Mucosally transplanted mesenchymal stem cells stimulate intestinal healing by promoting angiogenesis

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Abstract

Mesenchymal stem cell (MSC) therapy is an emerging field of regenerative medicine; however, it is often unclear how these cells mediate repair. Here, we investigated the use of MSCs in the treatment of intestinal disease and modeled abnormal repair by creating focal wounds in the colonic mucosa of prostaglandin-deficient mice. These wounds developed into ulcers that infiltrated the outer intestinal wall. We determined that penetrating ulcer formation in this model resulted from increased hypoxia and smooth muscle wall necrosis. Prostaglandin I₂ (PGI₂) stimulated VEGF-dependent angiogenesis to prevent penetrating ulcers. Treatment of mucosally injured WT mice with a VEGFR inhibitor resulted in the development of penetrating ulcers, further demonstrating that VEGF is critical for mucosal repair. We next used this model to address the role of transplanted colonic MSCs (cMSCs) in intestinal repair. Compared with intravenously injected cMSCs, mucosally injected cMSCs more effectively prevented the development of penetrating ulcers, as they were more efficiently recruited to colonic wounds. Importantly, mucosally injected cMSCs stimulated angiogenesis in a VEGF-dependent manner. Together, our results reveal that penetrating ulcer formation results from a reduction of local angiogenesis and targeted injection of MSCs can optimize transplantation therapy. Moreover, local MSC injection has potential for treating diseases with features of abnormal angiogenesis and repair.

Authors

Nicholas A. Manieri, Madison R. Mack, Molly D. Himmelrich, Daniel L. Worthley, Elaine M. Hanson, Lars Eckmann, Timothy C. Wang, Thaddeus S. Stappenbeck

Engineering the gut microbiota to treat hyperammonemia

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Abstract

Increasing evidence indicates that the gut microbiota can be altered to ameliorate or prevent disease states, and engineering the gut microbiota to therapeutically modulate host metabolism is an emerging goal of microbiome research. In the intestine, bacterial urease converts host-derived urea to ammonia and carbon dioxide, contributing to hyperammonemia-associated neurotoxicity and encephalopathy in patients with liver disease. Here, we engineered murine gut microbiota to reduce urease activity. Animals were depleted of their preexisting gut microbiota and then inoculated with altered Schaedler flora (ASF), a defined consortium of 8 bacteria with minimal urease gene content. This protocol resulted in establishment of a persistent new community that promoted a long-term reduction in fecal urease activity and ammonia production. Moreover, in a murine model of hepatic injury, ASF transplantation was associated with decreased morbidity and mortality. These results provide proof of concept that inoculation of a prepared host with a defined gut microbiota can lead to durable metabolic changes with therapeutic utility.

Authors

Ting-Chin David Shen, Lindsey Albenberg, Kyle Bittinger, Christel Chehoud, Ying-Yu Chen, Colleen A. Judge, Lillian Chau, Josephine Ni, Michael Sheng, Andrew Lin, Benjamin J. Wilkins, Elizabeth L. Buza, James D. Lewis, Yevgeny Daikhin, Ilana Nissim, Marc Yudkoff, Frederic D. Bushman, Gary D. Wu

BMP-driven NRF2 activation in esophageal basal cell differentiation and eosinophilic esophagitis

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Abstract

Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells, especially in tissues that are constantly replenished like the esophagus. Disruption of this balance is associated with pathological conditions, including eosinophilic esophagitis (EoE), in which basal progenitor cells become hyperplastic upon proinflammatory stimulation. However, how basal cells respond to the inflammatory environment at the molecular level remains undetermined. We previously reported that the bone morphogenetic protein (BMP) signaling pathway is critical for epithelial morphogenesis in the embryonic esophagus. Here, we address how this pathway regulates tissue homeostasis and EoE development in the adult esophagus. BMP signaling was specifically activated in differentiated squamous epithelium, but not in basal progenitor cells, which express the BMP antagonist follistatin. Previous reports indicate that increased BMP activity promotes Barrett’s intestinal differentiation; however, in mice, basal progenitor cell–specific expression of constitutively active BMP promoted squamous differentiation. Moreover, BMP activation increased intracellular ROS levels, initiating an NRF2-mediated oxidative response during basal progenitor cell differentiation. In both a mouse EoE model and human biopsies, reduced squamous differentiation was associated with high levels of follistatin and disrupted BMP/NRF2 pathways. We therefore propose a model in which normal squamous differentiation of basal progenitor cells is mediated by BMP-driven NRF2 activation and basal cell hyperplasia is promoted by disruption of BMP signaling in EoE.

