Targeting gut dysbiosis against inflammation and impaired autophagy in Duchenne muscular dystrophy

Abstract Nothing is known about the potential implication of gut microbiota in skeletal muscle disorders. Here, we provide evidence that fecal microbiota composition along with circulating levels of short‐chain fatty acids (SCFAs) and related metabolites are altered in the mdx mouse model of Duchenne muscular dystrophy (DMD) compared with healthy controls. Supplementation with sodium butyrate (NaB) in mdx mice rescued muscle strength and autophagy, and prevented inflammation associated with excessive endocannabinoid signaling at CB1 receptors to the same extent as deflazacort (DFZ), the standard palliative care for DMD. In LPS‐stimulated C2C12 myoblasts, NaB reduces inflammation, promotes autophagy, and prevents dysregulation of microRNAs targeting the endocannabinoid CB1 receptor gene, in a manner depending on the activation of GPR109A and PPARγ receptors. In sum, we propose a novel disease‐modifying approach in DMD that may have benefits also in other muscular dystrophies.

Dear Dr. Iannotti, Thank you for submitting your work to EMBO Molecular Medicine. First of all, I would like to apologize for the slow process. We have now heard back from two of the three referees who agreed to evaluate your manuscript. Unfortunately, after a series of reminders, we did not manage to obtain a report from Referee #3. In the interest of time, I prefer to make a decision now rather than further delay the process. If we receive the comments from Referee #3, we will send them to you, and you can address the issues raised by Referee #3 together with those raised by the other two referees. You will see from the comments below that the referees think the presented findings are interesting and novel. They raise, however, several important points, which should be convincingly addressed in a revision of this work.
I think the referees' recommendations are rather clear, and there is no need for me to reiterate their comments. Importantly, Referee #2 pointed out that several critical control experiments are missing, which must be carefully addressed.
All other issues raised by the reviewers need to be addressed as well. We would welcome the submission of a revised version within three months for further consideration. Please note that EMBO Molecular Medicine strongly supports a single round of revision. As acceptance or rejection of the manuscript will depend on another round of review, your responses should be as complete as possible.
EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection. Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it to update us on the status.
We are aware that many laboratories cannot function at full efficiency during the current COVID-19/SARS-CoV-2 pandemic and have therefore extended our "scooping protection policy" to cover the period required for a full revision to address the experimental issues. Please let me know should you need additional time and also if you see a paper with related content published elsewhere.

1.
A more comprehensive overview of the known biological roles sodium butyrate plays should be included to help readers. Sodium Butyrate is a known HDAC inhibitor but has poor pharmaceutical potential due to first pass hepatic clearance. The finding of normal levels of plasma Butyrate by GC mass is somewhat contradictory to the overall story.
2. Referencing of previous work on autophagy in DMD should be expanded. For example, overactivation of p-AKT and mTOR, both potential downstream targets of CB1 and PPAR, have been previously reported to reduce autophagy in DMD. This seems like a logical reference and work that you have expanded upon. Notes: 1. Iannotti reference to C2C12 cell characterization on page 5 is inappropriate. C2C12 cells have been used/referenced by many other groups prior to 2010. Figure 6B had altered bar graph colors from others.

