Review Reports - The Intricacies of Inflammatory Bowel Disease: A Preliminary Study of Redox Biology in Intestinal Organoids

Round 1

Reviewer 1 Report

The authors have experimentally evaluated redox status, especially glutathione levels on both healthy and IBD organoids. The GSH/GSSG ratio also calculate to understand the glutathione levels in both healthy and IBD organoids.

However, there have some concerns that I have with this manuscript at the present stage.

1. The authors need to mention the exact types of IBD. Is this UC or Crohn’s disease? What is the reason to select the duodenum organoid instead of the ileum or colon organoid?

2. The authors need to correct the cell number used in the lentiviral transfection study. Line 142, Materials and Methods section.

3. The authors need to add a reference against the statement mentioned in the discussion section, line 265.

4. The discussion section should be more precise.

Author Response

Dear reviewer,

Thank you very much for your comments on our manuscript. Thank you for noticing several mistakes we made in our initial submission, which we have corrected now. We have also changed our discussion and split it into a ‘discussion’ and a separate ‘conclusions’ section to make it more precise and increase readability. Please see the specific changes and explanations below. We hope everything seems ok in your opinion now.

However, there have some concerns that I have with this manuscript at the present stage.

The authors need to mention the exact types of IBD. Is this UC or Crohn’s disease? What is the reason to select the duodenum organoid instead of the ileum or colon organoid?

We have included information about canine IBD in the introduction, showing that there is no discrimination between CD and UC in dogs. Additionally, we have stated in the methods section “Cultivation of intestinal organoids” that inflammation as part of IBD is most prominent in the duodenum of dogs and histologic analysis is usually based upon biopsies thereof. Thus, using duodenal biopsies for the establishment of organoids is the easiest way as they can be taken at the same time as diagnostic samples.

The authors need to correct the cell number used in the lentiviral transfection study. Line 142, Materials and Methods section.

The mistake has been corrected and the number is now in superscript.

The authors need to add a reference against the statement mentioned in the discussion section, line 265.

Thank you for noticing this incorrect citing. The reference showing this result was cited at the end of the following sentence and we have now cited the publication at the correct place. Additionally, we have added the information that this was in regard to Crohn’s disease patients’ organoids only.

The discussion section should be more precise.

We have decided to omit half of one paragraph about the incorporation of nerves and blood vessels into organoids as this does have little added value for our manuscript. We have moved about 40% of the manuscript’s discussion into a “Conclusions” section to make a clearer discrimination between the actual discussion of our results and the conclusions drawn from our study and the respective literature.

Reviewer 2 Report

The intricacies of inflammatory bowel disease: redox biology in intestinal organoids,
examines canine duodenal-derived organoids from normal and IBD tissues.
The goal is to understand differences in the oxidant/antioxidant status defined as
the GSH/GSSG ratio and the mitochondrial redox status. These are measured
before and after challenge with various levels of hydrogen peroxide. The measures
of the GSH/GSSG ratio and Mitotracker analysis are OK. I am not sure that this is
very informative. I am provided with no real background here to expect differences
between normal and IBD. Do these organoids have NOX1 or DUOX2 expression,
and can these be stimulated in the cultures? Duox2 is often highly expressed in
human IBD relative to normal. If these organoids
have been cultured to stimulate stem cell properties, they may express NOX1, too.
I would be more impressed with how the GSH/GSSG ratio and mitotracker values
changed under these circumstances. As for mitochondria, there are agents that will
stimulate electron leakage off the electron transport chain. Using this route to
stimulate oxidative stress would be a little more physiological and report on
mitochondrial properties. My main complaint is that the significant effects in panel D
are observed at the high end of the hydroperoxide levels, beyond anything
physiological.

Finally, please state the number of samples examined for the data in each panel.

Author Response

Finally, please state the number of samples examined for the data in each panel.

Dear reviewer,

Thank you very much for your very insightful comments on our manuscript and stating that our experiments are alright. Please see our comments regarding your specific comments below:

As we wanted to simply test for the antioxidant system in healthy vs. IBD-derived organoids as a preliminary study and see whether we could recapitulate the altered morphology of IBD-derived organoids reported previously in humans (inverted polarity), we did not analyse the expression of ROS-generating proteins like NOX1 or DUOX2. Rather, we focussed on the analysis of the antioxidant system, as in vivo, most of the ROS involved in IBD that goes beyond the levels of physiologic signalling will most probably originate from the surrounding tissue and immune cells that contribute to the inflammatory state of the tissue. However, we are definitely interested in including expression analysis of these and other proteins involved in the generation and elimination of ROS in our follow-up studies.

Regarding the high concentrations of H₂O₂ in our assays, we have added a sentence in our discussion to point out the fact that these concentrations are likely non-physiological, even though it is hard to measure the exact concentrations present at the point of inflammation in vivo. Others before us used similar concentrations (and in some experiments even higher) in combination with the Grx1-roGFP redox sensor to reach a kind of plateau-phase in order to fully examine the potential of glutaredoxin-based ROS-elimination (see Hatori et al., 2020, for comparison).

We have included the information that we have done all experiments in three times with several organoids being evaluated at each concentration in the figure legends.

We hope we could make all changes to your satisfaction!

Round 2

Reviewer 1 Report

The authors have made all the corrections suggested by reviewers, the manuscript can be accepted for publication.

