Reviewer ’s report  
Title: Gut microbiota -mediated generation of saturated fatty acids elicits inflammation in the 
liver in murine high -fat diet -induced steatohepatitis  
 
Version: 0 Date: 01 May 2017  
 
Reviewer: Robert Schierwagen  
 
Reviewer's report:  
The study performed by Yamada et al. describes the changes of the microbiota and lipid 
metabolism in an experimental model of NASH. Therefore, the authors used a recently reported 
steatohepatitis -inducing high fat diet. Additionally, the authors treated an other group of animals 
with antibiotics supplementary to the high fat diet. The main findings of this study are the 
observed changes saturated fatty acid and n -6 fatty acid pathways, as well as an increase of 
migratory macrophages in the liver. However, th e results are not fully convincing. Furthermore, 
the study design, especially the choice of the prophylactic antibiotic group and the drawn 
conclusions from the comparison with the high fat diet group is questionable.  
 
Major critics:  
- The authors correctly describe that all three observed groups had completely different 
microbiomes and metabolic profiles. However, afterwards the authors directly compare the 
high fat diet group to the group that additionally received antibiotics. These  comparisons are 
not suitable, since the treatment antibiotics lead to a complete different metabolic phenotype, 
which also has no similarities with the control group when it comes to the gut microbiome 
and the metabolic profile.  
- The observations made on  macrophages are not convincing. The shown representative FACS 
analyses do not resemble the quantification bar graphs next to them. In the representative 
FACS analyses it looks like there is also a decrease of resident and migratory macrophages in 
the +Abx  group. And even if macrophage numbers are not decreased by antibiotic treatment, 
how do the authors explain the significant lower levels of inflammatory markers compared to 
the NASH model?  
- The authors state several times that antibiotic treatment improv es STHD -01 induced effects. 
Since treatment with antibiotics has been started even before the start of the diet, this is a 
prophylactic approach and therefore this statement cannot be kept up. Also this statement is 
critical, since the phenotype of the Abx  group is so different. A therapeutic approach in 
which the antibiotic treatment starts after the development of the disease would be more 
favourable, since this better adapts to the clinical routine.  
- The results describing Figure 1C state that a signifi cant number of ballooning and Mallory 
bodies was observed. How was this assessed? Furthermore, the pathological findings 
(ballooning, Mallory bodies, fibrotic extensions and infiltrating inflammatory cells) should 
be marked in the shown representative stai nings. Especially, since the resolution and size of 
the images in the current manuscript could be improved. More favourable, specific stainings 
should be performed for key pathological observations.  
- What is the advantage of the STHD -01 model in comparis on to the established models like 
MCD diet with respect to the treatment time of 10 weeks?  
 
Minor critics:  
- In the introduction, it is stated that 10 million patients have NAFLD and 1 million patients 
have NASH. However, it is not specified to which coun try these numbers are related.  
- The section about HCC in the introduction should be removed, since HCC is not a part of this 
study. In the used dietary model HCC was identified after 36 weeks of diet, but for this study 
only 10 weeks of diet were perform ed. 
- The section where the STHD -ß1 diet is introduced contains the wrong reference number. The 
authors should review the assignment of the reference numbers to the corresponding 
references. Furthermore, the same reference is used in three consecutive sent ences. This 
should be avoided.  
- Why were feces obtained at week 16 and not at the end of the study?      
- In the section about statistical analysis it is stated that students t -test or Mann -Whitney U -test 
was used. However, I could not found if students t-test was actually used in one of the 
comparisons. This should be reviewed.  
- There is a discrepancy in the methods. In the methods section it is written that mice were fed 
for 10 weeks and in the results section it is stated that they were fed 9 weeks.  
- In Figure 1, the abbreviations CAZ and MNZ should be spelled in full in the figure legends.  
- Figure 4 is not arranged correctly. The graph about Tnf mRNA should be Figure 4D as stated 
in the text.  
- In the legends to Figure 4 a significance level of p<0. 01 is introduced even though it does not 
apply in this figure.  
 