Authors

Ming Jiang, Wei-Yao Ku, Zhongren Zhou, Evan S. Dellon, Gary W. Falk, Hiroshi Nakagawa, Mei-Lun Wang, Kuancan Liu, Jun Wang, David A. Katzka, Xiaopeng Lan, Jeffrey H. Peters, Jianwen Que

Annexin A1–containing extracellular vesicles and polymeric nanoparticles promote epithelial wound repair

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Abstract

Epithelial restitution is an essential process that is required to repair barrier function at mucosal surfaces following injury. Prolonged breaches in epithelial barrier function result in inflammation and further damage; therefore, a better understanding of the epithelial restitution process has potential for improving the development of therapeutics. In this work, we demonstrate that endogenous annexin A1 (ANXA1) is released as a component of extracellular vesicles (EVs) derived from intestinal epithelial cells, and these ANXA1-containing EVs activate wound repair circuits. Compared with healthy controls, patients with active inflammatory bowel disease had elevated levels of secreted ANXA1-containing EVs in sera, indicating that ANXA1-containing EVs are systemically distributed in response to the inflammatory process and could potentially serve as a biomarker of intestinal mucosal inflammation. Local intestinal delivery of an exogenous ANXA1 mimetic peptide (Ac2-26) encapsulated within targeted polymeric nanoparticles (Ac2-26 Col IV NPs) accelerated healing of murine colonic wounds after biopsy-induced injury. Moreover, one-time systemic administration of Ac2-26 Col IV NPs accelerated recovery following experimentally induced colitis. Together, our results suggest that local delivery of proresolving peptides encapsulated within nanoparticles may represent a potential therapeutic strategy for clinical situations characterized by chronic mucosal injury, such as is seen in patients with IBD.

Authors

Giovanna Leoni, Philipp-Alexander Neumann, Nazila Kamaly, Miguel Quiros, Hikaru Nishio, Hefin R. Jones, Ronen Sumagin, Roland S. Hilgarth, Ashfaqul Alam, Gabrielle Fredman, Ioannis Argyris, Emile Rijcken, Dennis Kusters, Chris Reutelingsperger, Mauro Perretti, Charles A. Parkos, Omid C. Farokhzad, Andrew S. Neish, Asma Nusrat

Defective goblet cell exocytosis contributes to murine cystic fibrosis–associated intestinal disease

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Abstract

Cystic fibrosis (CF) intestinal disease is associated with the pathological manifestation mucoviscidosis, which is the secretion of tenacious, viscid mucus that plugs ducts and glands of epithelial-lined organs. Goblet cells are the principal cell type involved in exocytosis of mucin granules; however, little is known about the exocytotic process of goblet cells in the CF intestine. Using intestinal organoids from a CF mouse model, we determined that CF goblet cells have altered exocytotic dynamics, which involved intrathecal granule swelling that was abruptly followed by incomplete release of partially decondensated mucus. Some CF goblet cells exhibited an ectopic granule location and distorted cellular morphology, a phenotype that is consistent with retrograde intracellular granule movement during exocytosis. Increasing the luminal concentration of bicarbonate, which mimics CF transmembrane conductance regulator–mediated anion secretion, increased spontaneous degranulation in WT goblet cells and improved exocytotic dynamics in CF goblet cells; however, there was still an apparent incoordination between granule decondensation and exocytosis in the CF goblet cells. Compared with those within WT goblet cells, mucin granules within CF goblet cells had an alkaline pH, which may adversely affect the polyionic composition of the mucins. Together, these findings indicate that goblet cell dysfunction is an epithelial-autonomous defect in the CF intestine that likely contributes to the pathology of mucoviscidosis and the intestinal manifestations of obstruction and inflammation.

