Evidence for cross-species transmission of human coronavirus OC43 through bioinformatics and modeling infections in porcine intestinal organoids

Cross-species transmission of coronaviruses has been continuously posing a major challenge to public health. Pigs, as the major animal reservoirs for many zoonotic viruses, frequently mediate viral transmission to humans. This study comprehensively mapped the relationship between human and porcine coronaviruses through in-depth bioinformatics analysis. We found that human coronavirus OC43 and porcine coronavirus PHEV share a close phylogenetic relationship, evidenced by high genomic homology, similar codon usage patterns and comparable tertiary structure in spike proteins. Inoculation of infectious OC43 viruses in organoids derived from porcine small and large intestine demonstrated that porcine intestinal organoids (pIOs) are highly susceptible to human coronavirus OC43 infection and support infectious virus production. Using transmission electron microscopy, we visualized OC43 viral particles in both intracellular and extracellular compartments, and observed abnormalities of multiple organelles in infected organoid cells. Robust OC43 infections in pIOs result in a significant reduction of organoids viability and widespread cell death. This study bears essential implications for better understanding the evolutionary origin of human coronavirus OC43, and provides a proof-of-concept for using pIOs as a model to investigate cross-species transmission of human coronavirus.</p

Pro-inflammatory cytokines stimulate CFTR-dependent anion secretion in pancreatic ductal epithelium

Background: Loss of CFTR-dependent anion and fluid secretion in the ducts of the exocrine pancreas is thought to contribute to the development of pancreatitis, but little is known about the impact of inflammation on ductal CFTR function. Here we used adult stem cell-derived cell cultures (organoids) obtained from porcine pancreas to evaluate the effects of pro-inflammatory cytokines on CFTR function. Methods: Organoids were cultured from porcine pancreas and used to prepare ductal epithelial monolayers. Monolayers were characterized by immunocytochemistry. Epithelial bicarbonate and chloride secretion, and the effect of IL-1β, IL-6, IFN-γ, and TNF-α on CFTR function was assessed by electrophysiology. Results:Immunolocalization of ductal markers, including CFTR, keratin 7, and zonula occludens 1, demonstrated that organoid-derived cells formed a highly polarized epithelium. Stimulation by secretin or VIP triggered CFTR-dependent anion secretion across epithelial monolayers, whereas purinergic receptor stimulation by UTP, elicited CFTR-independent anion secretion. Most of the anion secretory response was attributable to bicarbonate transport. The combination of IL-1β, IL-6, IFN-γ, and TNF-α markedly enhanced CFTR expression and anion secretion across ductal epithelial monolayers, whereas these cytokines had little effect when tested separately. Although TNF-α triggered apoptotic signaling, epithelial barrier function was not significantly affected by cytokine exposure. Conclusions: Pro-inflammatory cytokines enhance CFTR-dependent anion secretion across pancreatic ductal epithelium. We propose that up-regulation of CFTR in the early stages of the inflammatory response, may serve to promote the removal of pathogenic stimuli from the ductal tree, and limit tissue injury.</p

CFTR function is impaired in a subset of patients with pancreatitis carrying rare CFTR variants

Background: Many affected by pancreatitis harbor rare variants of the cystic fibrosis (CF) gene, CFTR, which encodes an epithelial chloride/bicarbonate channel. We investigated CFTR function and the effect of CFTR modulator drugs in pancreatitis patients carrying CFTR variants. Methods: Next-generation sequencing was performed to identify CFTR variants. Sweat tests and nasal potential difference (NPD) assays were performed to assess CFTR function in vivo. Intestinal current measurement (ICM) was performed on rectal biopsies. Patient-derived intestinal epithelial monolayers were used to evaluate chloride and bicarbonate transport and the effects of a CFTR modulator combination: elexacaftor, tezacaftor and ivacaftor (ETI). Results: Of 32 pancreatitis patients carrying CFTR variants, three had CF-causing mutations on both alleles and yielded CF-typical sweat test, NPD and ICM results. Fourteen subjects showed a more modest elevation in sweat chloride levels, including three that were provisionally diagnosed with CF. ICM indicated impaired CFTR function in nine out of 17 non-CF subjects tested. This group of nine included five carrying a wild type CFTR allele. In epithelial monolayers, a reduction in CFTR-dependent chloride transport was found in six out of 14 subjects tested, whereas bicarbonate secretion was reduced in only one individual. In epithelial monolayers of four of these six subjects, ETI improved CFTR function. Conclusions: CFTR function is impaired in a subset of pancreatitis patients carrying CFTR variants. Mutations outside the CFTR locus may contribute to the anion transport defect. Bioassays on patient-derived intestinal tissue and organoids can be used to detect such defects and to assess the effect of CFTR modulators.</p

Evidence for cross-species transmission of human coronavirus OC43 through bioinformatics and modeling infections in porcine intestinal organoids

Cross-species transmission of coronaviruses has been continuously posing a major challenge to public health. Pigs, as the major animal reservoirs for many zoonotic viruses, frequently mediate viral transmission to humans. This study comprehensively mapped the relationship between human and porcine coronaviruses through in-depth bioinformatics analysis. We found that human coronavirus OC43 and porcine coronavirus PHEV share a close phylogenetic relationship, evidenced by high genomic homology, similar codon usage patterns and comparable tertiary structure in spike proteins. Inoculation of infectious OC43 viruses in organoids derived from porcine small and large intestine demonstrated that porcine intestinal organoids (pIOs) are highly susceptible to human coronavirus OC43 infection and support infectious virus production. Using transmission electron microscopy, we visualized OC43 viral particles in both intracellular and extracellular compartments, and observed abnormalities of multiple organelles in infected organoid cells. Robust OC43 infections in pIOs result in a significant reduction of organoids viability and widespread cell death. This study bears essential implications for better understanding the evolutionary origin of human coronavirus OC43, and provides a proof-of-concept for using pIOs as a model to investigate cross-species transmission of human coronavirus.</p

Differential thermostability and response to cystic fibrosis transmembrane conductance regulator (CFTR) potentiators of human and mouse F508del-CFTR

Source data for manuscript (Bose et al., 2019) accepted for publication by the American Journal of Physiology - Lung Cellular and Molecular Physiolog

Differential thermostability and response to cystic fibrosis transmembrane conductance regulator (CFTR) potentiators of human and mouse F508del-CFTR

Source data for manuscript (Bose et al., 2019) accepted for publication by the American Journal of Physiology - Lung Cellular and Molecular Physiolog

Rescue of chloride and bicarbonate transport by elexacaftor-ivacaftor-tezacaftor in organoid-derived CF intestinal and cholangiocyte monolayers

Background: In cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion precipitates the accumulation of viscous mucus in the lumen of respiratory and gastrointestinal epithelial tissues. We investigated whether the combination of elexacaftor (ELX), ivacaftor (IVA) and tezacaftor (TEZ), apart from its well-documented effect on chloride transport, also restores Phe508del-CFTR-mediated bicarbonate transport. Methods: Epithelial monolayers were cultured from intestinal and biliary (cholangiocyte) organoids of homozygous Phe508del-CFTR patients and controls. Transcriptome sequencing was performed, and bicarbonate and chloride transport were assessed in the presence or absence of ELX/IVA/TEZ, using the intestinal current measurement technique. Results: ELX/IVA/TEZ markedly enhanced bicarbonate and chloride transport across intestinal epithelium. In biliary epithelium, it failed to enhance CFTR-mediated bicarbonate transport but effectively rescued CFTR-mediated chloride transport, known to be requisite for bicarbonate secretion through the chloride-bicarbonate exchanger AE2 (SLC4A2), which was highly expressed by cholangiocytes. Biliary but not intestinal epithelial cells expressed an alternative anion channel, anoctamin-1/TMEM16A (ANO1), and secreted bicarbonate and chloride upon purinergic receptor stimulation. Conclusions: ELX/IVA/TEZ has the potential to restore both chloride and bicarbonate secretion across CF intestinal and biliary epithelia and may counter luminal hyper-acidification in these tissues

Impact of hypoxia and AMPK on CFTR-mediated bicarbonate secretion in human cholangiocyte organoids

Cholangiocytes express cystic fibrosis transmembrane conductance regulator (CFTR), which is involved in bicarbonate secretion for the protection against bile toxicity. During liver transplantation, prolonged hypoxia of the graft is associated with cholangiocyte loss and biliary complications. Hypoxia is known to diminish CFTR activity in the intestine, but whether it affects CFTR activity in cholangiocytes remains unknown. Thus, the aim of this study is to investigate the effect of hypoxia on CFTR activity in intrahepatic cholangiocyte organoids (ICOs) and test drug interventions to restore bicarbonate secretion. Fifteen different human ICOs were cultured as monolayers and ion channel [CFTR and anoctamin-1 (ANO1)] activity was determined using an Ussing chamber assay with or without AMP kinase (AMPK) inhibitor under hypoxic and oxygenated conditions. Bile toxicity was tested by apical exposure of cells to fresh human bile. Overall gene expression analysis showed a high similarity between ICOs and primary cholangiocytes. Under oxygenated conditions, both CFTR and ANO1 channels were responsible for forskolin and uridine- 50-triphosphate (UTP) UTP-activated anion secretion. Forskolin stimulation in the absence of intracellular chloride showed ion transport, indicating that bicarbonate could be secreted by CFTR. During hypoxia, CFTR activity significantly decreased (P = 0.01). Switching from oxygen to hypoxia during CFTR measurements reduced CFTR activity (P = 0.03). Consequently, cell death increased when ICO monolayers were exposed to bile during hypoxia compared with oxygen (P = 0.04). Importantly, addition of AMPK inhibitor restored CFTR-mediated anion secretion during hypoxia. ICOs provide an excellent model to study cholangiocyte anion channels and drug-related interventions. Here, we demonstrate that hypoxia affects cholangiocyte ion secretion, leaving cholangiocytes vulnerable to bile toxicity. The mechanistic insights from this model maybe relevant for hypoxia-related biliary injury during liver transplantation