Reviewer’s report
Title: 
RIP3 knockdown inhibits necroptosis of human intestinal epithelial cells
via TLR4/MyD88/NF-κB signaling and ameliorates murine colitis
Version: 
0 
Date: 
11 Jan 2022
Reviewer's report:
Title: RIP3 knockdown inhibits necroptosis of intestinal epithelial cells in
ulcerative colitis via TLR4/MyD88/NF-κB signaling pathway
Summary
TNF signaling has been seen to be a viable drug target for inflammatory diseases
including Ulcerative Colitis. The authors are interested in a TNF receptor
activated kinase (RIP3, RIPK3) that promotes cell death. They first show that
UC patients with more severe disease have elevated levels of RIP3. They then
show that in intestinal epithelial cells treated with TNF have reduced cell
viability and RIP3 inhibition with GSK872 but not knockdown partially rescue
the pro-death effect of TNF. Necroptosis is also increased after TNF treatment
and is reduced by both GSK872 mediated inhibition and knockdown of RIPK3.
Using a similar paradigm of TNF treatment of intestinal epithelial cells in vitro
the authors shown that many of the downstream effects of TNF on cytokine
production, NFKB activation and ROS production are reduced after RIP3
knockdown or RIP3 inhibition (GSK872). The authors then give RIP3 mice
colitis and see differing results as it pertains to disease activity, "gross
morphological damage", and cytokine levels. Furthermore, RIP3 knockout mice
with colitis have similar levels of CD68 macrophages, decreased neutrophil
activation (MPO) and reduced NFKB activation compared to wild type mice
with colitis.
Major Comments
1. Figure 4/5: As the mechanism of action for SASP (Sulfasalazine) is not well
understood (PMID: 16114981) it is unclear why this inhibitor was used in this
study. Furthermore, the response of RIP3 KO (and NEC-1 treated) mice to DSS
already been shown PMID: 25367573, PMID: 32599618 & PMID: 28256024.
2. Figure 4: There is no correlation between DAI and CMDI for NEC-1, SASP
and KO mice. The data for aspects such as mouse weight, colon length, and
histology scores should be included in their own panels for Figure 4.
3. The number of samples in each group and the number of times experiments
were performed are missing. This could be stated in either the methods or
legends.
4. A graph and statistical significance are needed for figure 1B. Furthermore, it is
unclear how many patients were analyzed.
5. A graph and statistical significance are needed for figure 2E, 3D, 3E. 
6. Figure 5: ZO-1 signal is too faint to make any conclusions. Either a new stain
needs to be performed or the image needs to be magnified.
Minor Comments
1. Figure 4 C: In the methods CMDI "colon gross morphological damage index"
should be defined as to what it is comprised of.
2. Lines 66-67: It is unclear what "proliferation inhibitory effect" is referring to.
3. Line 176: it is unclear where the cells came from and what they are. Does
CTCC = ATCC? if so the catalog number doesn't match with any ATCC
products. If the catalog number is in fact "CTCC-001-0074" where were the cells
purchased from needs to be stated.
4. Line 207: A reference for the severity classification system should be added.
5. Line 220-221: The sequences for the GAPDH primers should be added and
the font is not the same as the rest of the manuscript.
6. Line 273-274: A reference for the knockout mice should be added.
7. As RIP3 functions in the cytosol to induce apoptosis and RIP3 is seen to be
nuclear in grade 3 patients this should be discussed in the discussion.
Publons Reviewer Recognition
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find the details of processing in Publons’ privacy policy
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Yes
Declaration of competing interests
Please complete 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 of 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 applying 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 financial 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
Reviewer Publication Consent.
 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
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the
editorial office before completing the review.
If you wish, you can include your
name in your published report. Please note you must decide whether to include
your name at the start of the process and confirm this decision whenever
prompted. If you change your decision later, we will not be able to publish your
name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
Is the study design appropriate to answer the research question (including the use
of appropriate controls), and are the conclusions supported by the evidence
presented?
No
Are the methods sufficiently described to allow the study to be repeated?
No
Is the use of statistics and treatment of uncertainties appropriate?
No
Is the presentation of the work clear?
Yes
Are the images in this manuscript (including electrophoretic gels and blots) free
from apparent manipulation?
Yes
Reviewer’s report
Title: 
RIP3 knockdown inhibits necroptosis of human intestinal epithelial cells
via TLR4/MyD88/NF-κB signaling and ameliorates murine colitis
Version: 
0 
Date: 
11 Jan 2022
Reviewer's report:
Title: RIP3 knockdown inhibits necroptosis of intestinal epithelial cells in
ulcerative colitis via TLR4/MyD88/NF-κB signaling pathway
Summary
TNF signaling has been seen to be a viable drug target for inflammatory diseases
including Ulcerative Colitis. The authors are interested in a TNF receptor
activated kinase (RIP3, RIPK3) that promotes cell death. They first show that
UC patients with more severe disease have elevated levels of RIP3. They then
show that in intestinal epithelial cells treated with TNF have reduced cell
viability and RIP3 inhibition with GSK872 but not knockdown partially rescue
the pro-death effect of TNF. Necroptosis is also increased after TNF treatment
and is reduced by both GSK872 mediated inhibition and knockdown of RIPK3.
