over differentiation. We assessed proliferation, cell shedding, and enterocyte differentiation markers 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 were even longer in Slfn3KO mice. Mitotic marker, Phh3+, and the proliferation markers Lgr5, FoxL1, and platelet-derived growth factor-α (PDGFRα) were increased after resection in male WT, but this was blunted in male Slfn3KO mice. Cell-shedding regulators Villin1 and TNFα were downregulated in female mice and male WT mice only, whereas Gelsolin and EGFR increased expression in all mice. Slfn3 expression increased after resection in WT mice, whereas other Slfn family members 1, 2, 5, 8, and 9 had varied expressions that were affected also by sex difference and loss of Slfn3. Differentiation markers sucrase isomaltase, Dpp4, Glut2, and SGLT1 were all decreased, suggesting that enterocytic differentiation effort is incompatible with 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. Therefore, Slfn3 may be an important regulator not only of “normal” enterocytic differentiation but also in response to bowel resection.

NEW & NOTEWORTHY The differentiating stimulus of Slfn3 signaling restrains an increase in mucosal mass after bowel resection, and there is a Slfn3-sex interaction regulating differentiation gene expression and intestinal adaptation. This current study highlights the combinatory effects of gender and Slfn3 genotype on the gene expression changes that contribute to the adaptation in intestinal cellular milleu (i.e. villus and crypt structure) which are utilized to compensate for the stress-healing response that the animals display in intestinal adaptation.

INTRODUCTION

Short bowel syndrome (SBS) is a leading cause of morbidity and mortality in which inadequate mucosal surface area impairs nutrient absorption. SBS in children can be caused by necrotizing enterocolitis, gastroschisis, mesenteric tumor, intestinal obstruction, and malrotation, resulting in the blocking of the blood supply (1–5), whereas adult SBS may be associated with repetitive surgery for Crohn’s disease or ischemic bowel (6, 7). Most patients are initially managed with total parental nutrition (TPN). However, long-term treatment with TPN can lead to parental nutrition-associated liver disease and increases the risk of catheter-related septicemia (8). Approximately one-third of patients die over the next 2–5 yr, often related to their inability to achieve enteral autonomy (2). Therefore, current therapies need to aim to restore enteral autonomy.

The rodent protein schlafen 3 (Slfn3) is a part of a growth regulatory gene family. One important role of Slfn3 and its human analog SLFN12 is to regulate the differentiation, development, and maturation of small intestinal epithelial cells and T lymphocytes (9–16). Slfn3 mRNA expression is in thymus, lymph nodes, spleen, PBMC, bone marrow, intestine, colon, skeletal muscle, kidney, lung, liver, testis, heart, and brain of rats and mice (11, 17). Various stimuli such as repetitive deformation and butyrate increase Slfn3 and SLFN12 expression in rat intestinal IEC-6 and human Caco2 cells, respectively, and in turn induce the expression of differentiation markers dipeptidyl peptidase 4 (Dpp4) and villin 1 (Vil1) in the IEC-6 cells and sucrase isomaltase (SI) in the Caco2 cells (16, 18). There is a direct positive correlation between Slfn3 or SLFN12 expression with the expression of the intestinal epithelial cell differentiation markers that were also downregulated when siRNA to Slfn3 or SLFN12 was applied (16, 18). Additionally, using laser capture dissection, the mRNA expression of Slfn3 is approximately fivefold higher in the villi of the jejunal mucosa than in the crypts, which similarly correlated to Dpp4, SI, and Glut2 having five- to ninefold higher expressions also in the villi versus the crypts (13). The correlative expression of Slfn3 and the differentiation markers requires the Slfn3 P-loop region in the NH₂ terminus (10, 13, 16). In previous work, we investigated the role of Slfn3 in intestinal differentiation through a Slfn3 knockout (Slfn3KO) mouse model. Slfn3KO mice have less maturing weight gain and decreased villus length and crypt depth, as well as sex-dependent variances in metabolic pathway genes, the intestinal differentiation markers Glut2 and SGLT1, and the intestinal differentiation pathway genes Notch2 and Cdx2 (19). Loss of Slfn3 also affects expression of other Slfn family members, increasing Slfn1 and Slfn5 and decreasing Slfn4, Slfn8, and Slfn9 in the ileal mucosa (20). Although Slfn3 is known to regulate homeostatic development and differentiation of intestinal epithelial cells, the impact of Slfn3 on the adaptation to massive bowel resection has not previously been studied.

To model SBS, we utilized a 50% bowel resection with a defunctionalizing short limb Roux-en-Y anastomosis in mice. Murine massive bowel resection has the advantage of manifesting critical features in parallel with the human condition (21–23). We performed these resections on wild-type (WT) and Slfn3KO mice and harvested the tissue 10 days later. We chose this time point because intestinal adaptation is occurring with accelerated proliferation and renewal of the residual intestinal cells (24, 25). The intestinal adaptation response includes an increase in villus height and crypt depth as a consequence of the accelerated mucosal cell proliferation, hypertrophy of the smooth muscle cells, and the lengthening and dilation of the remnant intestine (25–27). Likewise, we measured proliferation and cell-shedding gene markers and canonical intestinal differentiation markers that are known to be regulated by Slfn3 to determine its role within the intestinal adaptation phase after bowel resection. Quantifying changes in intestinal epithelial cell differentiation (via functional differentiation marker expression) is a fundamental part of restoring nutrient and fluid absorptive capacity of the intestinal cells during intestinal adaptation. Ultimately, the expressions of differentiation markers are the hallmarks that need to be achieved clinically for patients with SBS. Some typical treatments for SBS focus on stimulating proliferation. The use of glucagonlike peptide 2 (GLP-2) and the GLP-2 analog teduglutide has been used to promote growth in the intestine of SBS patients (28, 29). However, treatment of human Caco-2 cells with teduglutide promoted proliferation but then inhibited functional differentiation marker expression of villin1, sucrase isomaltase, glucose transporter 2, and dipeptidyl peptidase 4 (30). Therefore, it is essential to tease out the regulation defining functional differentiation following residual intestinal cell proliferation after bowel resection in order to shape the appropriate intervention strategy.

