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: 
10 Jan 2022
Reviewer's report:
Please include all comments for the authors in this box rather than uploading
your report as an attachment. Please only upload as attachments annotated
versions of manuscripts, graphs, supporting materials or other aspects of your
report which cannot be included in a text format.
Please overwrite this text when adding your comments to the authors.
In this study, Duan et al use a combination of in vitro and in vivo experiments to
explore inhibition of RIP3, a downstream target of TNF-a, in human intestinal
epithelial cells and DSS murine colitis model. The authors show that RIP3
protein levels were increased and correlated with histologic inflammation in
patients with ulcerative colitis. The authors then inhibited RIP3 (via inhibitor
GSK872 or knockdown plasmid) in human intestinal epithelial cells in vitro
which resulted in reduction in TNF-a induced apoptosis and cell proliferation
inhibition as well as reduction in proinflammatory cytokines (IL-16, IL-17, IFN-
g) and reactive oxygen species (ROS) production. Finally, the authors showed
that the RIP3 inhibitor (Nec-1) can improve disease activity and histologic
inflammation in murine DSS colitis through various downstream mechanisms:
1) promoting tight junction ZO-1 expression 2) increasing proportion of T
regulatory cells 3) inhibiting TLR4/MyD88/NF-
κ
B signaling pathway, ROS
generation, and proinflammatory cytokine secretion. This is a interesting, novel,
and impactful study that provides further evidence of the role of necroptosis in
intestinal inflammation and suggest that RIP3 inhibition may be a therapeutic
target in inflammatory bowel disease. I have the following critiques and
recommendations:
1. Title: "RIP3 knockdown inhibits necroptosis of intestinal epithelial cells in
ulcerative colitis via TLR4/MyD88/NF-
κ
B signaling pathway." The title should
be more precise to reflect the actual findings. Consider "RIP3 knockdown
inhibits necroptosis of human intestinal epithelial cells via TLR4/MyD88/NF-
κ
B
signaling and ameliorates murine colitis."
2. Methods/Results: "Subsequently, we collected clinical colon tissues from
patients with mild, moderate, and severe UC based on the modified
Truelove&Witts severity classification." The description of patient recruitment
and clinical samples is extremely lacking. How many ulcerative colitis patients
and colon biopsies samples were included in this study? Furthermore, the authors
should include a Table 1 of clinical characteristics of included ulcerative colitis
patients (including age, sex, ethnicity, ulcerative colitis extent by Montreal
classification (E1, E2, E3) and medications (mesalamine, sulfasalazine, anti-
TNF biologics, etc). The authors should clarify which modified Truelove&Witts
scores corresponded to mild, moderate, or severe. 
3. Results: Figure 1B should clarify why there are 2 sets of mild (I), moderate
(II), and severe (II).
4. Results: "The expression of RIP3 in human colon is directly proportional to
the severity of UC." The authors should provide a more quantitative analysis to
show correlation between RIP3/MLKL protein expression with ulcerative colitis
severity (Truelove&Witts severity). I suggest they perform a linear regression of
RIP3/MLKL protein expression versus Truelove&Witts severity scores and
calculate Pearson/Spearman coefficients to show effect size and statistical
significance of proposed correlations. 
5. Methods/Discussion: The authors should clarify why they chose the specific
RIP3 inhibitors (GSK872 and Nec-1) in this study as there are other known RIP3
inhibitors. 
6. Results/Discussion: As a positive control, the authors treated DSS colitis mice
with sulfasalazine (SASP), a known treatment for human ulcerative colitis. Did
the sulfasalazine treated DSS mice group have any reductions in protein
expression of RIP3? It is important to report and discuss whether known
treatments for ulcerative colitis may already exert their anti-inflammatory effects
through reduction in colonic RIP3 levels. 
7. Discussion: The RIP3 inhibition pathway in this study only applies to colonic
inflammation as the authors did not use a small bowel (enteritis) model. The
authors should acknowledge this limitation and need for further studies as Paneth
cells (which express RIP3) are mostly expressed in the small bowel and it is
unclear if this pathway applies in patients with Crohn's disease.