Are the methods appropriate and well described?  
If not, pl ease specify what is required in your comments to the authors.  
Yes 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
Yes 
Are the conclusions drawn adequately suppo rted by the data shown?  
If not, please explain in your comments to the authors.  
No 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
I am able to assess the statistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Acceptable  
Declaration of competing interests  
Please c omplete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication o f this  
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently ap plying for any patents relating to the content of the  
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
5. Do you have any other f inancial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no  competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be  posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be  included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
Title: Gut microbiota -mediated generation of saturated fatty acids elicits inflammation in the 
liver in murine high -fat diet -induced steatohepatitis  
 
Version: 0 Date: 01 May 2017  
 
Reviewer: Robert Schierwagen  
 
Reviewer's report:  
The study performed by Yamada et al. describes the changes of the microbiota and lipid 
metabolism in an experimental model of NASH. Therefore, the authors used a recently reported 
steatohepatitis -inducing high fat diet. Additionally, the authors treated an other group of animals 
with antibiotics supplementary to the high fat diet. The main findings of this study are the 
observed changes saturated fatty acid and n -6 fatty acid pathways, as well as an increase of 
migratory macrophages in the liver. However, th e results are not fully convincing. Furthermore, 
the study design, especially the choice of the prophylactic antibiotic group and the drawn 
conclusions from the comparison with the high fat diet group is questionable.  
 
Major critics:  
- The authors correctly describe that all three observed groups had completely different 
microbiomes and metabolic profiles. However, afterwards the authors directly compare the 
high fat diet group to the group that additionally received antibiotics. These  comparisons are 
not suitable, since the treatment antibiotics lead to a complete different metabolic phenotype, 
which also has no similarities with the control group when it comes to the gut microbiome 
and the metabolic profile.  
- The observations made on  macrophages are not convincing. The shown representative FACS 
analyses do not resemble the quantification bar graphs next to them. In the representative 
FACS analyses it looks like there is also a decrease of resident and migratory macrophages in 
the +Abx  group. And even if macrophage numbers are not decreased by antibiotic treatment, 
how do the authors explain the significant lower levels of inflammatory markers compared to 
the NASH model?  
- The authors state several times that antibiotic treatment improv es STHD -01 induced effects. 
Since treatment with antibiotics has been started even before the start of the diet, this is a 
prophylactic approach and therefore this statement cannot be kept up. Also this statement is 
critical, since the phenotype of the Abx  group is so different. A therapeutic approach in 
which the antibiotic treatment starts after the development of the disease would be more 
favourable, since this better adapts to the clinical routine.  
- The results describing Figure 1C state that a signifi cant number of ballooning and Mallory 
bodies was observed. How was this assessed? Furthermore, the pathological findings 
(ballooning, Mallory bodies, fibrotic extensions and infiltrating inflammatory cells) should 
be marked in the shown representative stai nings. Especially, since the resolution and size of 
the images in the current manuscript could be improved. More favourable, specific stainings 
should be performed for key pathological observations.  
- What is the advantage of the STHD -01 model in comparis on to the established models like 
MCD diet with respect to the treatment time of 10 weeks?  
 
Minor critics:  
- In the introduction, it is stated that 10 million patients have NAFLD and 1 million patients 
have NASH. However, it is not specified to which coun try these numbers are related.  
- The section about HCC in the introduction should be removed, since HCC is not a part of this 
study. In the used dietary model HCC was identified after 36 weeks of diet, but for this study 
only 10 weeks of diet were perform ed. 
- The section where the STHD -ß1 diet is introduced contains the wrong reference number. The 
authors should review the assignment of the reference numbers to the corresponding 
references. Furthermore, the same reference is used in three consecutive sent ences. This 
should be avoided.  
- Why were feces obtained at week 16 and not at the end of the study?      
- In the section about statistical analysis it is stated that students t -test or Mann -Whitney U -test 
was used. However, I could not found if students t-test was actually used in one of the 
comparisons. This should be reviewed.  
- There is a discrepancy in the methods. In the methods section it is written that mice were fed 
for 10 weeks and in the results section it is stated that they were fed 9 weeks.  
- In Figure 1, the abbreviations CAZ and MNZ should be spelled in full in the figure legends.  
- Figure 4 is not arranged correctly. The graph about Tnf mRNA should be Figure 4D as stated 
in the text.  
- In the legends to Figure 4 a significance level of p<0. 01 is introduced even though it does not 
apply in this figure.  
 