METHODS

Mice and Surgery

Slfn3KO mice (global knockout) were acquired from Dr. Akira at the Laboratory of Host Defense, Research Institute for Microbial Diseases, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamadakoa, Suita, Osaka, 565-0871, Japan. The University of North Dakota Institutional Animal Care and Use Committee approved these studies under protocol nos. 1807-7 C and 1807-8 C. These mice are on a C57Bl/6 background, and mice aged 8–16 wk were used. Genotyping was performed as previously described (19, 20). Mice undergoing surgery received a liquid diet (Vivonex Pediatric) 72 h before and after surgery. Buprenorphine SR (1 mg/kg) was injected subcutaneously for pain relief. Mice were anesthetized with 1–2% isoflurane, and 50% of the small bowel was removed starting 5 cm from the pyloric orifice of the stomach. A Roux-en-Y anastomosis with a 2- to 3-cm-long defunctionalized ileal limb was used to join the remaining duodenum to the ileum using an 8-0 Nylon suture (Unify; ADSurgical). The intestinal mucosa was scraped from the 50% resected small bowel and was flash-frozen in liquid nitrogen or placed in 4% paraformaldehyde (Electron Microscopy Grade; Electron Microscopy Sciences, Hatfield, PA) in phosphate-buffered saline (PBS) for 40–48 h for histology. The small bowel was returned to the body cavity, the abdominal wall musculature was sutured using a 5-0 Polysorb Braided Absorbable suture (Covidien), and the skin was sutured using a 3-0 Perma-hand silk thread (Ethicon). Eight to 10 days after surgery, mice were euthanized between the times of 8 and 11 AM, and the intestinal mucosa was harvested from the proximal duodenum, Roux-en-Y limb, and distal ileum. Mucosa was flash-frozen in liquid nitrogen, or the intestine was placed in 4% paraformaldehyde for histology.

Histology, Design-Based Stereology, and Immunolabeling

Tissue samples in 4% paraformaldehyde were transferred to PBS (with 0.5 mM MgCl₂·6H₂O and 0.9 mM CaCl₂) and then put through a 30% sucrose gradient series. The initial resected control and duodenum-ileum border segments were imbedded in Neg50 Colorless imbedding compound (Richard Allan Scientific, San Diego, CA). The tissue was cut en face at a 35-µm interval to generate sections appropriate for conducting design-based stereological quantification. Sections were permeabilized with block solution (3% donkey serum, 1% bovine serum albumin, 0.1% Triton X-100 in PBS) to reduce nonspecific binding. The sections were immunolabeled with phospho-histone H3 (Ser¹⁰) antibody (Phh3, cat. no. 06-570; Millipore, Temecula, CA; diluted 1:100 from a 200 µg/mL stock to give a final concentration of 2 µg/mL antibody in block solution; block solution: 3% donkey serum, 1% bovine serum albumin, 0.1% Triton X-100, 1× phosphate-buffered saline) and detected with horseradish peroxidase-conjugated secondary antibody using the VectaStain Elite kit with diamino benzidine (DAB) as the chromogen substrate and the recommended manufacturer’s instructions (Vector Laboratories, Burlingame, CA). Labeled sections were counterstained with Gill’s Hematoxylin Solution No. 1 (Electron Microscopy Sciences) and Eosin Y solution (0.25% alcoholic; Fisher Scientific) before being taken through a standard ethanol dehydration-xylene series. Counterstained slides were permanently mounted (Vectamount; Vector Laboratories) and imaged for stereology on an Olympus BX51WI bright-field microscope with a motorized XYZ stage. For unbiased quantification of positive nuclei, the investigator masked to sex and genotype and the StereoInvestigator 11.0 software and the optical fractionator workflow (MBF Bioscience, Williston, VT) were used. Initial contour tracings were generated at low power (×2) around the intestinal mucosa and villus structures. Positive nuclei in the C segment were counted (at ×0) using an optical dissector frame set at 250 × 250 µm and a grid size of 727.2 × 861.9 µm in every eighth section, with a section counting interval of 280 µm. Counting parameters were set based on initial overcounting optimization for a subset of the animals so that the coefficient of error was ≤ 0.1, and all subsequent samples were counted with the same parameters. Positive nuclei in the A segment were counted (at ×40) using an optical dissector frame set at 100 × 100 µm and a grid size of 300 × 300 µm in every eighth section, with a section counting interval of 280 µm based on initial overcounting optimization. The total number of positive nuclei were estimated with the StereoInvestigator optical fractionator formula (n = 1/ssf·1/asf·1/hsf·ΣQ−), where ssf is the section-sampling fraction (30), asf is the area-sampling fraction (area sampled/total area), and hsf is the height-sampling fraction (counting frame height/35 μm), and ΣQ− (total particle count) with a guard zone distance is set at 5 µm.