2.
Referee #2 (Comments on Novelty/Model System for Author): The manuscript describes a potential intervention therapy to attenuate the symptoms of the accelerating disease in DMD. The data are clear and very interesting. There are controls missing but if the authors can complement I am in strong favor of rapid publication.
Referee #2 (Remarks for Author): In this paper, entitled "Targeting gut dysbiosis against inflammation and impaired autophagy in Duchenne muscular", by Hilal Kalkan et al, the authors explore the effects of altering the observed gut dysbiosis and its relevance to the mouse model of Duchenne Muscular Dystrophy (DMD). The authors report evidence that faecal microbiota together with circulating levels of the metabolites acetate, propionate, and butyrate are altered in the "mdx" mouse model. Introducing sodium butyrate (NaB) in "mdx" transgenic mice rescues muscle strength and autophagy. In addition, the butyrate also prevented inflammation associated with an increased endocannabinoid signaling at CB1 receptors, similar to deflazacort (DFZ), the standard palliative care for DMD. This phenomenon is also corroborated in cell line experiments. Specifically, Sodium butyrate reduces inflammation and prevents dysregulation of microRNA targeting the endocannabinoid CB1 receptor gene in a manner depending on the activation of GPR109A and PPARγ receptors. The manuscript provides interesting insights into the pathogenesis of DMD. General comment. The manuscript is well written, and the figures overall support the interpretation of their findings. The problem is what the authors do not show and the choice of the timepoint of recording of impact on SCFA treatment. That SCFA has beneficial effects on skeletal muscle growth have been reported and should be included (Lahiri et al 2019) Specifically Figure 2. The data presented in figure 2 are convincing but why did the authors not include the DFZ in WT mice? Please include. Moreover, the lack of including treatment of interest in the WT recipient mice is missing in many figures and needs to be corrected when appropriate. In addition, while minor, SCFA has been shown to reduce the production of HPA-mediated increase in cortisol levels (Neuropsychopharmacology (2020) 45:2257-2266; https://doi.org/10.1038/s41386-020-0732-xcortisol). The authors may want to comment on this.  . Inflammation and Autophagy recording. Again, overall convincing data. But the time point of recording is somewhat surprising as the inflammation is assumed to peak earlier (around two months, see (see 2020) 10:14070 | https://doi.org/10.1038/s41598-020-70987-y). At three months of age, inflammation is declining. Can the authors provide data from two Months old mice? In addition, the authors must include the monitoring TGFb1 and mRNA and protein level. Figure 5. Again, convincing data but lacking WT mice exposed to DFZ and NaB. Please include as a control Figure 9. Interesting preliminary finding but sample size too small. In addition, no muscle tissue from donor patients is presented. The authors must expand the sample size. Alternatively, consider taking out from the study.
Referee #1 (Comments on Novelty/Model System for Author): "This study follows a logical progression which builds upon our understanding of DMD inflammatory and autophagy dysregulation which has been previously reported. Novelty and impact are somewhat impacted because previous studies have shown that activation of AKT and mTOR pathways (downstream of CB1 receptor activation) suppress autophagy". We thank the reviewer for the positive comment. We appreciate his/her suggestion to remark on the concept that AKT and mTOR pathways are linked to CB1 receptor signalling (see lines 401-403; page 8).

Referee #1 (Remarks for Author):
"This study by Kalkan et al. is well written and with well thought out experiments and data presentation. The work pushes our understanding of the potential impact altered gut microbiota may be having on reducing autophagy in the skeletal muscle of dystrophin deficient mice and for Duchenne muscular dystrophy patients. Overall I believe the work is close to being ready for publication. I have included some thoughts below which should be considered". We thank the reviewer once again for the positive comment.
1. A more comprehensive overview of the known biological roles sodium butyrate plays should be included to help readers. Sodium Butyrate is a known HDAC inhibitor but has poor pharmaceutical potential due to first pass hepatic clearance. The finding of normal levels of plasma Butyrate by GC mass is somewhat contradictory to the overall story. We understand the concern raised by the reviewer. However, there is a consistent number of studies demonstrating and supporting the future development of probiotics and next-generation probiotics (NGS) using butyrate-producing bacterial species to treat human diseases. et al, 2022). Of course, we are not proposing to use butyrate per se as a treatment, but we believe that our data may lead to proposing butyrate-producing strategies, such as the enhancement of the relative abundance of butyrate-producing 21st Nov 2022 1st Authors' Response to Reviewers commensal bacteria by employing suitable prebiotics, or the use of butyrate-producing probiotics, as a potential co-treatment for DMD.