Author Response

Dear reviewer,

Thank you for your reviews and the comments improving our manuscript leading to your endorsement of publication.

Reviewer 2 Report

I don't see the added sentence pointing out the possible non-physiological hydrogen peroxide concentrations in the copy of the paper I received. Please, add it as mentioned in your response. I would like you to change the title to

The intricacies of inflammatory bowel disease: a preliminary study of redox biology in intestinal organoids

to emphasize that this is a test of principle and not your final statement on the redox status of the organoids.

As to hydrogen peroxide levels in cells. Most agree they would seldom reach 1micromolar and basal levels might achieve 0.1micromolar. One paper suggests neutrophils might achieve 10 micromolar in the extracellular environment [Sobotta MC, Barata AG, Schmidt U, Mueller S, Millonig G, Dick TP. Exposing cells to H2O2: a quantitative comparison between continuous low-dose and one-time high-dose treatments. Free Radic Biol Med. 2013 Jul;60:325-35. doi: 10.1016/j.freeradbiomed.2013.02.017. Epub 2013 Feb 26. PMID: 23485584.]

The next reference and several others suggest the 1 micromolar upper bound in cells is achieved by addition of 10-100micromolar to cultures [ Lim JB, Langford TF, Huang BK, Deen WM, Sikes HD. A reaction-diffusion model of cytosolic hydrogen peroxide. Free Radic Biol Med. 2016 Jan;90:85-90. doi: 10.1016/j.freeradbiomed.2015.11.005. Epub 2015 Nov 10. PMID: 26561774. Antunes F, Cadenas E. Cellular titration of apoptosis with steady state concentrations of H(2)O(2): submicromolar levels of H(2)O(2) induce apoptosis through Fenton chemistry independent of the cellular thiol state. Free Radic Biol Med. 2001 May 1;30(9):1008-18. doi: 10.1016/s0891-5849(01)00493-2. PMID: 11316581.]

These next references I have listed only to point out that NOX1 is focus of study in organoids of intestinal origin. NOX1 has been suggested to function in the WNT pathway. As a source of oxidants, it can be expressed in the organoid cultures and with mitochondria are endogenous sources that may be stimulated by the WNT ligands included in organoid cultures (Wnt-3A and Rspondin1), wnt-3a in reference [Kajla S, Mondol AS, Nagasawa A, Zhang Y, Kato M, Matsuno K, Yabe-Nishimura C, Kamata T. A crucial role for Nox 1 in redox-dependent regulation of Wnt-β-catenin signaling. FASEB J. 2012 May;26(5):2049-59. doi: 10.1096/fj.11-196360. Epub 2012 Jan 25. PMID: 22278940]. This avoids the need for co-cultures with immune cells. If the cultures can be made to express DUOX2, use of LPS could be used to induce it. And, this is suggested to be a link that shapes the microbiota.

Darby TM, Naudin CR, Luo L, Jones RM. Lactobacillus rhamnosus GG-induced Expression of Leptin in the Intestine Orchestrates Epithelial Cell Proliferation. Cell Mol Gastroenterol Hepatol. 2020;9(4):627-639. doi: 10.1016/j.jcmgh.2019.12.004. Epub 2019 Dec 23. PMID: 31874255; PMCID: PMC7160578.

Liu D, Marie JC, Pelletier AL, Song Z, Ben-Khemis M, Boudiaf K, Pintard C, Leger T, Terrier S, Chevreux G, El-Benna J, Dang PM. Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation. Cell Mol Gastroenterol Hepatol. 2022;13(4):1073-1093. doi: 10.1016/j.jcmgh.2022.01.003. Epub 2022 Jan 12. PMID: 35031518; PMCID: PMC8873962.

Schwerd T, Bryant RV, Pandey S, Capitani M, Meran L, Cazier JB, Jung J, Mondal K, Parkes M, Mathew CG, Fiedler K, McCarthy DJ; WGS500 Consortium; Oxford IBD cohort study investigators; COLORS in IBD group investigators; UK IBD Genetics Consortium; Sullivan PB, Rodrigues A, Travis SPL, Moore C, Sambrook J, Ouwehand WH, Roberts DJ, Danesh J; INTERVAL Study; Russell RK, Wilson DC, Kelsen JR, Cornall R, Denson LA, Kugathasan S, Knaus UG, Serra EG, Anderson CA, Duerr RH, McGovern DP, Cho J, Powrie F, Li VS, Muise AM, Uhlig HH. NOX1 loss-of-function genetic variants in patients with inflammatory bowel disease. Mucosal Immunol. 2018 Mar;11(2):562-574. doi: 10.1038/mi.2017.74. Epub 2017 Nov 1. PMID: 29091079; PMCID: PMC5924597.

Author Response

Dear reviewer,

Thank you once again for your comments.

We have added the explanation for using higher levels of hydrogen peroxide than one might expect to occur physiologically in the discussion, including the fact that using organoids embedded in ECM might change the diffusion of H₂O₂ completely. Additionally, we have explained that the goal was to analyze the organoids’ full potential to deal with redox stress rather than focus on low physiological levels only. Thank you very much for the valuable references you provided in this regard.

We have also included a reference in the discussion mentioning the expression of DUOX2 and NOX1 as potential enzymes of interest for further research. To keep the discussion as short as possible, we have omitted some sentences in the conclusions section.

In the end, we also changed the title following your suggestion to make clear that this is a preliminary study.