For fluorescent imaging of Phh3-positive nuclei, 10-µm sections were permeabilized with block solution as for stereology labeling using the same primary antibody. Bound antibody was detected with cy3-conjugated secondary antibody (cat. no. 711-166-152; Jackson ImmunoResearch, West Grove, PA; diluted 1:200 from a 750 µg/mL stock to give a final concentration of 3.75 µg/ml antibody), and nuclei were labeled with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, cat. no D9542-5MG, Sigma; diluted 1:1,000 from a 5 mg/mL stock to give a final concentration of 5 µg/mL). Images were collected on a Nikon Eclipse 80i compound fluorescent scope (UND Imaging Core) using a SPOT II camera and software. All composites were generated using Adobe Photoshop (CS6 Extended).

For immunolabeling of sucrase isomaltase, we used sections parallel to those used for the Phh3 immunolabeling. Sections were blocked as previously described and immunolabeled for sucrase isomaltase (SI) using a rabbit polyclonal antibody (cat. no RQ4659, NSJ Bioreagents; diluted 1:100 from a 100 µg/mL stock to give a final concentration of 1 µg/mL antibody in block solution) at 1 µg/mL. Secondary antibody used was donkey anti-rabbit IgG-FITC (cat. no 711-095-152, Jackson ImmunoResearch; diluted 1:200 from a 750 µg/mL stock to give a final concentration of 3.75 µg/mL antibody). Bound antibody detection and imaging were conducted as described for Phh3.

Parallel sections at 30-µm thickness were labeled for apoptotic nuclei using the indirect TUNEL method with the ApopTag Fluorescein in situ detection kit (cat. no. S7111; Millipore/Sigma), following the manufacturer’s recommended instructions. All nuclei were labeled with DAPI and imaged with standard fluorescence microscopy.

RNA Isolation and qPCR

Total RNA was isolated and cDNA synthesis prepared as previously described (19, 20). A Bio-Rad CFX96 Touch Real-Time PCR Detection System and the PrimeTime Gene Expression Master Mix from Integrated DNA Technology (IDT, Coralville, IA) were utilized for quantitative PCR (qPCR), analysis as previously described (19, 20). We utilized the hypoxanthine phosphoribosyltransferase 1 (HPRT) as a reference control gene and the threshold cycle (C_T) values and the $2^{{}^{–\Delta \Delta C_T}}$ method to determine expression levels. Primer/probe sets used from Bio-Rad (Hercules, CA), including mouse RPLP0, mouse Slfn3, mouse sucrase isomaltase (SI), mouse Dpp4, mouse Vil1, mouse Cdx2, and mouse Glut2 (Slc2a2), are proprietary and were previously described (19), except for mouse Bmi1 (assay ID: qMmuCEP0043063), mouse Pdgfra (assay ID: qMmuCIP0033780), mouse Gsn (assay ID: qMmuCIP0033929), mouse Egfr (assay ID: qMmuCIP0030060), and mouse Tnf (assay ID: qMmuCEP0028054). The following primer/probe sets for mouse HPRT and mouse Glut1 (Slc2a1) were purchased from IDT, and their sequences were previously published (19). The following primer/probe sequences were also from IDT: mouse Lgr5: probe-5′/56-FAM/AGC TAC CCG/ZEN/CCA GTC TCC TAC AT/3IABkFQ/-3′, forward 5′CTC CAA CCT CAG CGT CTT C-3′, reverse 5′-GTC AAA GCA TTT CCA GCA AGA-3′; and mouse FoxL1: probe 5′-/5Cy5/TCG CTA TAT CTC TAA GTC CTG CCC TCA G/3IAbRQSp/-3′, forward 5′GTA CAG TGA CCA CCA CAA GTC-3′, reverse 5′-CAT GAT CCA GTT GAA CCT CCA-3′; The qPCR cycle conditions were one cycle of 2 min at 95°C, 50 cycles of 10 s at 95°C, and 45 s at 55°C for the annealing temperature (19, 20).

Statistics

Quantitative PCR results were compared by two-way ANOVA with Uncorrected Fisher’s LSD (GraphPad Prism Software, version 7; GraphPad, San Diego, CA). Quantitative morphometric results of the histology villus/crypt/muscularis externa and design-based stereology were assessed by an unpaired, two-tailed t test. Data are represented as means ± SE.

RESULTS

Male Villus Length and Female Crypt Depth Are Increased More in Slfn3KO Mice Compared with WT Mice after Bowel Resection

A 50% bowel resection was performed on WT and Slfn3KO mice, with a Roux-en-Y anastomosis of the distal duodenum to the distal ileum and intestinal mucosa collected and labelled as depicted in Fig. 1A. Male WT mice had an average percent weight loss of 23.7% ± 0.23%, whereas male Slfn3KO weight decreased 15.0% ± 4.4%, although this was not significantly different (Fig. 1B). Female WT versus Slfn3KO mice had a significantly different decreases in percent weight loss between genotypes of 16.7% ± 2.8% versus 21% ± 3.1% (Fig. 1C). Although the mice had weight loss due to bowel resection, the survival curves were not significantly different between WT and Slfn3KO mice (Fig. 1, D and E). Additionally, we observed no rectal bleeding or diarrheal stool in the mice. Morphometric analysis demonstrated an increase in male and female mice of villus length and crypt depth of the postoperative distal duodenum segment C in comparison with the resected duodenal segment A (Fig. 2, A and C, and Supplemental Fig. S1; all Supplemental Material for this article can be found online at https://doi.org/10.6084/m9.figshare.12971381). In the Slfn3KO mice, this increase in villus length was even greater for males when baseline corrections were made by deducting the initial resected segment A (Fig. 2B and Supplemental Fig. S1). The crypt depth was significantly greater in only the Slfn3KO female mice (Fig. 2D). Muscularis externa thickness decreased in male Slfn3KO and female WT mice to a greater extent than in the Slfn3KO female mice after bowel resection (Fig. 2, E and F).