Are the methods appropriate and well described?  
If not, pl ease specify what is required in your comments to the authors.  
Yes 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
Yes 
Are the conclusions drawn adequately suppo rted by the data shown?  
If not, please explain in your comments to the authors.  
No 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
I am able to assess the statistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Acceptable  
Declaration of competing interests  
Please c omplete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication o f this  
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently ap plying for any patents relating to the content of the  
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
5. Do you have any other f inancial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no  competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be  posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be  included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
Title: Gut microbiota -mediated generation of saturated fatty acids elicits inflammation in the 
liver in murine high -fat diet -induced steatohepatitis  
 
Version: 0 Date: 01 May 2017  
 
Reviewer: Robert Schierwagen  
 
Reviewer's report:  
The study performed by Yamada et al. describes the changes of the microbiota and lipid 
metabolism in an experimental model of NASH. Therefore, the authors used a recently reported 
steatohepatitis -inducing high fat diet. Additionally, the authors treated an other group of animals 
with antibiotics supplementary to the high fat diet. The main findings of this study are the 
observed changes saturated fatty acid and n -6 fatty acid pathways, as well as an increase of 
migratory macrophages in the liver. However, th e results are not fully convincing. Furthermore, 
the study design, especially the choice of the prophylactic antibiotic group and the drawn 
conclusions from the comparison with the high fat diet group is questionable.  
 
Major critics:  
- The authors correctly describe that all three observed groups had completely different 
microbiomes and metabolic profiles. However, afterwards the authors directly compare the 
high fat diet group to the group that additionally received antibiotics. These  comparisons are 
not suitable, since the treatment antibiotics lead to a complete different metabolic phenotype, 
which also has no similarities with the control group when it comes to the gut microbiome 
and the metabolic profile.  
- The observations made on  macrophages are not convincing. The shown representative FACS 
analyses do not resemble the quantification bar graphs next to them. In the representative 
FACS analyses it looks like there is also a decrease of resident and migratory macrophages in 
the +Abx  group. And even if macrophage numbers are not decreased by antibiotic treatment, 
how do the authors explain the significant lower levels of inflammatory markers compared to 
the NASH model?  
- The authors state several times that antibiotic treatment improv es STHD -01 induced effects. 
Since treatment with antibiotics has been started even before the start of the diet, this is a 
prophylactic approach and therefore this statement cannot be kept up. Also this statement is 
critical, since the phenotype of the Abx  group is so different. A therapeutic approach in 
which the antibiotic treatment starts after the development of the disease would be more 
favourable, since this better adapts to the clinical routine.  
- The results describing Figure 1C state that a signifi cant number of ballooning and Mallory 
bodies was observed. How was this assessed? Furthermore, the pathological findings 
(ballooning, Mallory bodies, fibrotic extensions and infiltrating inflammatory cells) should 
be marked in the shown representative stai nings. Especially, since the resolution and size of 
the images in the current manuscript could be improved. More favourable, specific stainings 
should be performed for key pathological observations.  
- What is the advantage of the STHD -01 model in comparis on to the established models like 
MCD diet with respect to the treatment time of 10 weeks?  
 
Minor critics:  
- In the introduction, it is stated that 10 million patients have NAFLD and 1 million patients 
have NASH. However, it is not specified to which coun try these numbers are related.  
- The section about HCC in the introduction should be removed, since HCC is not a part of this 
study. In the used dietary model HCC was identified after 36 weeks of diet, but for this study 
only 10 weeks of diet were perform ed. 
- The section where the STHD -ß1 diet is introduced contains the wrong reference number. The 
authors should review the assignment of the reference numbers to the corresponding 
references. Furthermore, the same reference is used in three consecutive sent ences. This 
should be avoided.  
- Why were feces obtained at week 16 and not at the end of the study?      
- In the section about statistical analysis it is stated that students t -test or Mann -Whitney U -test 
was used. However, I could not found if students t-test was actually used in one of the 
comparisons. This should be reviewed.  
- There is a discrepancy in the methods. In the methods section it is written that mice were fed 
for 10 weeks and in the results section it is stated that they were fed 9 weeks.  
- In Figure 1, the abbreviations CAZ and MNZ should be spelled in full in the figure legends.  
- Figure 4 is not arranged correctly. The graph about Tnf mRNA should be Figure 4D as stated 
in the text.  
- In the legends to Figure 4 a significance level of p<0. 01 is introduced even though it does not 
apply in this figure.  
 
Are the methods appropriate and well described?  
If not, pl ease specify what is required in your comments to the authors.  
Yes 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
Yes 
Are the conclusions drawn adequately suppo rted by the data shown?  
If not, please explain in your comments to the authors.  
No 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
I am able to assess the statistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Acceptable  
Declaration of competing interests  
Please c omplete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication o f this  
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently ap plying for any patents relating to the content of the  
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
5. Do you have any other f inancial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no  competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be  posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be  included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant