Broad-spectrum antibiotics alter the microbiome, increase intestinal fxr, and decrease hepatic steatosis in zebrafish short bowel syndrome.

Short bowel syndrome (SBS) is associated with changes in the intestinal microbiome and marked local and systemic inflammation. There is also a late complication of SBS, intestinal failure associated liver disease (IFALD) in which hepatic steatosis progresses to cirrhosis. Most patients with SBS arrive at massive intestinal resection after a contaminating intraabdominal catastrophe and have a history of exposure to broad-spectrum antibiotics. We therefore investigated whether the administration of broad-spectrum antibiotics in conjunction with SBS in zebrafish (ZF) would replicate these systemic effects observed in humans to identify potentially druggable targets to aid in the management of SBS and resulting IFALD. In zebrafish with SBS, broad-spectrum antibiotics altered the microbiome, decreased inflammation, and reduced the development of hepatic steatosis. After two weeks of broad-spectrum antibiotics, these fish exhibited decreased alpha diversity, with less variation in microbial community composition between SBS and sham fish. Additionally, administration of broad-spectrum antibiotics was associated with decreased expression of intestinal toll-like receptor 4 (tlr4), increased expression of the intestinal gene encoding the Farnesoid X receptor (fxr), decreased expression of downstream hepatic cyp7a1, and decreased development of hepatic steatosis. SBS in zebrafish reproducibly results in increased epithelial surface area as occurs in human patients who demonstrate intestinal adaptation, but antibiotic administration in zebrafish with SBS reduced these gains with increased cell death in the intervillus pocket that contains stem/progenitor cells. These alternate states in SBS zebrafish might direct the development of future human therapies.NEW & NOTEWORTHY In a zebrafish model that replicates a common clinical scenario, systemic effects of the administration of broad-spectrum antibiotics in a zebrafish model of SBS identified two alternate states that led to the establishment of fat accumulation in the liver or its absence. Broad-spectrum antibiotics given to zebrafish with SBS over 2 wk altered the intestinal microbiome, decreased intestinal and hepatic inflammation, and decreased hepatic steatosis.

Spleen Organoid Units Generate Functional Human and Mouse Tissue-Engineered Spleen in a Murine Model.

Introduction: Splenectomy is common after trauma or hematologic disease, and alters immune protection against pathogens, which may lead to fulminant infection with high mortality. Yet the spleen has demonstrable regenerative capacity and cells might be recovered and reimplanted at the time of injury or excision to avoid these risks. Methods: Tissue-engineered spleen (TESp) was generated from ActinGFP mice (mTESp) or human donor spleen (hTESp) through implantation of spleen organoid units (spleen OU), in NOD/SCID mice with concurrent splenectomy, on a biodegradable scaffold. Explants were evaluated and blood smears were obtained to investigate the presence of target cells or Howell-Jolly bodies, which are erythrocyte sequelae of asplenia. Results: TESp was generated from mouse (mTESp) and human (hTESp) donor cells with essential splenic components: red and white pulp with trabeculae. mTESp and hTESp demonstrated green fluorescent protein- or lamin-positive costaining with proliferating cell nuclear antigen, CD4, and CD11c, identifying proliferative donor cells and key immune components of the spleen of donor origin. Animals with hTESp and mTESP combined with splenectomy had significantly fewer Howell-Jolly bodies on blood smears than controls. Conclusion: TESp from mouse and human donor cells can be generated by 4 weeks and contains donor immune cells identified by CD4 and CD11c. TESp reduces postsplenectomy erythrocyte inclusions, indicating possible function. Impact Statement Overwhelming postsplenectomy infection is rare but highly mortal. Tissue-engineered spleen (TESp) was generated from murine (mTESp) and human (hTESp) donors and appeared histologically similar to native spleen. Both mTESp and hTESp demonstrated proliferative cells of donor spleen origin. Importantly, functional cells were demonstrated on imaging with a corresponding reduction in the number of erythrocyte inclusions in blood smears that are typically identified in patients with asplenia and indicate a lack of clearance by functional spleen tissue. Taken together, these findings indicate that this approach might be clinically relevant as a future human therapy.

Wnt signaling inhibition by monensin results in a period of Hippo pathway activation during intestinal adaptation in zebrafish.

Intestinal adaptation (IA) is a critical response to increase epithelial surface area after intestinal loss. Short bowel syndrome (SBS) may follow massive intestinal resection in human patients, particularly without adequate IA. We previously validated a model in zebrafish (ZF) that recapitulates key SBS pathophysiological features. Previous RNA sequencing in this model identified upregulation of genes in the Wnt and Hippo pathways. We therefore sought to identify the timeline of increasing cell proliferation and considered the signaling that might underpin the epithelial remodeling of IA in SBS. SBS was created in a ZF model as previously reported and compared with sham fish with and without exposure to monensin, an ionophore known to inhibit canonical Wnt signaling. Rescue of the monensin effects was attempted with a glycogen synthase kinase 3 inhibitor that activates wnt signaling, CHIR-99021. A timeline was constructed to identify peak cellular proliferation, and the Wnt and Hippo pathways were evaluated. Peak stem cell proliferation and morphological changes of adaptation were identified at 7 days. Wnt inhibition diminished IA at 2 wk and resulted in activation of genes of the Wnt/ß-catenin and Yes-associated protein (YAP)/Hippo pathway. Increased cytoplasmic YAP was observed in monensin-treated SBS fish. Genes of the WASP-interacting protein (WIP) pathway were elevated during Wnt blockade. In conclusion, cellular proliferation and morphological changes accompany SBS even in attempted Wnt blockade. Wnt/ß-catenin, YAP/Hippo pathway, and WIP pathway genes increase during early Wnt blockade. Further understanding of the effects of Wnt and YAP pathway signaling in proliferating stem cells might enrich our knowledge of targets to assist IA. NEW & NOTEWORTHY Intestinal adaptation is a critical response to increase epithelial surface area after large intestinal losses. Inhibition of Wnt/ß-catenin signaling impairs intestinal adaptation in a zebrafish model of short bowel syndrome. There is a subsequent upregulation in genes of the Yes-associated protein/Hippo and WIP pathway. These may be targets for future human therapies, as patients are salvaged by the compensation of increased intestinal epithelial surface area through successful intestinal adaptation.