For instance, a randomized clinical study by Zhao et al. demonstrated that supplementation of a mix of dietary fibres to individuals with type 2 diabetes -T2D-improved glycemic parameters, accompanied by an increased abundance of acetate-and butyrate-producing bacteria and increased faecal levels of acetate and butyrate (Zhao et al, 2018). In another study, performed also in individuals with T2D, a mix of bacteria species producing butyrate together with inulin increased butyrate levels and improved oral glucose tolerance and glycated haemoglobin levels (Perraudeau et al, 2020). Again other investigators reported the therapeutic value of bacteria-producing butyrate in patients with Crohn's and/or inflammatory bowel diseases (Geirnaert et al, 2017; Parada Venegas et al, 2019). Similar effects were also seen in patients with obesity-associated metabolic disorders (XU
We also agree with the reviewer about the apparent discrepancy in butyrate levels we found between control and mdx mice. However, we do see a trend for a decrease in butyrate levels in the faeces of mdx mice (Fig. EV2) and, perhaps most importantly, we find, butyrate in the plasma, a significant decrease in 3-hydroxy-butyrate (Fig, EV1), which can be produced from butyrate after first-pass hepatic clearance, possibly suggesting an overall reduced production of butyrate by gut microbiota. Importantly both the trend in the faeces and the decrease of 3-hydroxy-butyrate in the plasma were reverted by DFZ. It is also worth mentioning that also in other studies it is reported that under specific pathological circumstances, the butyrate concentration in plasma does not change either after exogenous supplementation, although the whole body turnover is significantly modified (Zhao et al, 2018; Perry et al, 2016). These authors, in particular, state that circulating or intestinal butyrate content does not always reflect its endogenous production and turnover. However, future studies to define butyrate production and turnover as well as the influence of environmental factors in DMD are certainly needed.
2. Referencing of previous work on autophagy in DMD should be expanded. For example, overactivation of p-AKT and mTOR, both potential downstream targets of CB1 and PPAR, have been previously reported to reduce autophagy in DMD. This seems like a logical reference and work that you have expanded upon. We thank the reviewer for the constructive suggestion, and we have integrated the text of the discussion to mention these relevant findings (see lines 401-403; page 8). Notes: 1. Iannotti reference to C2C12 cell characterization on page 5 is inappropriate. C2C12 cells have been used/referenced by many other groups prior to 2010. According to this request, we changed the reference (line 570, page 11). Figure 6B had altered bar graph colors from others. We apologize for the mistake. The figure has been revised.

2.
Referee #2 (Comments on Novelty/Model System for Author): "The manuscript describes a potential intervention therapy to attenuate the symptoms of the accelerating disease in DMD. The data are clear and very interesting. There are controls missing but if the authors can complement I am in strong favor of rapid publication". We thank the reviewer for the positive comment and constructive suggestions.
The manuscript is well written, and the figures overall support the interpretation of their findings. The problem is what the authors do not show and the choice of the timepoint of recording of impact on SCFA treatment. That SCFA has beneficial effects on skeletal muscle growth have been reported and should be included (Lahiri et al 2019). We thank the reviewer for the positive comment and constructive suggestions she/he made throughout the manuscript. According to her/his specific request, the study of Lahiri et al. has been emphasized (line 73, page 2). Specifically 1. Figure 2. The data presented in figure 2 are convincing but why did the authors not include the DFZ in WT mice? Please include. According to this request, the experimental group wt+DFZ has been included in Fig. 2.

2.
Moreover, the lack of including treatment of interest in the WT recipient mice is missing in many figures and needs to be corrected when appropriate. According to this request, the effect of DFZ and NaB in healthy mice is now shown in Figures 3, 4 and 5. 3.
In addition, while minor, SCFA has been shown to reduce the production of HPA-mediated increase in cortisol levels (Neuropsychopharmacology (2020) 45:2257-2266; https://doi.org/10.1038/s41386-020-0732-xcortisol). The authors may want to comment on this. According to this request, we mentioned the study of Dalile et al. as well as others in the discussion (lines 377-386, page 8). Figure 3. The functional data are clear. But the authors must include the NaB and DFZ on WT mice as an important control. According to this request, we included the missing control groups in Fig. 3