Using a similar paradigm of TNF treatment of intestinal epithelial cells in vitro
the authors shown that many of the downstream effects of TNF on cytokine
production, NFKB activation and ROS production are reduced after RIP3
knockdown or RIP3 inhibition (GSK872). The authors then give RIP3 mice
colitis and see differing results as it pertains to disease activity, "gross
morphological damage", and cytokine levels. Furthermore, RIP3 knockout mice
with colitis have similar levels of CD68 macrophages, decreased neutrophil
activation (MPO) and reduced NFKB activation compared to wild type mice
with colitis.
Major Comments
1. Figure 4/5: As the mechanism of action for SASP (Sulfasalazine) is not well
understood (PMID: 16114981) it is unclear why this inhibitor was used in this
study. Furthermore, the response of RIP3 KO (and NEC-1 treated) mice to DSS
already been shown PMID: 25367573, PMID: 32599618 & PMID: 28256024.
2. Figure 4: There is no correlation between DAI and CMDI for NEC-1, SASP
and KO mice. The data for aspects such as mouse weight, colon length, and
histology scores should be included in their own panels for Figure 4.
3. The number of samples in each group and the number of times experiments
were performed are missing. This could be stated in either the methods or
legends.
4. A graph and statistical significance are needed for figure 1B. Furthermore, it is
unclear how many patients were analyzed.
5. A graph and statistical significance are needed for figure 2E, 3D, 3E. 
6. Figure 5: ZO-1 signal is too faint to make any conclusions. Either a new stain
needs to be performed or the image needs to be magnified.
Minor Comments
1. Figure 4 C: In the methods CMDI "colon gross morphological damage index"
should be defined as to what it is comprised of.
2. Lines 66-67: It is unclear what "proliferation inhibitory effect" is referring to.
3. Line 176: it is unclear where the cells came from and what they are. Does
CTCC = ATCC? if so the catalog number doesn't match with any ATCC
products. If the catalog number is in fact "CTCC-001-0074" where were the cells
purchased from needs to be stated.
4. Line 207: A reference for the severity classification system should be added.
5. Line 220-221: The sequences for the GAPDH primers should be added and
the font is not the same as the rest of the manuscript.
6. Line 273-274: A reference for the knockout mice should be added.
7. As RIP3 functions in the cytosol to induce apoptosis and RIP3 is seen to be
nuclear in grade 3 patients this should be discussed in the discussion.
Publons Reviewer Recognition
. Springer Nature can send verification of this
review directly to Publons (a subsidiary of Clarivate Analytics). If you would
like to take advantage of this service, please click on the “Yes” option below.
Your name, email address, title of the reviewed manuscript, name of the journal,
and date of your review submission (the “Review Data”) will then be transmitted
to Publons after the final decision on the manuscript has been made. If you have
already registered at Publons, they will notify you of the receipt of this review
and update your profile 
as per your settings and their policy. If you are not
registered with Publons, you will receive an email from them asking you to
register in order for them to be able to recognize your review on your new
profile page. Publons may use the Review Data to generate derivative metadata
for the benefit of Publons and you as a reviewer, carefully considering the
sensitivity of such information. For example, Publons may verify your record as
a reviewer by updating your profile published on its webservice if you have
registered for such service or help editors to identify candidate reviewers. Please
find the details of processing in Publons’ privacy policy
https://publons.com/about/terms
Yes
Declaration of competing interests
Please complete 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 of 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 applying 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 financial 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
Reviewer Publication Consent.
 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
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the
editorial office before completing the review.
If you wish, you can include your
name in your published report. Please note you must decide whether to include
your name at the start of the process and confirm this decision whenever
prompted. If you change your decision later, we will not be able to publish your
name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
Is the study design appropriate to answer the research question (including the use
of appropriate controls), and are the conclusions supported by the evidence
presented?
No
Are the methods sufficiently described to allow the study to be repeated?
No
Is the use of statistics and treatment of uncertainties appropriate?
No
Is the presentation of the work clear?
Yes
Are the images in this manuscript (including electrophoretic gels and blots) free
from apparent manipulation?
