The proposed studies will test the hypothesis that induced imbalance in Wnt/?-catenin signaling results in Paneth cell depletion, dysbiosis of the resident microbiota, inflammation, enhanced susceptibility to infection by pathogenic bacteria, and that these effects can be reversed or ameliorated using a specific modulator of the Wnt/?-catenin signaling pathway. Anatomically restricted to the small bowel, Paneth cells release ?-defensins and host defense molecules, and maintenance of Paneth cell homeostasis is essential to small intestinal health and integrity. The Wnt signaling cascade has a critical role i gut ontogeny, and Wnt signaling mediated by ?- catenin p300 coactivator complexes is critical for Paneth cell differentiation. ?-catenin/CBP-mediated transcription maintains stem cell potency;in contrast, ?-catenin/p300 transcription complexes initiate differentiation programs of more limited proliferative capacity. The primary ?-catenin co-activators CBP and p300 share extensive similarity but interact with ?-catenin via their distinctive N termini, which has enabled development of small molecule antagonists that specifically inhibit their association with ?-catenin and participation in ?-catenin-mediated transcription. For example, ICG-001 promotes Wnt-dependent differentiation by binding to CBP specifically, but not to p300, blocking CBP/?-catenin transcription without 1) interfering with p300, 2) altering levels of ?-catenin, but still ) allowing CBP to participate in other transcription complexes. Conversely, IQ-1 antagonizes the ?-catenin-p300 interaction but not CBP associations, providing a means for inducing selective Paneth cell deficiency. Thus, we will test the hypothesis that antagonism of ?- catenin/p300 signaling in mice with IQ-1 induces Paneth cell depletion and dysfunction. The impact of disrupting Paneth cell homeostasis will be determined by monitoring the time course of IQ-1 induced Paneth cell deficiency and coincident changes in expression of Paneth cell specific markers and induced inflammation evident as infiltration and translocation of myeloid and lymphoid cells. Effects of Paneth cell deficiency on the composition of the prokaryotic and eukaryotic microbiota and on barrier integrity inferred from translocation of gut bacteria in vivo will be characterized. After establishing Paneth cell deficiency, the effects of drug withdrawal and of ICG-001 administration to correct co-activator imbalance and restore Paneth cell differentiation and barrier integrity will be determined, and Paneth cell autonomous responses to IQ-1 and ICG- 001 will be determined on crypt organoids ex vivo. The effect of reestablishing Paneth cell lineage allocation and crypt homeostasis on reversing prokaryotic and eukaryotic dysbiosis also will be determined. We anticipate that IQ-1-induced Paneth cell depletion will evoke proinflammatory responses, induce dysbiosis, and enhance susceptibility to bacterial translocation and that ICG-001 will selectively enable p300/?-catenin driven transcription to promote Wnt-dependent Paneth cell differentiation and accelerate recovery of small bowel homeostasis.
The objectives of this research proposal are to test the hypothesis that induced imbalance in Wnt/?-catenin signaling results in Paneth cell depletion, inflammation, dysbiosis of the resident prokaryotic and eukaryotic microbiota, disruption of enteric barrier integrity, and that these effects can be reversed or ameliorated using a specific modulator of the Wnt/?-catenin signaling pathway to correct co-activator imbalance and Wnt/?-catenin signaling to restore Paneth cell differentiation and barrier integrity. The proposed aims are innovative, because there is no precedent for impairment of Paneth cell differentiation by reversible antagonism of essential ?- catenin co-activator interactions in the Wnt signaling pathway, and the proposed research is significant to the mucosal immunology field, because it is broadly innovative in characterizing inflammation, barrier dysfunction, and prokaryotic and eukaryotic dysbiosis by reversible, selective antagonism of the Paneth cell lineage differ- entiation. The research represents a significant and innovative aspect of mucosal immunity and holds promise of future translational applications in settings of acute or chronic inflammation.
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