Review Reports - Chestnut Shell Extract Modulates Immune Parameters in the Rainbow Trout Oncorhynchus mykiss

Round 1

Reviewer 1 Report

From my understanding authors have used 3 fish per experiment and make the mean. Then, repeat this experiment 3 times. Now you make the mean+-SD of these 3 experiemtns. In my opinion this is wrong since you make the statistics for the 3 experiments (technical replicate), not for the 9 fish (Biological replicate).

Author Response

From my understanding authors have used 3 fish per experiment and make the mean. Then, repeat this experiment 3 times. Now you make the mean+-SD of these 3 experiments. In my opinion this is wrong since you make the statistics for the 3 experiments (technical replicate), not for the 9 fish (Biological replicate).

Answer. We agree with the referee, we have not been clear in formulating the sentence. The experiments were replicated 3 times, each time with 3 different fish samples (n=9). We have clarified this in the revised manuscript.

Reviewer 2 Report

My concerns have been addressed. However, please find below a list of things to be corrected before accepting the manuscript for publication:

- Line 145: the non-adherent fraction of PBLs also include thrombocytes, granulocytes and monocytes, please indicate

- use the same description for your leulocytes throughout the text (ie: non-adherent leukocytes..)

- Line 147: protocol for intestinal leukocytes preparation was the same? Please indicate.

- Please indicate what is the control in figures 7, 8, and 9.

Author Response

My concerns have been addressed. However, please find below a list of things to be corrected before accepting the manuscript for publication:

- Line 145: the non-adherent fraction of PBLs also include thrombocytes, granulocytes and monocytes, please indicate

Answer. We specified it.

- use the same description for your leulocytes throughout the text (ie: non-adherent leukocytes..)

A. Done.

- Line 147: protocol for intestinal leukocytes preparation was the same? Please indicate.

A. The protocol for the intestinal leukocytes preparation is described in detail in the section 5.7 Cell culture

- Please indicate what is the control in figures 7, 8, and 9.

A. Cntr = control. We specified it in the legends. As described in the section ‘5.12 qPCR’ it represents the negative control.

Reviewer 3 Report

In this manuscript, phenolic compounds are extracted from chestnut shells and tested for cytotoxicity and modulation of immune responses of rainbow trout leukocytes in vitro. For extraction several solvents were used, and the authors provide an analysis of active compounds in the extracts achieved by the different solvents and data on the antioxidant activity of the different extracts. During in vitro testing, these extracts were supplemented to leukocyte cultures derived from peripheral blood and from the intestine of rainbow trout. The rationale of the study is to explore possible antioxidant and immune modulating properties of chestnut shell extracts in fish as a first step for an application of these compounds in the treatment of infectious diseases in fish. In this respect the study has its value and will be interesting for readers working on the development of alternative fish therapeutics.

The results presented here provide a good determination of the phenols present in chestnut shell extracts, which compounds can be obtained by various solvents, and they provide a first indication of antioxidant and immune modulating properties of the extracts in rainbow trout. The current study could be a good starting point for further studies, which, for instance test for any activity of the compounds in vivo during feeding experiments.

For the in vitro studies, peripheral blood leukocytes were depleted from adherent cells and by this enriched for lymphocytes. This cell preparation was then used to study superoxide anion production and phagocytosis, mainly an activity of adherent phagocytic cells. Since rainbow trout lymphocytes also display phagocytic activity, the authors were nevertheless able to generate meaningful measurements.

I have just a request for some minor amendments:

Tables 1 and 2: please provide some information how many different replicate extractions were considered in order to calculate the mean and SD values for the measurements.

Fig. 2: please provide information on which leukocyte preparation was used for this graph: peripheral blood (BPL) or intestinal leukocytes and from how many fish (n)?

Fig 4: cellular uptake of chestnut shell extract compounds: The kinetic of the CSE/ CA concentration should be addressed in the discussion. After 60 min of incubation, CSE/GA concentration in the cell matches the concentration in the medium, after 180 min of incubation a reduced amount of CSE/GA is found in gut leukocytes and a reduced amount of CSE in PBL. Could this be attributed to the effect that GA is highly metabolized in the cells as mentioned in the discussion for CaCo 2 cells? Please consider this in the discussion of the manuscript.