Authors

Jinghua Liu, Nancy M. Walker, Akifumi Ootani, Ashlee M. Strubberg, Lane L. Clarke

Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation

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Abstract

The intracellular protein HMGB1 is released from cells and acts as a damage-associated molecular pattern molecule during many diseases, including inflammatory bowel disease (IBD); however, the intracellular function of HMGB1 during inflammation is poorly understood. Here, we demonstrated that cytosolic HMGB1 regulates apoptosis by protecting the autophagy proteins beclin 1 and ATG5 from calpain-mediated cleavage during inflammation. Colitis in mice with an intestinal epithelial cell–specific Hmgb1 deletion and patients with IBD were both characterized by increased calpain activation, beclin 1 and ATG5 cleavage, and intestinal epithelial cell (IEC) death compared with controls. In vitro cleavage assays and studies of enteroids verified that HMGB1 protects beclin 1 and ATG5 from calpain-mediated cleavage events that generate proapoptotic protein fragments. Together, our results indicate that HMGB1 is essential for mitigating the extent and severity of inflammation-associated cellular injury by controlling the switch between the proautophagic and proapoptotic functions of beclin 1 and ATG5 during inflammation. Moreover, these studies demonstrate that HMGB1 is pivotal for reducing tissue injury in IBD and other complex inflammatory disorders.

Authors

Xiaorong Zhu, Jeannette S. Messer, Yunwei Wang, Fanfei Lin, Candace M. Cham, Jonathan Chang, Timothy R. Billiar, Michael T. Lotze, David L. Boone, Eugene B. Chang

Neuroepithelial circuit formed by innervation of sensory enteroendocrine cells

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Abstract

Satiety and other core physiological functions are modulated by sensory signals arising from the surface of the gut. Luminal nutrients and bacteria stimulate epithelial biosensors called enteroendocrine cells. Despite being electrically excitable, enteroendocrine cells are generally thought to communicate indirectly with nerves through hormone secretion and not through direct cell-nerve contact. However, we recently uncovered in intestinal enteroendocrine cells a cytoplasmic process that we named neuropod. Here, we determined that neuropods provide a direct connection between enteroendocrine cells and neurons innervating the small intestine and colon. Using cell-specific transgenic mice to study neural circuits, we found that enteroendocrine cells have the necessary elements for neurotransmission, including expression of genes that encode pre-, post-, and transsynaptic proteins. This neuroepithelial circuit was reconstituted in vitro by coculturing single enteroendocrine cells with sensory neurons. We used a monosynaptic rabies virus to define the circuit’s functional connectivity in vivo and determined that delivery of this neurotropic virus into the colon lumen resulted in the infection of mucosal nerves through enteroendocrine cells. This neuroepithelial circuit can serve as both a sensory conduit for food and gut microbes to interact with the nervous system and a portal for viruses to enter the enteric and central nervous systems.

Authors

Diego V. Bohórquez, Rafiq A. Shahid, Alan Erdmann, Alex M. Kreger, Yu Wang, Nicole Calakos, Fan Wang, Rodger A. Liddle

Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett’s metaplasia

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Abstract

Metaplasia can result when injury reactivates latent developmental signaling pathways that determine cell phenotype. Barrett’s esophagus is a squamous-to-columnar epithelial metaplasia caused by reflux esophagitis. Hedgehog (Hh) signaling is active in columnar-lined, embryonic esophagus and inactive in squamous-lined, adult esophagus. We showed previously that Hh signaling is reactivated in Barrett’s metaplasia and overexpression of Sonic hedgehog (SHH) in mouse esophageal squamous epithelium leads to a columnar phenotype. Here, our objective was to identify Hh target genes involved in Barrett’s pathogenesis. By microarray analysis, we found that the transcription factor Foxa2 is more highly expressed in murine embryonic esophagus compared with postnatal esophagus. Conditional activation of Shh in mouse esophageal epithelium induced FOXA2, while FOXA2 expression was reduced in Shh knockout embryos, establishing Foxa2 as an esophageal Hh target gene. Evaluation of patient samples revealed FOXA2 expression in Barrett’s metaplasia, dysplasia, and adenocarcinoma but not in esophageal squamous epithelium or squamous cell carcinoma. In esophageal squamous cell lines, Hh signaling upregulated FOXA2, which induced expression of MUC2, an intestinal mucin found in Barrett’s esophagus, and the MUC2-processing protein AGR2. Together, these data indicate that Hh signaling induces expression of genes that determine an intestinal phenotype in esophageal squamous epithelial cells and may contribute to the development of Barrett’s metaplasia.

