RESUMO
The treatment of defective glycosylation in clinical practice has been limited to patients with rare and severe phenotypes associated with congenital disorders of glycosylation (CDG). Carried by approximately 5% of the human population, the discovery of the highly pleiotropic, missense mutation in a manganese transporter ZIP8 has exposed under-appreciated roles for Mn homeostasis and aberrant Mn-dependent glycosyltransferases activity leading to defective N-glycosylation in complex human diseases. Here, we test the hypothesis that aberrant N-glycosylation contributes to disease pathogenesis of ZIP8 A391T-associated Crohn's disease. Analysis of N-glycan branching in intestinal biopsies demonstrates perturbation in active Crohn's disease and a genotype-dependent effect characterized by increased truncated N-glycans. A mouse model of ZIP8 391-Thr recapitulates the intestinal glycophenotype of patients carrying mutations in ZIP8. Borrowing from therapeutic strategies employed in the treatment of patients with CDGs, oral monosaccharide therapy with N-acetylglucosamine ameliorates the epithelial N-glycan defect, bile acid dyshomeostasis, intestinal permeability, and susceptibility to chemical-induced colitis in a mouse model of ZIP8 391-Thr. Together, these data support ZIP8 391-Thr alters N-glycosylation to contribute to disease pathogenesis, challenging the clinical paradigm that CDGs are limited to patients with rare diseases. Critically, the defect in glycosylation can be targeted with monosaccharide supplementation, providing an opportunity for genotype-driven, personalized medicine.
RESUMO
Lack of understanding of the pathophysiology of gastrointestinal (GI) complications in type 1 diabetes (T1D), including altered intestinal transcriptomes and protein expression represents a major gap in the management of these patients. Human enteroids have emerged as a physiologically relevant model of the intestinal epithelium but establishing enteroids from individuals with long-standing T1D has proven difficult. We successfully established duodenal enteroids using endoscopic biopsies from pediatric T1D patients and compared them with aged-matched enteroids from healthy subjects (HS) using bulk RNA sequencing (RNA-seq), and functional analyses of ion transport processes. RNA-seq analysis showed significant differences in genes and pathways associated with cell differentiation and proliferation, cell fate commitment, and brush border membrane. Further validation of these results showed higher expression of enteroendocrine cells, and the proliferating cell marker Ki-67, significantly lower expression of NHE3, lower epithelial barrier integrity, and higher fluid secretion in response to cAMP and elevated calcium in T1D enteroids. Enteroids established from pediatric T1D duodenum identify characteristics of an abnormal intestinal epithelium and are distinct from HS. Our data supports the use of pediatric enteroids as an ex-vivo model to advance studies of GI complications and drug discovery in T1D patients.
Assuntos
Diabetes Mellitus Tipo 1 , Duodeno , Mucosa Intestinal , Humanos , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Criança , Duodeno/metabolismo , Duodeno/patologia , Feminino , Masculino , Proliferação de Células , Adolescente , Células Enteroendócrinas/metabolismo , Células Enteroendócrinas/patologia , Trocador 3 de Sódio-Hidrogênio/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética , Diferenciação Celular , Organoides/metabolismo , Organoides/patologia , Antígeno Ki-67/metabolismoRESUMO
BACKGROUND & AIMS: Diarrhea occurs in up to 50% of cases of COVID-19. Nonetheless, the pathophysiologic mechanism(s) have not been determined. METHODS: This was examined using normal human enteroid monolayers exposed apically to live SARS-CoV-2 or non-replicating virus-like particles (VLPs) bearing the 4 SARS-CoV-2 structural proteins or irradiated virus, all of which bound and entered enterocytes. RESULTS: Live virus and VLPs increased secretion of multiple cytokines and reduced mRNAs of ACE2, NHE3, and DRA. Interleukin (IL)-6 plus IL-8 alone reduced NHE3 mRNA and protein and DRA mRNA. Neither VLPs nor IL-6 plus IL-8 alone altered Cl- secretion, but together they caused Cl- secretion, which was Ca2+-dependent, CFTR-independent, blocked partially by a specific TMEM16 A inhibitor, and entirely by a general TMEM16 family inhibitor. VLPs and irradiated virus, but not IL-6 plus IL-8, produced Ca2+ waves that began within minutes of VLP exposure, lasted for at least 60 minutes, and were prevented by pretreatment with apyrase, a P2Y1 receptor antagonist, and general TMEM16 family inhibitor but not by the specific TMEM16A inhibitor. CONCLUSIONS: The pathophysiology of COVID-19 diarrhea appears to be a unique example of a calcium-dependent inflammatory diarrhea that is caused by direct viral effects plus the virus-induced intestinal epithelial cytokine secretion.