Yes
Please review this paper.
user
Reviewer’s report
Title: 
RIP3 knockdown inhibits necroptosis of human intestinal epithelial cells
via TLR4/MyD88/NF-κB signaling and ameliorates murine colitis
Version: 
0 
Date: 
11 Jan 2022
Reviewer's report:
Title: RIP3 knockdown inhibits necroptosis of intestinal epithelial cells in
ulcerative colitis via TLR4/MyD88/NF-κB signaling pathway
Summary
TNF signaling has been seen to be a viable drug target for inflammatory diseases
including Ulcerative Colitis. The authors are interested in a TNF receptor
activated kinase (RIP3, RIPK3) that promotes cell death. They first show that
UC patients with more severe disease have elevated levels of RIP3. They then
show that in intestinal epithelial cells treated with TNF have reduced cell
viability and RIP3 inhibition with GSK872 but not knockdown partially rescue
the pro-death effect of TNF. Necroptosis is also increased after TNF treatment
and is reduced by both GSK872 mediated inhibition and knockdown of RIPK3.
Using a similar paradigm of TNF treatment of intestinal epithelial cells in vitro
the authors shown that many of the downstream effects of TNF on cytokine
production, NFKB activation and ROS production are reduced after RIP3
knockdown or RIP3 inhibition (GSK872). The authors then give RIP3 mice
colitis and see differing results as it pertains to disease activity, "gross
morphological damage", and cytokine levels. Furthermore, RIP3 knockout mice
with colitis have similar levels of CD68 macrophages, decreased neutrophil
activation (MPO) and reduced NFKB activation compared to wild type mice
with colitis.
Major Comments
1. Figure 4/5: As the mechanism of action for SASP (Sulfasalazine) is not well
understood (PMID: 16114981) it is unclear why this inhibitor was used in this
study. Furthermore, the response of RIP3 KO (and NEC-1 treated) mice to DSS
already been shown PMID: 25367573, PMID: 32599618 & PMID: 28256024.
2. Figure 4: There is no correlation between DAI and CMDI for NEC-1, SASP
and KO mice. The data for aspects such as mouse weight, colon length, and
histology scores should be included in their own panels for Figure 4.
3. The number of samples in each group and the number of times experiments
were performed are missing. This could be stated in either the methods or
legends.
4. A graph and statistical significance are needed for figure 1B. Furthermore, it is
unclear how many patients were analyzed.
5. A graph and statistical significance are needed for figure 2E, 3D, 3E. 
6. Figure 5: ZO-1 signal is too faint to make any conclusions. Either a new stain
needs to be performed or the image needs to be magnified.
Minor Comments
1. Figure 4 C: In the methods CMDI "colon gross morphological damage index"
should be defined as to what it is comprised of.
2. Lines 66-67: It is unclear what "proliferation inhibitory effect" is referring to.
3. Line 176: it is unclear where the cells came from and what they are. Does
CTCC = ATCC? if so the catalog number doesn't match with any ATCC
products. If the catalog number is in fact "CTCC-001-0074" where were the cells
purchased from needs to be stated.
4. Line 207: A reference for the severity classification system should be added.
5. Line 220-221: The sequences for the GAPDH primers should be added and
the font is not the same as the rest of the manuscript.
6. Line 273-274: A reference for the knockout mice should be added.
7. As RIP3 functions in the cytosol to induce apoptosis and RIP3 is seen to be
nuclear in grade 3 patients this should be discussed in the discussion.
Publons Reviewer Recognition
. Springer Nature can send verification of this
review directly to Publons (a subsidiary of Clarivate Analytics). If you would
like to take advantage of this service, please click on the “Yes” option below.
Your name, email address, title of the reviewed manuscript, name of the journal,
and date of your review submission (the “Review Data”) will then be transmitted
to Publons after the final decision on the manuscript has been made. If you have
already registered at Publons, they will notify you of the receipt of this review
and update your profile 
as per your settings and their policy. If you are not
registered with Publons, you will receive an email from them asking you to
register in order for them to be able to recognize your review on your new
profile page. Publons may use the Review Data to generate derivative metadata
for the benefit of Publons and you as a reviewer, carefully considering the
sensitivity of such information. For example, Publons may verify your record as
a reviewer by updating your profile published on its webservice if you have
registered for such service or help editors to identify candidate reviewers. Please
find the details of processing in Publons’ privacy policy
https://publons.com/about/terms
Yes
Declaration of competing interests
Please complete 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 of 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 applying 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 financial 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
Reviewer Publication Consent.
 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
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the
editorial office before completing the review.
If you wish, you can include your
name in your published report. Please note you must decide whether to include
your name at the start of the process and confirm this decision whenever
prompted. If you change your decision later, we will not be able to publish your
name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
Is the study design appropriate to answer the research question (including the use
of appropriate controls), and are the conclusions supported by the evidence
presented?
No
Are the methods sufficiently described to allow the study to be repeated?
No
Is the use of statistics and treatment of uncertainties appropriate?
No
Is the presentation of the work clear?
Yes
Are the images in this manuscript (including electrophoretic gels and blots) free
from apparent manipulation?
Yes
assistant