8. Discussion: Vitamin D and Vitamin D receptor (VDR) signaling has been
shown to have immunoprotective effects on inflammatory bowel disease
(Gubatan, J. and Moss, A.C., 2018. Vitamin D in inflammatory bowel disease:
more than just a supplement. Current opinion in gastroenterology, 34(4), pp.217-
225). Interestingly, the VDR signaling pathway has been previously shown to
suppress necroptosis in intestinal epithelial cells by binding RIPK1/3 necrosomes
(Shi, Y., Cui, X., Sun, Y., Zhao, Q. and Liu, T., 2020. Intestinal vitamin D
receptor signaling ameliorates dextran sulfate sodium
‐
induced colitis by
suppressing necroptosis of intestinal epithelial cells. The FASEB Journal, 34(10),
pp.13494-13506.) The authors should acknowledge in their discussion that
endogenous ligands/signaling such as vitamin D/VDR may also inhibit RIP3-
mediated necroptosis. 
9. Discussion: Finally, the authors should briefly discuss the current therapeutic
landscape of known RIP3 pathway inhibitors in autoimmune diseases including
inflammatory bowel disease. Have there been any clinical trials inhibiting
necroptosis and targeting RIP3 pathways? Have RIP3 and related pathways been
previously targeted in terms of drug development?
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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
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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.
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I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
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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?
Yes
Are the methods sufficiently described to allow the study to be repeated?
Yes
Is the use of statistics and treatment of uncertainties appropriate?
Yes
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: 
10 Jan 2022
Reviewer's report:
Please include all comments for the authors in this box rather than uploading
your report as an attachment. Please only upload as attachments annotated
versions of manuscripts, graphs, supporting materials or other aspects of your
report which cannot be included in a text format.
Please overwrite this text when adding your comments to the authors.
In this study, Duan et al use a combination of in vitro and in vivo experiments to
explore inhibition of RIP3, a downstream target of TNF-a, in human intestinal
epithelial cells and DSS murine colitis model. The authors show that RIP3
protein levels were increased and correlated with histologic inflammation in
patients with ulcerative colitis. The authors then inhibited RIP3 (via inhibitor
GSK872 or knockdown plasmid) in human intestinal epithelial cells in vitro
which resulted in reduction in TNF-a induced apoptosis and cell proliferation
inhibition as well as reduction in proinflammatory cytokines (IL-16, IL-17, IFN-
g) and reactive oxygen species (ROS) production. Finally, the authors showed
that the RIP3 inhibitor (Nec-1) can improve disease activity and histologic
inflammation in murine DSS colitis through various downstream mechanisms:
1) promoting tight junction ZO-1 expression 2) increasing proportion of T
regulatory cells 3) inhibiting TLR4/MyD88/NF-
κ
B signaling pathway, ROS
generation, and proinflammatory cytokine secretion. This is a interesting, novel,
and impactful study that provides further evidence of the role of necroptosis in
intestinal inflammation and suggest that RIP3 inhibition may be a therapeutic
target in inflammatory bowel disease. I have the following critiques and
recommendations:
1. Title: "RIP3 knockdown inhibits necroptosis of intestinal epithelial cells in
ulcerative colitis via TLR4/MyD88/NF-
κ
B signaling pathway." The title should
be more precise to reflect the actual findings. Consider "RIP3 knockdown
inhibits necroptosis of human intestinal epithelial cells via TLR4/MyD88/NF-
κ
B
signaling and ameliorates murine colitis."
2. Methods/Results: "Subsequently, we collected clinical colon tissues from
patients with mild, moderate, and severe UC based on the modified
Truelove&Witts severity classification." The description of patient recruitment
and clinical samples is extremely lacking. How many ulcerative colitis patients
and colon biopsies samples were included in this study? Furthermore, the authors
should include a Table 1 of clinical characteristics of included ulcerative colitis
patients (including age, sex, ethnicity, ulcerative colitis extent by Montreal
classification (E1, E2, E3) and medications (mesalamine, sulfasalazine, anti-
TNF biologics, etc). The authors should clarify which modified Truelove&Witts
scores corresponded to mild, moderate, or severe. 
3. Results: Figure 1B should clarify why there are 2 sets of mild (I), moderate
(II), and severe (II).
4. Results: "The expression of RIP3 in human colon is directly proportional to
the severity of UC." The authors should provide a more quantitative analysis to
show correlation between RIP3/MLKL protein expression with ulcerative colitis
severity (Truelove&Witts severity). I suggest they perform a linear regression of
RIP3/MLKL protein expression versus Truelove&Witts severity scores and
calculate Pearson/Spearman coefficients to show effect size and statistical
significance of proposed correlations. 
5. Methods/Discussion: The authors should clarify why they chose the specific
RIP3 inhibitors (GSK872 and Nec-1) in this study as there are other known RIP3
inhibitors. 
6. Results/Discussion: As a positive control, the authors treated DSS colitis mice
with sulfasalazine (SASP), a known treatment for human ulcerative colitis. Did
the sulfasalazine treated DSS mice group have any reductions in protein
expression of RIP3? It is important to report and discuss whether known
treatments for ulcerative colitis may already exert their anti-inflammatory effects
through reduction in colonic RIP3 levels. 
7. Discussion: The RIP3 inhibition pathway in this study only applies to colonic
inflammation as the authors did not use a small bowel (enteritis) model. The
authors should acknowledge this limitation and need for further studies as Paneth
cells (which express RIP3) are mostly expressed in the small bowel and it is
unclear if this pathway applies in patients with Crohn's disease.
8. Discussion: Vitamin D and Vitamin D receptor (VDR) signaling has been
shown to have immunoprotective effects on inflammatory bowel disease
(Gubatan, J. and Moss, A.C., 2018. Vitamin D in inflammatory bowel disease:
more than just a supplement. Current opinion in gastroenterology, 34(4), pp.217-
225). Interestingly, the VDR signaling pathway has been previously shown to
suppress necroptosis in intestinal epithelial cells by binding RIPK1/3 necrosomes
(Shi, Y., Cui, X., Sun, Y., Zhao, Q. and Liu, T., 2020. Intestinal vitamin D
receptor signaling ameliorates dextran sulfate sodium
‐
induced colitis by
suppressing necroptosis of intestinal epithelial cells. The FASEB Journal, 34(10),
pp.13494-13506.) The authors should acknowledge in their discussion that
endogenous ligands/signaling such as vitamin D/VDR may also inhibit RIP3-
mediated necroptosis. 
9. Discussion: Finally, the authors should briefly discuss the current therapeutic
landscape of known RIP3 pathway inhibitors in autoimmune diseases including
inflammatory bowel disease. Have there been any clinical trials inhibiting
necroptosis and targeting RIP3 pathways? Have RIP3 and related pathways been
previously targeted in terms of drug development?
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?
Yes
Are the methods sufficiently described to allow the study to be repeated?
Yes
Is the use of statistics and treatment of uncertainties appropriate?
Yes
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: 
10 Jan 2022
Reviewer's report:
Please include all comments for the authors in this box rather than uploading
your report as an attachment. Please only upload as attachments annotated
versions of manuscripts, graphs, supporting materials or other aspects of your
report which cannot be included in a text format.
Please overwrite this text when adding your comments to the authors.