In addition, there were a few suggestions to language use, which are added directly to the text.

Author Response

I have just a request for some minor amendments:

Tables 1 and 2: please provide some information how many different replicate extractions were considered in order to calculate the mean and SD values for the measurements.

Answer. The extractions were replicated twice in one year for 3 years (n=6). We have clarified this in the revised manuscript.

Fig. 2: please provide information on which leukocyte preparation was used for this graph: peripheral blood (BPL) or intestinal leukocytes and from how many fish (n)?

A. The graph is representative of both blood and intestinal cell cultures and n=3. We have clarified this in the legend of the revised manuscript.

A. We agree with the referee on the hypothesis that GA can be quickly metabolized like CaCo2 cells and we have clarified it in the discussion section of the revised manuscript.

In addition, there were a few suggestions to language use, which are added directly to the text.

A. Thank you. We have included them in the revised version

Reviewer 4 Report

The manuscript must be revised and improved, clarifying following points:

Line 37-42: Delete, the authors should focus (mainly) on fish. Findings from higher vertebrates may not be translated to fish.

Line 52: Are fish vaccines too expensive? Please clarify.

Line 56-58. Probably said before.

Line 89: Uptake capacity: This study has not found the exact uptake capacity of given polyphenols (PF) in the study cells

Results: Do 100°C affect chemical stability of polyphenols?

Table 2 should be moved to appear first in the Results section.

Line 125: What is carboxils?

Sections 2.3. Line 145: What are the results from the cell isolation? ´The authors should present some micrographs of cells. What are small and large lymphocytes? Do you mean leucocytes?

All figure legends: Be sure to correctly describe whether the results are a representative from e.g. three replicate experiments, or based on three technical replicates, or pooled samples etc. This must be done.

Cellular uptake: It is not clear whether DMSO facilitate uptake of PF in cells. DMSO is know to destabilise the cell membrane, and any uptake may be due to destabilised membrane structures and NOT specific uptake. To study uptake of a given substance, more thorough study protocols has to be followed. It appears that there may be a certain increase of uptake between 15 and 60 mins, whether this is caused by receptor mediated uptake or simply diffusion is not clear from the results. What is the reason that the cell contains much lower amounts at 180 mins compared to earlier time points? Inhibitors (endocytosis) or antagonists should be included in the study. As it is now, there are too much uncertainties to say that the process is cellular uptake. I suggest that the authors remove this topic.

Respiratory burst: The optical density is quite high (up to OD= ca. 7). This means that the colour from the reaction is very intense. Most microplate readers have exact detections only in a given interval. Please check your microplate reader.

Discussion: Line 296-302. Delete, may consider move to introduction if not already mentioned.

Line 386: B-cells in fish are also phagocytic

Line 404: What is "statistically significant variations"?

Line 421+: Some text repetitions. Consider deletion.

Line 436+ : Reference needed.

M&M: Section 5.2: Where there any microbial contamination BEFORE doing PF extraction?

Section 5.7: What was the yield and purity of cells after cell isolation? Leucocytes or lymphocytes? Probably a mixture.

Section 5.9: See above, regarding cellular uptake, DMSO ans study protocol

Section 5.11 and 5.12: The authors describe that non-adherent cells were used in further experiments, but in the phagocytosis qPCR assays the cells were plated. Please explain.

5.12: 2000 microgram Zymosan A per ml. This is a very high concentration. Please explain why. What is the reason why Zymosan was used as the second stimulant (after PF stimulation). Zymosan do stimulate cells and may in this case stimulate blood cells more or less that intestinal leucocytes. Is the differential gene expression a result from Zymosan treatment?

5.12: All accession numbers for the primers/genes must be included.

Author Response

answers in the attached file

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Done.