Figure 1.

Schematic of 50% bowel resection and weight loss and survival curves. A: regions A and B indicate segments where intestinal mucosa was collected from duodenum and ileum regions of the resected segment, respectively. Regions C, D, and E are the distal duodenum, limb, and ileum, where intestinal mucosa was harvested 10 days postoperative. B and C: male (B) and female (C) %weight loss day 0 through day 10 postoperative. *P < 0.05 compared with Slfn3-knockout (Slfn3KO) as analyzed with the mixed-effects analysis model (GraphPad Prism version 8) D and E: Male (D) and female (E) survival curves after bowel resection.

Figure 2.

Villus length and crypt depth increase after 50% bowel resection is enhanced in Slfn3-knockout (Slfn3KO) mice. Quantitation of villus length (A and B), crypt depth (C and D), and muscularis externa thickness (E and F) from regions A and C. Results are from 3 mice/group with an average of 83 villus, 236 crypt, and 230 muscularis externa measured per mouse group. *P < 0.05 to respective A region, #P < 0.05 compared with wild type (WT) of the same region.

Proliferation Markers Lgr5, FoxL1, and PDGFRα Increase after Bowel Resection in WT Mice but to a Significantly Lesser Extent in Male Slfn3KO Mice

An increase in villus length suggests an increase in proliferation of intestinal epithelial cells. Stereological analysis revealed an increase in Phh3-immunoreactive nuclei in the mucosa of the postoperative distal duodenal segment C in comparison with the resected duodenal segment A (Fig. 3, B–D). There were more Phh3+ nuclei along the length of the villus, whereas the A regions only had Phh3+ cells near the crypts (Fig. 3, A and B). Additionally, we evaluated markers of intestinal stem cells that are specific for whether cells proliferate faster (Lgr5) or slower (Bmi1) and whether the cells are pro-proliferative (FoxL1) or are induced to mature and differentiate (PDGFRα) (Fig. 4 and Supplemental Fig. S2) (31–33). In general, Lgr5, FoxL1, and PDGFRα expression increased after bowel resection (segment C) in WT mice but to a significantly lesser extent in male Slfn3KO mice (Fig. 4, A–C). Interestingly, both WT and Slfn3KO female mice had increased expressions of Lgr5 and PDGFRα after bowel resection (segment C; Fig. 4A), but then only FoxL1 expression had a blunted upregulation in the female Slfn3KO mice compared with the female WT mice and was similar in expression to the male WT and Slfn3KO mice (Fig. 4B). The Bmi1 stem cell marker for slower proliferation was significantly increased only in the male Slfn3KO mice compared with the WT mice in just segment C (Fig. 4D). For the distal postoperative D and/or E segments, there was an increased expression of Lgr5 in male Slfn3KO and female WT mice, FoxL1 in male and female WT mice, and PDGFRα in male WT and female WT and Slfn3KO mice (Supplemental Fig. S2).

Figure 3.

Phh3 labeling increases along the length of the villi after bowel resection. A: sections generated from male and female wild-type and Slfn3-knockout (Slfn3KO) mice were immunolabeled for Phh3 (red), a marker of mitotic cells, and DAPI (blue) to identify nuclei. A: preoperative segment A; arrows indicate Phh3-positive nuclei associated with the crypt region. B: postoperative segment C; arrows indicate Phh3-positive nuclei along the villus. Scale bars, 100 µm. C: ratio of the Phh3+ cells to the measured contour area over the counting frame. D: ratio of segment C to segment A was determined for data based on the ratio of Phh3+ cells relative to the contour area/counting frame, and then the data were natural log transformed prior to statistical analysis (n = 3, *P < 0.05 compared with respective A region).

Figure 4.

Sex- and genotype-dependent changes in stem cell markers after bowel resection. Total RNA was isolated from intestinal mucosa regions of wild-type (WT) and Slfn3-knockout (Slfn3KO) mice before and after 50% bowel resection. Stem cell marker expression was measured by quantitative PCR (qPCR), using HPRT as a reference control gene for Lgr5 (A), FoxL1 (B), platelet-derived growth factor-α (PDGFRα; C), and Bmi1 (D) in region A vs. C (n = 10–14; *P < 0.05 compared with respective A region and #P < 0.05 compared with WT of the same region).

Regulators of Epithelial Cell Shedding, Gelsolin and EGFR, Were Upregulated after Massive Bowel Resection

Villus length is also determined by the level of epithelial cell turnover or shedding. Therefore, we evaluated the expression of the following antiapoptotic and cell turnover regulators: villin1 (Vil1) and villin-family member gelsolin (GSN) (34–36). Vil1 significantly decreased in all postoperative segments, except for male Slfn3KO segment C (Fig. 5A and Supplemental Fig. S3). GSN significantly increased in the postoperative segment C for male and female Slfn3KO and WT mice, but only in segment D of male WT and female Slfn3KO mice (Fig. 5B and Supplemental Fig. S3). Two other factors involved with cell shedding are epidermal growth factor receptor (EGFR) and tumor necrosis factor α (TNFα) (37–39). EGFR is a driver of intestinal growth and differentiation and suppresses constitutive intestinal epithelial cell shedding through the binding of its ligand EGF (37). Prolonged TNFα exposure to the intestinal epithelial cells leads to cell death and shedding (38). There was a significant increase in EGFR in segment C of male WT and Slfn3KO mice and the female Slfn3KO mice in comparison with segment A (Fig. 5C). TNFα expression levels were significantly elevated in the preoperative segment A for the Slfn3KO male mice. This expression was then decreased after surgery in the complementary segment C for the male Slfn3KO mice (Fig. 5D). Finally, the male WT mice had a greater increase in TNFα expression than the Slfn3KO mice in the postoperative segment D in comparison with the resected segment B (Supplemental Fig. S3).

