SARS-COV-2 induced Diarrhea is inflammatory, Ca 2+ Dependent and involves activation of calcium activated Cl channels

  1. Mark Donowitz
  2. Chung-Ming Tse
  3. Karol Dokladny
  4. Manmeet Rawat
  5. Ivy Horwitz
  6. Chunyan Ye
  7. Alison Kell
  8. Ruxian Lin
  9. Sun Lee
  10. Chenxu Guo
  11. Shang Jui Tsai
  12. Andrea Cox
  13. Stephen Gould
  14. Julie In
  15. Steven Bradfute
  16. Nicholas C. Zachos
  17. Olga Kovbasnjuk

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Abstract

Diarrhea occurs in 2-50% of cases of COVID-19 (∼8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for ∼24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by ∼8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca 2+ waves and an overall increase in intracellular Ca 2+ with a delay of ∼10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).

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    SciScore for 10.1101/2021.04.27.441695: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Forskolin, clotrimazole, BAPTA-AM (Sigma); IL-6, IL-8 (PepProtech); dextran Alexa Fluor 488 (10kDa) and Alexa Fluor 568 (70kDa) (Invitrogen); anti-dsRNA monoclonal antibodies (clone rJ2; Millipore, cat.# MABE1134); anti-spike rabbit polyclonal antibodies (ProSci, cat # 3525); anti-NHE3 antibodies (Novusbio, Cat# NBP1-82574); anti-ACE2 – monoclonal mouse IgG2A (R&D systems catalog # MAB933); Phalloidin and secondary fluorescent AlexaFluor antibodies from Invitrogen; Vero E6 cells (ATCC® CRL-1586™).
    IL-8
    suggested: (Thermo Fisher Scientific Cat# 53-8088-41, RRID:AB_2784756)
    anti-dsRNA
    suggested: (Millipore Cat# MABE1134, RRID:AB_2819101)
    anti-spike
    suggested: None
    anti-NHE3
    suggested: None
    anti-ACE2
    suggested: None
    mouse IgG2A
    suggested: (R and D Systems Cat# MAB933, RRID:AB_2223153)
    Experimental Models: Cell Lines
    SentencesResources
    Caco-2/BBE Culture and GCaMP6s Expression: Caco-2/BBe cells were cultured in Dulbecco’s Modified Eagle Medium (DMDM) supplemented with 25 mM NaHCO3, 0.1 mM non essential amino acids, 10% fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin in a 5% CO2/95% air atmosphere at 37C Plasmid GP-CMV-GCaMP6s (Addgene plasmid # 40753) was cloned into lentiviral vector pCDH-EF1-MCS-IRES (puro) and lentiviral particles produced.
    Caco-2/BBe
    suggested: None
    Caco-2 wild-type cells were transduced the the GCaMP6s lentiviral particles and puromycin selected (10 mg/ml).
    Caco-2
    suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
    VLP-producing cell lines: 293F cells were transfected with pS149 and selected in zeocin-containing media, creating the cell line Ftet1, which constitutively expresses the rtTAv16 Tet-on transcription factor from a CMV promoter-driven bicistronic ORF, upstream of a viral peptide and the bleomycin resistance gene.
    293F
    suggested: None
    To generate SARS-CoV-2 VLPs carrying the non-fusogenic yet receptor-binding S-2P-CSM protein, Ftet1/S226 cells were grown in suspension cultures in Freestyle media in the presence of 1 ug/ml doxycycline for 3-4 days.
    Ftet1/S226
    suggested: None
    Vero E6 cells were incubated in a 24 well plate until they reached 90% confluency.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Detection of SARS-CoV-2 by RT-qPCR: Presence of SARS-CoV-2 in enteroids and colonoids, as well as in apical and basolateral media were determined using primers and probes (N1, N2 and RP) from the CDC 2019 Real-time Reverse Transcriptase (RT)-PCR diagnostic panel (IDT).
    N2
    suggested: None
    Recombinant DNA
    SentencesResources
    Caco-2/BBE Culture and GCaMP6s Expression: Caco-2/BBe cells were cultured in Dulbecco’s Modified Eagle Medium (DMDM) supplemented with 25 mM NaHCO3, 0.1 mM non essential amino acids, 10% fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin in a 5% CO2/95% air atmosphere at 37C Plasmid GP-CMV-GCaMP6s (Addgene plasmid # 40753) was cloned into lentiviral vector pCDH-EF1-MCS-IRES (puro) and lentiviral particles produced.
    GP-CMV-GCaMP6s
    suggested: None
    pCDH-EF1-MCS-IRES
    suggested: None
    Caco-2 cells stably expressing GCaMP6s were transduced with lentiviral particles produced from lentiviral vector pLV-EF1aa IRES-3HA-CHRM3 (Blasticidin), as described40.
    pLV-EF1aa IRES-3HA-CHRM3
    suggested: None
    VLP-producing cell lines: 293F cells were transfected with pS149 and selected in zeocin-containing media, creating the cell line Ftet1, which constitutively expresses the rtTAv16 Tet-on transcription factor from a CMV promoter-driven bicistronic ORF, upstream of a viral peptide and the bleomycin resistance gene.
    pS149
    suggested: None
    Htet1 cells were converted to SARS-CoV-2 VLP-producing cell lines Ftet1/S226 and Ftet1/CG201 by transfection with Sleeping Beauty transposons that carry 5 separate genes: (1) a puromycin-resistance gene under control of a minimal EF1alpha promoter; (2) SARS-CoV-2 Envelope gene under control of the tetracycline/doxycycline-inducible TRE3G promoter; (3) SARS-CoV-2 Membrane gene under control of the TRE3G promoter; (4) SARS-CoV-2 Nucleocapsid gene under control of the TRE3G promoter; and either (5a) he SARS-CoV-2 Spike-2PP-CSM gene under control of the TRE3G promoter (pS226) or (5b) the SARS-CoV-2 Spike/D614G gene under control of the TRE3G promoter (pCG201) (Spike-2PP-CSM encodes a protein identical to Spike encoded by the Wuhan-1 strain of SARS-CoV-2, with the exception of diproline and cleavage site mutations (986KV987-986PP987 and 682RRAR685-to-682GSAG685); Spike-D614G encodes a protein identical to Spike encoded by the Wuhan-1 strain of SARS-CoV-2 with the exception of the D614G substitution mutation).
    pS226
    suggested: RRID:Addgene_133557)
    pCG201
    suggested: None
    Software and Algorithms
    SentencesResources
    Average fluorescence for the entire frame was generated in MetaMorph Basic (v7.7.3.0) and transferred to Microsoft Excel for normalization before being transferred to Prism 8/9 for graphing and analysis.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.04.27.441695v1 on bioRxiv