Author Response

there are no comments to reply

Reviewer 4 Report

Thanks for the revision. Some minor point before final submission:

Section 2.5

I still think it is premature to say that there is an uptake (active) of the polyphenols in cells. There are receptors in other vertebrate species, as far as I know. Whether you results mirror a receptor mediated uptake is not clear. I envise the authors to rephrase "uptake" with "the contents" of CSE and GA in leucocytes - leaving the phenomenon open for discussion.

For future studies, I envise having full (microbial) control of raw materials their products, and leaving out zymosan as a additional stimulant if there is a possibility to do that. In addition, higher quality micrographs of cells must be made. In the current manuscript, you can place the micrographs into "supplementary material" with legend.

Author Response

We have followed the referee suggestion and changed the work uptake. We have also added a phrase in the discussion regarding the polyphenol uptake mechanism and added a ref.

We thank the referee for her/his advise for future studies. The micrograph has been added to the manuscript ad supplementary material.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript describes a novel immunostimulant extract from Castanea sativa. The authors investigate three the uptake, superoxide anion production, cytokine production and phagocitosys activation after treating blood or intestinal leukocytes with the Castanea extract. The results are promising, however, the authors should correct several aspects before publication:

Major corrections:

- English editing should be deeply corrected

- Include cell images after purification, uptake and phagocytosis

- Further explain about blood leukocytes purification

- Include MTT results

- Indicate all figures with A and B instead of upper or lower panel

- For statistical analysis use SD instead of SEM, also indicate what is the meaning of the letters on the columns? Statistical analysis results should be better indicated.

- Figure 5 and 6 legends do not correspond to the genes indicated in the figure.

- Sentence 246-248 seems to indicate that a time-course was performed but it is not shown in the text. Please include.

- Further discuss about the benefits or disadvantages of ROS activation or inhibition. Also, extend the discussion about advantages and disadvantages of pro-inflmmatory and anti-inflammatory immune genes activation

- Include discussion about further antimicrobial applications in aquaculture

- Clearly indicate whether the chelnuts extracts used were suitable for cell culture or may carry some contaminants like bacteria or fungi. Toxins present in the extract may mask the immune response observed.

- Line 500: why do you use zymosan for immunoestimulation? If this is not a mistyping, this previous treatment is masking the immune response results. Please clarify, otherwise the results are not correct.

Minor corrections:

- Line 28: which both?

- Add reference to sentence line 47-49.

- Species name in Latin are repeated throughout the text. Please write down only once.

- Line 108: what do you mean with: “The values are the mean of three years.”

- Spell out MTT, NBT, HPLC, GC-MS.

- Gene name should be used the same throughout the text (i.e. TNF-a or TNFa)

- Sentence 261: what do you mean with inactive?

- Sentence 264-265: confusing sentence, please re-write it is not clear what you want to mean.

- Line 352: when did you use those commercial tannins?

- Line 354-356: change to qPCR section

- Line 356-357: change to cell culture section

- Line 482: instead of stimulate, do you mean quantification?

Author Response

Major corrections:

- English editing should be deeply corrected.

A. The manuscript has been revised by a mother tongue.

- Include cell images after purification, uptake and phagocytosis

A. During the cell purification procedure, the cells obtained from each step described in the “Cell culture” section were smeared on slides and stained with May-Grunwald Giemsa for microscopic observations. In the revised manuscript, images of blood and intestinal leukocytes after purification have been added. However, it was not possible to take pictures of the cells after uptake and phagocytosis because the light could have interfered with the assays.

- Further explain about blood leukocytes purification

A. In the revised manuscript a more detailed description about the purification of blood leukocytes has been added.

- Include MTT results

A. We have included in the revised manuscript the figure 3 showing the results of the MTT test

- Indicate all figures with A and B instead of upper or lower panel

A. Done

- For statistical analysis use SD instead of SEM, also indicate what is the meaning of the letters on the columns? Statistical analysis results should be better indicated.

A. We substituted SEM with SD, and specified the meaning of the letters on the columns.

- Figure 5 and 6 legends do not correspond to the genes indicated in the figure.