Figure 5.

Epithelial cell-shedding regulators gelsolin (GSN) and epidermal growth factor receptor (EGFR) are upregulated after bowel resection. Total RNA was isolated from intestinal mucosa regions of wild-type (WT) and Slfn3-knockout (Slfn3KO) mice before and after 50% bowel resection. Changes in gene expression were measured by quantitative PCR (qPCR), using HPRT as a reference control gene for villin 1 (Vil1; A), GSN (B), EGFR (C), and TNFα (D) in regions A vs. C (n = 9–14; *P < 0.05 to respective A region and #P < 0.05 to WT of the same region).

Finally, we evaluated the level of apoptosis by ApoTag immunohistochemistry. There were more cells stained with the ApoTag in the C segments after bowel resection than in the preoperative A segments for all mice (Fig. 6). Most of the ApoTag was present in the villi and muscularis externa in the C segment. The female WT mice did exhibit ApoTag staining in the crypts (Fig. 6D). The only ApoTag staining observed in the A segments was within the muscularis externa region (Fig. 6D).

Figure 6.

Apoptosis increases after Roux-en-Y bowel resection. Sections from postsurgical segments A and C were labeled using an indirect TUNEL assay (ApoTag). Representative positive regions are shown for the villus tip (A, A′), the intermediate villus region and crypt region (B, B′), and the muscularis externa (C, C′) from segment C. Apoptotic nuclei were notably absent in segment A but present postresection surgery. Arrows indicate regions with positive nuclei. The ApopTag-positive nuclei are shown in A–C, and the overlay with DAPI is in A′–C′. Scale bar, 100 µm for all. Semiquantitative assessment of apoptotic nuclei is shown in D, with overall assessment for the different regions indicated with a + or ++ for regions that had ApoTag present and – for regions with no ApoTag-stained cells.

Expression of Differentiation Markers and Glucose Transporters Responds Differently for Slfn3KO Mice

We wanted to determine whether the cells have fully differentiated after proliferation as they support the important functions of intestinal epithelial cells; therefore, we evaluated various differentiation markers and glucose transporters. In this study, Slfn3 RNA expression was increased in segment C of WT mice after bowel resection and in the male WT segment E (Fig. 7A). Functional differentiation marker Dpp4 was decreased in RNA expression for segment C in WT male and female WT and Slfn3KO mice; however, male Slfn3KO mice did not have a decrease in Dpp4 (Fig. 7B). Only female WT mice displayed an increase in Dpp4 in segment D compared with segment B (Supplemental Fig. S4). The differentiation transcription factor Cdx2 was slightly increased in postbowel resection segment C and significantly increased in segment E of the female Slfn3KO mice (Fig. 7, C and D). SI RNA expression was decreased in all of the postoperative segment, segments C, D, and E, in comparison with the resected segments A and B (Fig. 7, E and F). We measured SI protein expression by immunohistochemistry and found that WT male mice had no change in SI protein expression after bowel resection (Fig. 7G and Supplemental Fig. S5). The male Slfn3KO mice had lower SI protein in the resected A segment in comparison with WT male mice and after bowel resection the SI expression was increased in segment C compared with segment A (Fig. 7G and Supplemental Fig. S6). Additionally, the greatest levels of SI protein in the male Slfn3KO mice were observed from the middle to tip of the villus (Fig. 7G and Supplemental Fig. S6). However, female WT mice differed from male WT mice. They had greater levels of SI after bowel resection in the middle to tip of the villus (Fig. 7H and Supplemental Fig. S7). The female Slfn3KO mice did not have a significant difference in SI protein expression when segment A was compared with segment C; however, the levels of SI observed in segment C were significantly less than the female WT segment C SI protein levels (Fig. 7H and Supplemental Fig. S8).

Figure 7.

Sucrase isomaltase (SI) and markers dipeptidyl peptidase 4 (Dpp4) mRNA are reduced after bowel resection. Total RNA was isolated from intestinal mucosa regions of wild-type (WT) and Slfn3-knockout (Slfn3KO) mice before and after 50% bowel resection. A–F: gene expression was measured by quantitative PCR (qPCR), using HPRT as a reference control gene for Slfn3 (A), Dpp4 (B), Cdx2 (C and D), and SI (E and F) (n = 9–14; *P < 0.05 to respective A region, **P < 0.05 to respective B region, #P < 0.05 to WT of the same region, and $P < 0.05, E vs. D regions). G and H: immunohistochemistry scores for SI in (G) male and (H) female WT and Slfn3KO mice. [Scores were collected from 5 blinded reviewers, scoring 28–49 villi in regions of base, middle (Mid), and tip of the villi; these data are from 2 mice per sex/genotype group. Scoring was from 0 to 4, and Supplemental Fig. S9 was utilized as a scoring template.] *P < 0.05 to respective A region and #P < 0.05 to WT of the same region.

We investigated RNA expression for the glucose transporters Glut1, Glut2, and SGLT1. WT male and female mice displayed an increase in Glut1 and a corresponding decrease in Glut2 and SGLT1 in the postoperative C segment compared with the preoperative A segment (Fig. 8, A–C). The Slfn3KO mice had shifted expression patterns similar to the WT mice in the C segment, with statistical significance observed with the increased expression of Glut1 in males and the decreased expression of Glut2 and SGLT1 in females (Fig. 8, A–C). The postoperative D and E segments also exhibited decreases in Glut2 and SGLT1 compared with the preoperative B segment in both the WT and Slfn3KO mice (Supplemental Fig. S10). Conversely, Glut1 was only significantly increased in the female WT E segment (Supplemental Fig. S10).

Figure 8.

Glucose transporters Glut2 and SGLT1 decrease, whereas Glut1 increases after bowel resection in both wild-type (WT) and Slfn3-knockout (Slfn3KO) mice. Total RNA was isolated from intestinal mucosa regions of WT and Slfn3KO mice before and after 50% bowel resection. Gene expression was measured by quantitative PCR (qPCR), using HPRT as a reference control gene for Glut1 (A), Glut2 (B), and SGLT1 (C) in region A vs. C (n = 9–14, *P < 0.05 to respective A region, #P < 0.05 to WT of the same region).

Finally, we examined the expression levels of Slfn family members, as the loss of Slfn3 has previously been shown to decrease the expression of Slfn4, Slfn8, and Slfn9 and increase the expression of Slfn1 and Slfn5 in ileal mucosa (20). Slfn family member expression was affected by the loss of Slfn3, bowel resection, and sex difference (Figs. 9 and 10). The Slfn1 increase observed in the male Slfn3KO mice was significantly decreased due to bowel resection (Fig. 9, A and B). This was also observed significantly for female Slfn3KO mice in the D and E segments (Fig. 9B). Slfn2 also had a decrease in expression due to bowel resection but just in the male Slfn3KO mice in segments D and E (Fig. 9D). The female Slfn3KO mice, alternatively, increased Slfn2 expression after bowel resection in segment C (Fig. 9C). Slfn4 expression was increased in all mice after bowel resection (Fig. 9, E and F). Slfn5 increased after bowel resection greatly in the segment C after bowel resection for both male and female Slfn3KO mice (Fig. 10A). In the distal segments, Slfn5 remains high in the Slfn3KO mice after bowel resection, whereas female WT mice only had an increase in Slfn5 in segment E (Fig. 10B). Slfn8 had some trending increases in the Slfn3KO mice but was only significant in the female segment C (Fig. 10C), but the male Slfn3KO segment D had a decreased expression due to bowel resection (Fig. 10D). Finally, Slfn9 increased after bowel resection for male WT and female Slfn3KO mice in segment C, whereas the male Slfn3KO had a decreased expression of Slfn9 in segment C (Fig. 10E). Slfn9 had no significant changes in segments B, D, and E (Fig. 10F).

Figure 9.

Slfn1, -2, and -4 expression affected by bowel resection is dependent on sex and loss of Slfn3. Total RNA was isolated from intestinal mucosa regions of wild-type (WT) and Slfn3-knockout (Slfn3KO) mice before and after 50% bowel resection. Gene expression was measured by quantitative PCR (qPCR), using HPRT as a reference control gene for Slfn1 A and C segments (A), Slfn1 B, D, and E segments (B), Slfn2 A and C segments (C), Slfn2 B, D, and E segments (D), Slfn4 A and C segments (E), and Slfn4 B, D, E segments (F) (n = 7–13; *P < 0.05 to respective A region and #P < 0.05 to WT of the same region).

Figure 10.

Slfn5, -8, and -9 expression affected by bowel resection is dependent on sex and loss of Slfn3. Total RNA was isolated from intestinal mucosa regions of wild-type (WT) and Slfn3-knockout (Slfn3KO) mice before and after 50% bowel resection. Gene expression was measured by quantitative PCR (qPCR), using HPRT as a reference control gene for Slfn5 (A), Slfn8 (C), and Slfn9 (E) A and C segments and Slfn5 (B), Slfn8 (D), and Slfn9 (F) B, D, and E segments (n = 7–13; *P < 0.05 to respective A region, #P < 0.05 to WT of the same region, and $P < 0.05, E vs. D regions).

DISCUSSION

The present treatment goals of SBS are to increase absorption through intestinal adaptation and reversal procedures or by restoring intestinal length by transplant or lengthening procedures (36). Intestinal adaption results in an increase in intestinal epithelial cell proliferation but also enhanced programmed cell death, which means accelerated cell turnover (40). Therefore, this balanced regulation between cell proliferation and cell loss by apoptosis after surgery is complex. The precise factors that control and monitor this adaptive process remain unclear, as to the effects of this adaptation on the differentiation of the intestinal epithelium (41). Previously, we have shown how Slfn3 and its human analog SLFN12 regulate the functional differentiation, development, and maturation of small intestinal epithelial cells (9, 10, 12, 13, 15, 16). Specifically, we have shown that Slfn3 induced the intestinal differentiation marker villin through the transcription factor Cdx2 (15). Further studies have demonstrated that Slfn3 acts within the cytosol to trigger a secondary signal cascade that stimulates villin and sucrase isomaltase expression, and this mechanism of action is through the Slfn3 P-loop NTPases domain (10). The human ortholog of Slfn3 is SLFN12. SLFN12 drives expression sucrase isomaltase by binding to SerpinB12 through its ATP-binding region (9). SLFN12 binding to SerpinB12 then stimulates USP14 and UCHL5 deubiquitylase activity, which increases the levels of Cdx2 (9).

This current study further explores the role of Slfn3 in intestinal epithelial cell adaptation after 50% bowel resection with a defunctionalizing short Roux-en-Y anastomosis, with a focus on the cellular and molecular responses during intestinal adaptation. Overall, Slfn3KO mice display sex-dependent changes after bowel resection, including an increase in villus length, crypt depth, and proliferation markers (Phh3+), but with a blunted increase in Lgr5, FoxL1, and PDGFRα. Additionally, regulators for cell shedding revealed a consistent expression of Vil1, an increase in GSN and EGFR, and a decrease in TNFα for Slfn3KO mice compared with the differential changes seen in WT mice.

