Intestinal goblet cells sample and deliver lumenal antigens by regulated endocytic uptake and transcytosis

  1. Jenny K Gustafsson
  2. Jazmyne E Davis
  3. Tracy Rappai
  4. Keely G McDonald
  5. Devesha H Kulkarni
  6. Kathryn A Knoop
  7. Simon P Hogan
  8. James AJ Fitzpatrick
  9. Wayne I Lencer
  10. Rodney D Newberry  Is a corresponding author
  1. University of Gothenburg, Sweden
  2. Washington University in Saint Louis School of Medicine, United States
  3. University of Michigan, United States
  4. Washington University School of Medicine, United States
  5. Harvard Medical School, United States

Abstract

Intestinal goblet cells maintain the protective epithelial barrier through mucus secretion and yet sample lumenal substances for immune processing through formation of goblet cell associated antigen passages (GAPs). The cellular biology of GAPs and how these divergent processes are balanced and regulated by goblet cells remains unknown. Using high resolution light and electron microscopy, we found that in mice, GAPs were formed by an acetylcholine (ACh) dependent endocytic event remarkable for delivery of fluid phase cargo retrograde into the trans golgi network and across the cell by transcytosis - in addition to the expected transport of fluid phase cargo by endosomes to multi-vesicular bodies and lysosomes. While ACh also induced goblet cells to secrete mucins, ACh-induced GAP formation and mucin secretion were functionally independent and mediated by different receptors and signaling pathways, enabling goblet cells to differentially regulate these processes to accommodate the dynamically changing demands of the mucosal environment for barrier maintenance and sampling of lumenal substances.

Data availability

All data generated and analysed for this study are included in the manuscript and source data files for figure 8

Article and author information

Author details

  1. Jenny K Gustafsson

    Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden
    Competing interests
    The authors declare that no competing interests exist.
  2. Jazmyne E Davis

    Medicine, Washington University in Saint Louis School of Medicine, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Tracy Rappai

    Center for Cellular Imaging, Washington University in Saint Louis School of Medicine, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Keely G McDonald

    Medicine, Washington University in Saint Louis School of Medicine, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Devesha H Kulkarni

    Medicine, Washington University in Saint Louis School of Medicine, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Kathryn A Knoop

    Center for Cellular Imaging, Washington University in Saint Louis School of Medicine, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2007-3066
  7. Simon P Hogan

    Mary H. Weiser Food Allergy Center, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. James AJ Fitzpatrick

    Department of Neuroscience, Washington University School of Medicine, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Wayne I Lencer

    Department of Pediatrics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7346-2730
  10. Rodney D Newberry

    Medicine, Washington University in Saint Louis School of Medicine, St Louis, United States
    For correspondence
    rnewberry@wustl.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4152-5191

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (DK097317)

  • Rodney D Newberry

Stiftelserna Wilhelm och Martina Lundgrens

  • Jenny K Gustafsson

Åke Wiberg Stiftelse

  • Jenny K Gustafsson

Jeanssons Stiftelser

  • Jenny K Gustafsson

National Institute of Allergy and Infectious Diseases (AI131342)

  • Rodney D Newberry

National Institute of Diabetes and Digestive and Kidney Diseases (DK109006)

  • Kathryn A Knoop

National Institute of Allergy and Infectious Diseases (AI136515)

  • Rodney D Newberry

National Institute of Allergy and Infectious Diseases (AI140755)

  • Rodney D Newberry

National Institute of Allergy and Infectious Diseases (AI112626)

  • Simon P Hogan

National Institute of Diabetes and Digestive and Kidney Diseases (DK048106)

  • Wayne I Lencer

Crohn's and Colitis Foundation (34835)

  • Jenny K Gustafsson

Vetenskapsrådet (2014-00366)

  • Jenny K Gustafsson

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal procedures and protocols were performed in accordance with the Institutional Animal Care and Use Committee at Washington University School of Medicine (Animal Wellfare Assurance number: A-3381-01) and the Swedish animal welfare legislation and approved by the Swedish Laboratory Animal Ethical Committee in Gothenburg (Ethical permit ID number: 5.8.18-11053/2019.

Reviewing Editor

  1. Kelly G Ten Hagen, National Institutes of Health, United States

Publication history

  1. Received: February 8, 2021
  2. Accepted: October 20, 2021
  3. Accepted Manuscript published: October 22, 2021 (version 1)
  4. Version of Record published: November 16, 2021 (version 2)

Copyright

© 2021, Gustafsson et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jenny K Gustafsson
  2. Jazmyne E Davis
  3. Tracy Rappai
  4. Keely G McDonald
  5. Devesha H Kulkarni
  6. Kathryn A Knoop
  7. Simon P Hogan
  8. James AJ Fitzpatrick
  9. Wayne I Lencer
  10. Rodney D Newberry
(2021)
Intestinal goblet cells sample and deliver lumenal antigens by regulated endocytic uptake and transcytosis
eLife 10:e67292.
https://doi.org/10.7554/eLife.67292

Further reading

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    Background:

    Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients.

    Methods:

    To test whether dystrophin mutations lead to defective cardiac NaV1.5–Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays.

    Results:

    Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers.

    Conclusions:

    We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5–Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5–Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias.

    Funding:

    Supported by National Institutes of Health R01 HL122352 grant; ‘la Caixa’ Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.