A. Legends have been corrected

- Sentence 246-248 seems to indicate that a time-course was performed but it is not shown in the text. Please include.

A. Time-course was not performed. The sentence has been rephrased.

A. Done

- Include discussion about further antimicrobial applications in aquaculture

A. Done

A. We checked for the presence of bacteria and fungi. We reported it in the revised manuscript

A. As reported in the literature (Park et al., 2012) we stimulated the inflammation after pretreating the cells with the chestnut extract to see if the extract has a modulating effect on the immune response. Zymosane was used to induce inflammation (Mazur-Bialy and Pochec, 2016). We replaced “immune response” with “inflammation” in the text and added the two references.

Minor corrections:

- Line 28: which both?

A. The sentence has been corrected

- Add reference to sentence line 47-49.

A. Done

- Species name in Latin are repeated throughout the text. Please write down only once.

A. Done

- Line 108: what do you mean with: “The values are the mean of three years.”

A. The extraction was repeated during three years with consistent results and we report the mean. The sentence has been rephrased in the revised manuscript.

- Spell out MTT, NBT, HPLC, GC-MS.

A. Done

- Gene name should be used the same throughout the text (i.e. TNF-a or TNFa)

A. Done

- Sentence 261: what do you mean with inactive?

A. Degraded. We changed it in the revised manuscript.

- Sentence 264-265: confusing sentence, please re-write it is not clear what you want to mean.

A. Sentence re-written

- Line 352: when did you use those commercial tannins?

A. We used them for ATR-FTIR analysis. In the first version of the manuscript we decided to omit this analysis, but forgot to eliminate the product from the list of reagents. In this revised version, the ATR-FTIR analysis has been reintroduced to provide more information about the chemical composition of the chestnut extract as required by one of the referees.

- Line 354-356: change to qPCR section.

A. the name of the section has been changed

- Line 356-357: change to cell culture section.

A. the name of the section has been changed

- Line 482: instead of stimulate, do you mean quantification?

A. We wanted to stimulate the immune response and evaluate the effect of PECS on it.

Reviewer 2 Report

The results of this study showed that PECS are able to regulate the immune function of Oncorhynchus mykiss. The experiments were properly carried out and the data presented, using HPCL-DAD, cytotoxicity assay, cell culture and qPCR are detailed and interesting. However, some issues need to be addressed before it is recommended for publication in the journal.

Why choose EF1α gene as an internal reference, for most studies use actin, 18s rRNA gene in fish.

In fig. 5, 6, 7: Are all RT-PCRs the same RT-PCR reaction conditions? It is better to explain how to let the brightness of the electrophoresis band is consistent with qPCR.

Author Response

Why choose EF1α gene as an internal reference, for most studies use actin, 18s rRNA gene in fish.

A. As reported in a study by Ingerslev et al., (2006), EF1a in fish is the most stably expressed and appropriate as reference gene.

In fig. 5, 6, 7: Are all RT-PCRs the same RT-PCR reaction conditions? It is better to explain how to let the brightness of the electrophoresis band is consistent with qPCR.

A. All RT-PCRs had the same reaction conditions. The conditions developed for end-point PCR reactions, including amplification cycles, allowed to obtain unsaturated signals that showed differences among samples. Indeed, these results were consistent with those obtained from the qPCR as shown by the luminosity matching of the electrophoresis bands with the qPCR plots.

Reviewer 3 Report

This manuscript describes the role of extracts of chestnut on trout leucocytes. The study is well executed. The reading is easy and clear. However, the assumption that the effects are due to polyphenols is wrong and needs to reformulated throughout the manuscript.

Comments:

- I am not convinced that the role of chestnut extract has to be related to polyphenols. I know that these kinds of extracts are rich in polyphenols but they are not the only substances present. Therefore, the assumption that the role of extract is the same as polyphenols need to be revised. In this sense, there are many studies dealings with the role of plant extracts on fish immunity that are not referenced or included in the manuscript. The GS is a primary culture or a cell line? Please, give adequate reference.