First, WT and Slfn3KO mice after bowel resection displayed increases in proliferation, as quantified by increases in villus length and Phh3+ cell numbers. Furthermore, the increase in villus length was more pronounced after bowel resection in male Slfn3KO mice. This adaptive response has been shown previously to include an increase in villus height and crypt depth resulting from increased mucosal cell proliferation, in addition to smooth muscle hypertrophy, and dilation of the remaining intestine (25–27, 42–46). The use of Phh3 as a proliferation/mitotic marker is more specific and reproducible than K_i-67, as Phh3 only highlights cells that are in late G2 and M phases, whereas K_i-67 is expressed throughout the cell cycle, except in the G0 phase (47, 48). Interestingly, in our study, Phh3-immunoreactive epithelial cells were localized along the length of the villus in the postoperative C segment, which differs from normal biology in which mitotic cells are confined to the crypt region as displayed in the A segments. Similarly, conditional Apc-deficient (AhCre + Apcfl^/flMyc^+/+) mice have a significantly higher number of BrdU-labelled cells outside of the proliferation zone in comparison with the WT mice and double-mutant (AhCre + Apcfl^/fl Myc^fl/fl) mice, where BrdU was incorporated only within the crypt (49). Remarkably, although villus length was longer and Phh3+ cells were observed moving up the villus of the male Slfn3KO mice in comparison with the WT mice, we observed diminished expression of pro-proliferation markers Lgr5, FoxL1, and PDGFRα and an increase in Bmi1, which is known to drive a slower rate of proliferation (Fig. 11A, section 1). To reconcile these apparently opposing responses, we speculated that there was a potential imbalance in the regulation of proliferation versus cell shedding. To this end, we evaluated the expression of genes involved with intestinal epithelial cell shedding.

Figure 11.

Schematics of Slfn3 involvement in the adaptation of intestinal epithelial cells after bowel resection. A: section 1 displays graphically the expressions of Bmi1 increase and FoxL1, Lgr5, and platelet-derived growth factor-α (PDGFRα) decrease in the crypt of male Slfn3-knockout (Slfn3KO) mice compared with wild-type (WT) mice. Section 2 displays graphically the expression state of villin 1 (Vil1), gelsolin (GSN), and increased epidermal growth factor receptor (EGFR) expression in male WT versus Slfn3KO mice. Graphic was created with BioRender.com. B: Slfn3 is an upstream regulator contributing to the balance between proliferation and apoptosis versus villus length in normal intestinal epithelial cell physiology and turnover. In intestinal adaptation after 50% bowel resection, this balance is perturbed, and the absence of Slfn3 exacerbates this imbalance.

Vil1 and GSN are members of the villin family that are antiapoptotic factors and regulators of intestinal epithelial cell shedding (34–36). When either Vil1 or GSN are overexpressed in epithelial cell lines, apoptosis is delayed as the actin dynamics are maintained (50). Conversely, in double-knockout mice for Vil1 and GSN, there are apoptotic cells observed along the villus resulting from damaged mitochondria characterized by disorganized and collapsed cristae (51). The male Slfn3KO segment C maintained consistent expression of Vil1 and had an increase in GSN in comparison with resected A segment (Fig. 11A, section 2). Moreover, we observed an increase in EGFR expression, which suppresses constitutive intestinal epithelial cell shedding (Fig. 11A, section 2). Increased mucosal TNFα production is associated with increased rates of intestinal epithelial shedding, but in male Slfn3KO mice the TNFα expression levels were decreased from the high levels observed in the starting resected A segment (39, 52). Additionally, we observed an increase in ApoTag cells after bowel resection in the villi and muscularis externa regions. Observations of an increase in proliferation and apoptosis after bowel resection have been seen by others as well and are considered a part of the adaptation process (53–57). Therefore, the greater villus length observed in male Slfn3KO mice after bowel resection could be a combination of suppression of epithelial cell shedding from higher expression levels of Vil1, GSN, and EGFR, a decrease in TNFα, and the progression of Phh3+ proliferating cells into the villus, plus an adaption balance with an increase in enterocyte apoptosis.

After bowel surgery, enterocytes must differentiate to function properly in nutrient and fluid absorption, both of which are key challenges for patients with SBS. Previously, Sprague-Dawley rats with an intact bowel had differentiation marker expression increases or decreases correspondingly after in vivo intramucosal modulation of Slfn3 using adenoviral delivery or siRNA reduction of Slfn3 in rat jejunal mucosa (12, 13). Surprisingly, we observed a decrease in functional differentiation markers SI and Dpp4 and glucose transporters Glut2 and SGLT1 for both male and female WT and Slfn3KO mice in segment C. However, segment C of male Slfn3KO mice maintained Dpp4 levels in comparison with the resected A segment. These differences between the Slfn3 expression and differentiation marker expression of the current study and our previous observations point toward the stressful environment in intestinal adaptation after bowel resection. Notably, Slfn3 or homolog SLFN12 regulates intestinal differentiation markers, such as SI, transcriptionally and posttranscriptionally (9, 10, 12, 13). Additionally, we have reported that Roux-en-Y anastomosis of rat jejunum led to decreases in SI, Dpp4, and Glut2 protein expression in the defunctionalized limb (equivalent to segment D in this study) in the earliest phase of healing 3 days after surgery (13). Therefore, we further evaluated SI at the protein level before and after bowel resection (10 days postoperative). Interestingly, there was a differential protein expression of SI based on sex. Male WT and female Slfn3KO mice displayed no change in SI protein expression due to bowel resection, whereas male Slfn3KO and female WT mice had increased SI protein expression. Additionally, we noted that the increases in SI protein expression were focused in the middle to the tip of the villus. Van Beers et al. (58) demonstrated correlations between SI protein levels to their respective mRNA amounts in human duodenum. However, with interpatient variability in mRNAs levels, the correlation coefficient for SI was 0.33, suggesting a posttranscriptional regulation. Moreover, in situ hybridization and histology staining have shown that SI is present from the crypt to about one-third of the villus length in human duodenum (58, 59). In rat jejunum and ileum there is corresponding SI mRNA to protein activity that was greatest at the villus tip and decreased to the crypt (60, 61). Conversely, in mouse intestine, sucrase activity was greatest in duodenum and jejunum and from villus base to midvillus, with little to no activity at the tip (62). These data and ours demonstrate the complexity of SI mRNA and protein regulation. Specifically, this study informs us of a Slfn3-sex interaction involved in the regulation of SI in the course of intestinal healing after bowel resection.