5.
Please also include proper histopathology and relevant immunohistochemistry to demonstrate muscle tissue effects before and after treatment. Regrettably, we are not able to provide these results. During the execution of these experiments, there was an unexpected breakdown of the lasers of the confocal microscope which seemed like an easily solvable problem but it was not. After that, we did not have time to ask other collaborators to carry out the experiments in the allotted time. We deeply apologize for the inconvenience. However, we would like to draw the attention of this reviewer to the fact that in our previous study, we did produce histopathological and morphological studies in support of the fact that pharmacological and genetic blockade of CB1 receptors can indeed ameliorate muscle structure in mdx mice (Iannotti et al, 2018). Since we show in our present study that also butyrate produces its beneficial effects by counteracting CB1 receptor activity, we have no reason to believe that, had we had the possibility of performing these experiments, we would have found also morphological evidence of the protective action of butyrate.

6.
At present, it is not clear if the functional effects observed are direct in the skeletal muscle or an indirect effect by improved central coordination of peripheral muscle function. Our study demonstrates that daily supplementation with NaB to mdx mice changes the expression of key genes regulating inflammation and autophagy as well as the endocannabinoid system activity in skeletal muscles. Moreover, we also provide mechanistic insights into the effects of NaB obtained in LPS-stimulated C2C12 myoblasts. Therefore although it is clear that NaB exerts a beneficial effect on muscle tissues, we cannot exclude its action in the central nervous system, particularly as we could not detect changes in butyrate levels in the skeletal muscle of mdx mice. However, to the best of our knowledge, there is no evidence that NaB used at 100 mg/Kg could interfere with central coordination.
In the next years, our research activity will be focused on exploring the gut microbiota-brain axis in DMD. During the revision of this manuscript, F.A.I. received a grant from Duchenne Parent Project NL to carry out the study. Thus, we will keep in mind this precious comment and hope to provide novel insight into this subject.
7. Figure 4. Inflammation and Autophagy recording. Again, overall convincing data. But the time point of recording is somewhat surprising as the inflammation is assumed to peak earlier (around two months, see (see 2020) 10:14070 | https://doi.org/10.1038/s41598-020-70987-y). At three months of age, inflammation is declining. Can the authors provide data from two Months old mice? We understand and agree with the point raised by the reviewer. However, it is reported that in mdx mice older than eight weeks the inflammation is still persistent with levels of pro-inflammatory factors not diminished or in any case significantly high (Lagrota-Candido et al, 2002). However, to fulfil this request, we were able to produce preliminary data (n=3) demonstrating that treatment with sodium butyrate significantly reduces the expression of inflammation markers in 8-week-old mdx mice. For now, we have not inserted these data in the manuscript, but are ready to do so on request or to mention them as data not shown. In addition, the authors must include the monitoring TGFb1 and mRNA and protein level. To fulfil this request, we measured TGFb1 levels measured in the skeletal muscle of mice subject to our experimental conditions. mRNA levels of TGFb1 are shown below. Regarding the western blot analysis, we performed the experiments using a commercially available antibody that we purchased from Thermo Fisher (MA5-15065). However, using this antibody we could not detect any signal for TGF beta in our samples (see the representative blot below). We are inclined to believe that the quality of the antibody is most likely poor, given the presence of non-specific bands. The other option is that in most tissues of mdx mice the fibrotic process peaks later than 21-22 weeks as reported by many other investigators (Morrison et al, 2000; Au et al, 2011; Lagrota-Candido et al, 2002). For now, we have not inserted these data in the manuscript, but are ready to do so on request or to mention them as data not shown.  Figure 5. Again, convincing data but lacking WT mice exposed to DFZ and NaB. Please include as a control. According to this request, we included the missing experimental groups in Fig. 5 10. Figure 9. Interesting preliminary finding but sample size too small. In addition, no muscle tissue from donor patients is presented. The authors must expand the sample size. Alternatively, consider taking out from the study. Also according to this request, we have implemented the analysis using RNA samples isolated from primary skeletal muscle cells obtained from other two DMD donors. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed report from the referee, who agreed to re-assess it. As you will see, the referee is now overall supportive, and I am pleased to inform you that we will be able to accept your manuscript pending the following amendments: 1. Remove the yellow color font.
2. We updated our journal's competing interests policy in January 2022 and request authors to consider both actual and perceived competing interests. Please review the policy https://www.embopress.org/competing-interests and update your competing interests if necessary. Please use the heading "Disclosure statement and competing interests".
3. Remove the Author contribution section from the manuscript text.  Table EV2; - Figure EV3 should not be called out before Figure EV2. -There are callouts for Fig 9E-J but no such panels. -There is a callout for Suppl Table 3; should this be Table EV3? 6. Data availability -primary datasets produced in this study (sequencing data) need to be deposited in an appropriate public database (such as GEO etc). The accession numbers and database should be listed in a formal "Data Availability" section that follows the model below (see also https://www.embopress.org/page/journal/17574684/authorguide#dataavailability). # Data availability