- Based on this the title need to be reconsidered.

- I would like to see images of blood and intestinal leucocyte cultures. They should consist on mixed populations of cells with different cell-type percentages, size and metabolic activities. Therefore, the use of MTT is not an appropriate method to assess cell viability due to these different parameters. This MTT is valid when you have a single-cell type population.

- In Table 1 legend. “The values are the mean of three years.” Is this correct?

- There is not number of fish in table and figure legends.

- There is no statistics in Figure 2 to compare between time or leucocyte sources.

- In Fig.3-7 the statistics is done using the data from 3 separate assays but this needs to be done with several fish to know the biological significance, not the technical significance.

Author Response

Comments:

A. We agree with the referee that the chestnut extract is not solely composed by polyphenols. According to the literature (Vazquez et al., 2008) chestnut shell extract contains polyhenols (mainly tannins and lignins) and carbohydrates. We performed the ATR- FTIR analysis to determine the chemical composition of the chestnut extracts, however, in the first version of the manuscript we omitted it, to not overload the manuscript. The analysis has been reintroduced in the revised manuscript. In the light of these considerations, we have changed not only the title, but substituted polyphenols with chestnut shell extract, when appropriate.

GS is not mentioned anywhere in the manuscript. I can only suppose that the referee meant GA, that is Gallic acid used as standard in the experiments. The abbreviation is reported in the manuscript.

- Based on this the title need to be reconsidered.

A. The title has been changed

A. We agree with the referee, however, we found agreement between the MTT assay and cell counts. We added the images of leukocytes from the blood and intestine after purification, showing that the cell preparations were rather clean. More details on leukocytes purification have been added.

- In Table 1 legend. “The values are the mean of three years.” Is this correct?

A. Yes. The extractions have been repeated several times over a period of three years with consistent results.

- There is not number of fish in table and figure legends.

A. The number of fish has been added in the figure legends (the tables do not refer to animals).

- There is no statistics in Figure 2 to compare between time or leucocyte sources.

A. The letters on the columns were added in figure 2 to compare the cellular uptake over time.

- In Fig.3-7 the statistics is done using the data from 3 separate assays but this needs to be done with several fish to know the biological significance, not the technical significance.

A. fish numbers used for each experiment have been added.

Round 2

Reviewer 1 Report

The manuscript has been substantially improved, however further corrections indicated below should be carried out before being accepted:

Which leukocytes did you used throughout your study, total blood or intestinal leukocytes or non-adherent leukocytes? Please clarify because it is not clear in the results, discussion and mat and meths.

Correct SEM in the mat and meths section, statistical analysis, line 670

Figure 5 letters and numbers are two small, difficult to read

Presence is repeated in line 480

I can see that what you want to see is if pretreating the cells with CSE reduces the inflammation caused by zymosan, right? If so, where is the control of zymosan treatment alone? you should include this or clarify it.

You use all throughout the text rainbow trout and Oncorhynchus mykiss, as well as Castanea and chesnuts. please correct.

Reagents for cell culture and qPCR are repeated in chemicals and specific sections

- Sentence 264-265: confusing sentence, please re-write it is not clear what you want to mean.

A. Sentence re-written

Sentence has not been re-written

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Reviewer 2 Report

This research is rich in content, clear in description, reasonable in experimental design. So I recommended it for publication.

Reviewer 3 Report

The authors have amended some of my concerns. However, I still have some doubts.

- Fig 2 is not of good quality and cell-types are not clearly and well established and described. For example, I see macrophages and many thrombocytes that are not properly identified. However, this Fig is not really needed in the manuscript and should be deleted.

- From the manuscript and response I understand that they make 3 experiments with 3 fish (most probably pooled leucocytes). This is probably the reason to have so low SD/SEM bars. Data must be of separate and individual fish leucocytes, never pooled, never the mean and SEM of 3 experiments.

- In some figures the concentration is of CES in the X-axe legend, not of total phenolic compounds.

- Line 545. If you separated adherent and non-adherent blood leucocytes, which ones did you use for the experiments?