The absorption of glucose is critical for functional recovery and long-term management of SBS and primarily takes place through the unidirectional transporter SGLT1 on the brush-border membrane in both mice and humans (63). If levels of SGLT1 are low on the membrane, then SGLT1 will shuttle Glut2 to the brush border to help transport glucose (64). In our study, both SGLT1 and Glut2 RNA expression decreased after bowel resection, whereas the expression of Glut1 increased. This compensatory response of Glut1 upregulation in response to reduced SGLT1 and Glut2 has also been seen during nematode infection in the small intestine (63). Conversely, Lewis rats that underwent a 70% small bowel resection displayed an increase in SGLT1 mRNA expression 1 wk postoperative in the remaining jejunum and ileum distal to reanastomosis (65). These differences could reflect variations in animal species, diet, percent of resection, type of anastomosis, location, and timing postoperative of mRNA expression measurement for SGLT1.

The loss of Slfn3 in female mice after bowel resection did not always result in similar gene expression changes, as we observed in the male Slfn3KO mice. In fact, the following genes in female Slfn3KO mice changed similarly to wild-type mice: Lgr5, PDGFRα, Bmi1, Vil1, GSN, EGFR, TNFα, and Dpp4 (Fig. 11A), whereas the only genes to change similarly in female Slfn3KO in comparison with male Slfn3KO mice were FoxL1, SI, and the three glucose transporters. However, the SI protein in female Slfn3KO after bowel resection was not increased as it was in male Slfn3KO and female WT mice. The female WT mice only had a significant increase in Slfn3 in the C segment, whereas male WT mice were observed to have an increase in Slfn3 in all three postoperative segments: segments C, D, and E. Interestingly, there were also numerous differences in Slfn family member RNA expression due to bowel resection that was also influenced due to sex and the loss of Slfn3 (Figs. 9 and 10), which further suggests the potential complexity of intestinal mucosal regulation by Slfn family members. Dou and colleagues (66, 67) have also observed intestinal expression differences of P-glycoprotein (P-gp) between male and female Wistar rats that were dependent on food intake. Male rats had decreased intestinal P-gp mRNA and protein in the fed state compared with the fasted state, whereas female rats had an increase in intestinal P-gp (66, 67). They speculated that the sex-dependent food effect on intestinal P-gp could be due to food-stimulated sex hormones (66, 67). So, it could be possible that the female Slfn3KO mice adapt differentially from male Slfn3KO mice after bowel resection because of an inequality in sex hormone levels.

In conclusion, Slfn3 affects the morphological and gene expression response to 50% bowel resection in a sex-dependent manner (Fig. 11B). Massive bowel resection drives longer villi and deeper crypts, with the response being potentiated by the absence of Slfn3. This result could suggest that the differentiating stimulus of Slfn3 signaling may restrain the increase in mucosal mass. The network of stem cell proliferation and cell turnover gene expression is also impacted by the lack of Slfn3. Interestingly, even Slfn3 RNA expression is increased by bowel resection. Yet this does not translate to an increase in functional differentiation markers (SI, Dpp4) and glucose transporters at this stage of acute adaptation at the mRNA level. However, there does appear to be a Slfn3-sex interaction posttranscriptionally regulating SI protein expression. This indicates that the normal strength of the proliferation signals regulating proliferation are unbalanced postoperatively and even more pronounced in the absence of Slfn3. Overall, this current study highlights the combinatory effects of sex and Slfn3 genotype on the gene expression changes that contribute to intestinal adaptation.

SUPPLEMENTAL DATA

Supplemental Figs. S1–S10: https://doi.org/10.6084/m9.figshare.12971381.

GRANTS

This work was funded by National Institutes of Health (NIH) Grant RO1-DK-096137 (to M. D. Basson). Efforts by J. T. Lansing, J. Umthin, C. Brown, and D. C. Darland were supported by NIH Grant 2P20-GM-104360-06A1. Imaging studies were conducted in the UND Imaging Core Facility, supported by NIH Grant P20-GM-113123, DaCCoTA CTR NIH Grant U54-GM-128729, and UNDSMHS funds.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.E.V., D.C.D., and M.D.B. conceived and designed research; E.E.V., J.T.L., D.C.D., J.U., A.D.S. and C.B. performed experiments; E.E.V., D.C.D., J.U., and A.D.S. analyzed data; E.E.V., D.C.D., and M.D.B. interpreted results of experiments; E.E.V. and D.C.D. prepared figures; E.E.V. drafted manuscript; E.E.V., D.C.D., and M.D.B. edited and revised manuscript; E.E.V., J.T.L., D.C.D., J.U., A.D.S., C.B., and M.D.B. approved final version of manuscript.