Figure and
The datasets (and computer code) produced in this study are available in the following databases: 9. Our data editors have seen the manuscript, and they have made some comments and suggestions that need to be addressed (see attached). Please send back a revised version (in track change mode), as we will need to go through the changes.
10. As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts.
In the event of acceptance, this file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response, and all pertinent correspondence relating to the manuscript. Let us know whether you disagree with this and if you want to remove or keep any figures from it prior to publication.
Please note that the Authors checklist will be published at the end of the RPF.
Please submit your revised manuscript by December 21st, and ideally as soon as possible.

Kind regards, Jingyi
Jingyi Hou Editor EMBO Molecular Medicine ***** Reviewer's comments ***** Referee #2 (Comments on Novelty/Model System for Author): The authors have largely responded well to my comments. Yet, there are parts missing, though they give a reasonable explanation it's an interesting report that warrants publication in an area always looking for potential ways to improve the quality of life for patients with Duchenne muscle dystrophia I am pleased to inform you that your manuscript is accepted for publication and is now being sent to our publisher to be included in the next available issue of EMBO Molecular Medicine.
We would like to remind you that as part of the EMBO Publications transparent editorial process initiative, EMBO Molecular Medicine will publish a Review Process File online to accompany accepted manuscripts. If you do NOT want the file to be published or would like to exclude figures, please immediately inform the editorial office via e-mail.
Please read below for additional IMPORTANT information regarding your article, its publication and the production process.
Congratulations on your interesting work, Best wishes, Jingyi Jingyi Hou Editor EMBO Molecular Medicine Follow us on Twitter @EmboMolMed Sign up for eTOCs at embopress.org/alertsfeeds *** *** *** IMPORTANT INFORMATION *** *** *** SPEED OF PUBLICATION The journal aims for rapid publication of papers, using using the advance online publication "Early View" to expedite the process: A properly copy-edited and formatted version will be published as "Early View" after the proofs have been corrected. Please help the Editors and publisher avoid delays by providing e-mail address(es), telephone and fax numbers at which author(s) can be contacted.
Should you be planning a Press Release on your article, please get in contact with embomolmed@wiley.com as early as possible, in order to coordinate publication and release dates.

LICENSE AND PAYMENT:
All articles published in EMBO Molecular Medicine are fully open access: immediately and freely available to read, download and share.
EMBO Molecular Medicine charges an article processing charge (APC) to cover the publication costs. You, as the corresponding author for this manuscript, should have already received a quote with the article processing fee separately. Please let us know in case this quote has not been received.
Once your article is at Wiley for editorial production you will receive an email from Wiley's Author Services system, which will ask you to log in and will present you with the publication license form for completion. Within the same system the publication fee can be paid by credit card, an invoice, pro forma invoice or purchase order can be requested.