Synergistic Effect of Postbiotic Yeast ABB C22^® on Gut Inflammation, Barrier Function, and Protection from Rotavirus Infection in In Vitro Models

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript from Carrera Marcolin et al. describes the effects of treatment with the posbiotic mixture of yeasts ABB C22 on proniflammatory activity on intestinal epithelial and myeloid cells as well as the effects on rotavirus infection on intestinal epithelial cells. The results shown indicate that the combination of yeast strains in the product contribute to its anti-inflammatory effects and potentiates the antiviral response. Although there is novelty in the presented results and the field of postbiotic yeast is gaining interest as a new generation of products, there are several points that should be clarified before acceptance.

1- Authors show in Figure 1 the ratio of TNFa/IL-10 levels in superanatants of THP-1 cells exposed to heat-treated single yeast species or the mixture of the three strains contained in the ABB C22 mixture. Furthermore they show the ratio of LPS treated cells, pretreated with the different yeast products. Although results show evident differences in the effects of each treatment, it would be interesting to show the individual levels of TNFa and IL-10 in each condition, as well as the ratio to make clear in which case the variation of the ratio is because increase in one or both of the cytokines or even by a combination of increase and decrease depending on the case. 

2- In Figure 3 authors show the variations in transepithelial resistance (TEER) of an epithelial monolayer exposed to the ABB C22 product and comercial live S. boulardii as positive control, since it was reported that this yeast has positive effects on barrier function. The experiment starts upon formation of the monolayer and rise of the basal TEER and is sustained along 22 days. Authors should clarify  how many replicates of each condition are measuring. In this type of experiments several replicates should be included to have consistency. Furthermore, altough in M&M sections authors state that they will express the results as deltaTEER in ohm/cm2 the Figure seems to be expressed in relative values (normalized to t0??). This should be clarified. Besides these matters of units, which is not clear to me is how authors dealt with live S. boulardii in this experiment: how many ufc/ml were used? since this seems to be a follow up of individual transwells along time, how authors dealt with yeast growing along the experiment? did this changed pH of the medium? These factors may have change the monolayer environement and induced artefactual changes, In fact, although it is claimed that S. boulardii has positive effects on barrier function, in Figure 3 the effect which is shown is a decrease in TEER, indicating damage to barrier, which is not suitable for a positive control.

3-Author studied the effects of pretreatment of kidney cells with the yeast products on rotavirus replication using NSP3 and VP7 gene expresion as surrogate markers of viral replication. Although reduction in NSP3 expression is observed in all conditions, the effects of the VP7 gene expresion is observed in a few conditions without clear pattern (eg. single inactivated S. cereviseae shows the effect and also when combined with other inactivated yeast but not in the triple combination). It would be interesting to include some known inhibitor of viral replication as positive control for meaningful comparisons. Furthermore, the use of intestinal epithelial cells for viral replication would constitute a system closer to the in vivo situation. Besides, putative mechanisms that mediate the antiviral effects should be explored and discussed (eg. is the treatment with inactivated yeast triggering some known antiviral effector pathways such as type I IFN or inducing expresion of antiviral sensors such as RIG-1 or MDA5?)

4- I find the conclusion a bit overenthusiastic on the effects and mechanisms involved in the putative anti-diharreal properties of ABB C22. Authors should include a paragraph explaining the limitations of the study: mainly in vitro evidence that should be carefuly extrapolated to clinical situations. Furthermore conclusions should be based on the experimental evidence shown being less ambitious in the potential extrapolations.

Comments on the Quality of English Language

I am not a native English speaker. To the best of my knowledge, the Quality of English of the manuscript is in accordance to international standards of science communication.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors studied the effect of a postbiotic (i.e. ABB C22) consisting of a combination of three tyndallized yeasts on gut inflammation, gut epithelial barrier function and Rotavirus infection and found that ABB C22 exerted beneficial effects in this experimental setting.

In my opinion this manuscript is interesting and of potential clinical impact dealing with emerging therapeutic role of naturally-derived compounds in the gastrointestinal tract. The manuscript is well written, background is appropriate, methodology is correct,results are presented clearly and the conclusions are supported by the results.

Minor comment

1)    The Authors should slightly modify the title making it clear that the study has been conducted using in vitro experimental models

2)    The Authors should also make it clear that the beneficial  response to ABB C22 in vitro needs to be confirmed using an in vivo, or ex-vivo experimental model

3)    I would also add in the Discussion a short paragraph outlining that this study further supports the concepts that natural products (such as nutraceuticals) are a useful therapeutic tool in general and that ABB C22, as other natural products, might be of use not only for treating infectious diarrhoea but also in several conditions in which an inflamed, leaky intestine plays a pathogenic role (Romano L et al The potential therapeutic  role of Hericium erinaceus extract in  pathologic conditions involving the urogenital-gut axis: insights into the involved mechanisms and mediators. J Physiol Pharmacol 2024 Feb;75(1). doi: 10.26402/jpp.2024.1.01).

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Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have assessed most of the comments in the points raised to the original version, significantly improving the manuscript.

Comments on the Quality of English Language

Quality of English is acceptable for publication