Authors

David H. Wang, Anjana Tiwari, Monica E. Kim, Nicholas J. Clemons, Nanda L. Regmi, William A. Hodges, David M. Berman, Elizabeth A. Montgomery, D. Neil Watkins, Xi Zhang, Qiuyang Zhang, Chunfa Jie, Stuart J. Spechler, Rhonda F. Souza

Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature

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Abstract

Interactions between the host and gut microbial community likely contribute to Crohn disease (CD) pathogenesis; however, direct evidence for these interactions at the onset of disease is lacking. Here, we characterized the global pattern of ileal gene expression and the ileal microbial community in 359 treatment-naive pediatric patients with CD, patients with ulcerative colitis (UC), and control individuals. We identified core gene expression profiles and microbial communities in the affected CD ilea that are preserved in the unaffected ilea of patients with colon-only CD but not present in those with UC or control individuals; therefore, this signature is specific to CD and independent of clinical inflammation. An abnormal increase of antimicrobial dual oxidase (DUOX2) expression was detected in association with an expansion of Proteobacteria in both UC and CD, while expression of lipoprotein APOA1 gene was downregulated and associated with CD-specific alterations in Firmicutes. The increased DUOX2 and decreased APOA1 gene expression signature favored oxidative stress and Th1 polarization and was maximally altered in patients with more severe mucosal injury. A regression model that included APOA1 gene expression and microbial abundance more accurately predicted month 6 steroid-free remission than a model using clinical factors alone. These CD-specific host and microbe profiles identify the ileum as the primary inductive site for all forms of CD and may direct prognostic and therapeutic approaches.

Authors

Yael Haberman, Timothy L. Tickle, Phillip J. Dexheimer, Mi-Ok Kim, Dora Tang, Rebekah Karns, Robert N. Baldassano, Joshua D. Noe, Joel Rosh, James Markowitz, Melvin B. Heyman, Anne M. Griffiths, Wallace V. Crandall, David R. Mack, Susan S. Baker, Curtis Huttenhower, David J. Keljo, Jeffrey S. Hyams, Subra Kugathasan, Thomas D. Walters, Bruce Aronow, Ramnik J. Xavier, Dirk Gevers, Lee A. Denson

Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease

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Abstract

Microvillus inclusion disease (MVID) is a severe form of congenital diarrhea that arises from inactivating mutations in the gene encoding myosin Vb (MYO5B). We have examined the association of mutations in MYO5B and disruption of microvillar assembly and polarity in enterocytes. Stable MYO5B knockdown (MYO5B-KD) in CaCo2-BBE cells elicited loss of microvilli, alterations in junctional claudins, and disruption of apical and basolateral trafficking; however, no microvillus inclusions were observed in MYO5B-KD cells. Expression of WT MYO5B in MYO5B-KD cells restored microvilli; however, expression of MYO5B-P660L, a MVID-associated mutation found within Navajo populations, did not rescue the MYO5B-KD phenotype but induced formation of microvillus inclusions. Microvilli establishment required interaction between RAB8A and MYO5B, while loss of the interaction between RAB11A and MYO5B induced microvillus inclusions. Using surface biotinylation and dual immunofluorescence staining in MYO5B-KD cells expressing mutant forms of MYO5B, we observed that early microvillus inclusions were positive for the sorting marker SNX18 and derived from apical membrane internalization. In patients with MVID, MYO5B-P660L results in global changes in polarity at the villus tips that could account for deficits in apical absorption, loss of microvilli, aberrant junctions, and losses in transcellular ion transport pathways, likely leading to the MVID clinical phenotype of neonatal secretory diarrhea.

Authors

Byron C. Knowles, Joseph T. Roland, Moorthy Krishnan, Matthew J. Tyska, Lynne A. Lapierre, Paul S. Dickman, James R. Goldenring, Mitchell D. Shub