In this study, Duan et al use a combination of in vitro and in vivo experiments to
explore inhibition of RIP3, a downstream target of TNF-a, in human intestinal
epithelial cells and DSS murine colitis model. The authors show that RIP3
protein levels were increased and correlated with histologic inflammation in
patients with ulcerative colitis. The authors then inhibited RIP3 (via inhibitor
GSK872 or knockdown plasmid) in human intestinal epithelial cells in vitro
which resulted in reduction in TNF-a induced apoptosis and cell proliferation
inhibition as well as reduction in proinflammatory cytokines (IL-16, IL-17, IFN-
g) and reactive oxygen species (ROS) production. Finally, the authors showed
that the RIP3 inhibitor (Nec-1) can improve disease activity and histologic
inflammation in murine DSS colitis through various downstream mechanisms:
1) promoting tight junction ZO-1 expression 2) increasing proportion of T
regulatory cells 3) inhibiting TLR4/MyD88/NF-
κ
B signaling pathway, ROS
generation, and proinflammatory cytokine secretion. This is a interesting, novel,
and impactful study that provides further evidence of the role of necroptosis in
intestinal inflammation and suggest that RIP3 inhibition may be a therapeutic
target in inflammatory bowel disease. I have the following critiques and
recommendations:
1. Title: "RIP3 knockdown inhibits necroptosis of intestinal epithelial cells in
ulcerative colitis via TLR4/MyD88/NF-
κ
B signaling pathway." The title should
be more precise to reflect the actual findings. Consider "RIP3 knockdown
inhibits necroptosis of human intestinal epithelial cells via TLR4/MyD88/NF-
κ
B
signaling and ameliorates murine colitis."
2. Methods/Results: "Subsequently, we collected clinical colon tissues from
patients with mild, moderate, and severe UC based on the modified
Truelove&Witts severity classification." The description of patient recruitment
and clinical samples is extremely lacking. How many ulcerative colitis patients
and colon biopsies samples were included in this study? Furthermore, the authors
should include a Table 1 of clinical characteristics of included ulcerative colitis
patients (including age, sex, ethnicity, ulcerative colitis extent by Montreal
classification (E1, E2, E3) and medications (mesalamine, sulfasalazine, anti-
TNF biologics, etc). The authors should clarify which modified Truelove&Witts
scores corresponded to mild, moderate, or severe. 
3. Results: Figure 1B should clarify why there are 2 sets of mild (I), moderate
(II), and severe (II).
4. Results: "The expression of RIP3 in human colon is directly proportional to
the severity of UC." The authors should provide a more quantitative analysis to
show correlation between RIP3/MLKL protein expression with ulcerative colitis
severity (Truelove&Witts severity). I suggest they perform a linear regression of
RIP3/MLKL protein expression versus Truelove&Witts severity scores and
calculate Pearson/Spearman coefficients to show effect size and statistical
significance of proposed correlations. 
5. Methods/Discussion: The authors should clarify why they chose the specific
RIP3 inhibitors (GSK872 and Nec-1) in this study as there are other known RIP3
inhibitors. 
6. Results/Discussion: As a positive control, the authors treated DSS colitis mice
with sulfasalazine (SASP), a known treatment for human ulcerative colitis. Did
the sulfasalazine treated DSS mice group have any reductions in protein
expression of RIP3? It is important to report and discuss whether known
treatments for ulcerative colitis may already exert their anti-inflammatory effects
through reduction in colonic RIP3 levels. 
7. Discussion: The RIP3 inhibition pathway in this study only applies to colonic
inflammation as the authors did not use a small bowel (enteritis) model. The
authors should acknowledge this limitation and need for further studies as Paneth
cells (which express RIP3) are mostly expressed in the small bowel and it is
unclear if this pathway applies in patients with Crohn's disease.
8. Discussion: Vitamin D and Vitamin D receptor (VDR) signaling has been
shown to have immunoprotective effects on inflammatory bowel disease
(Gubatan, J. and Moss, A.C., 2018. Vitamin D in inflammatory bowel disease:
more than just a supplement. Current opinion in gastroenterology, 34(4), pp.217-
225). Interestingly, the VDR signaling pathway has been previously shown to
suppress necroptosis in intestinal epithelial cells by binding RIPK1/3 necrosomes
(Shi, Y., Cui, X., Sun, Y., Zhao, Q. and Liu, T., 2020. Intestinal vitamin D
receptor signaling ameliorates dextran sulfate sodium
‐
induced colitis by
suppressing necroptosis of intestinal epithelial cells. The FASEB Journal, 34(10),
pp.13494-13506.) The authors should acknowledge in their discussion that
endogenous ligands/signaling such as vitamin D/VDR may also inhibit RIP3-
mediated necroptosis. 
9. Discussion: Finally, the authors should briefly discuss the current therapeutic
landscape of known RIP3 pathway inhibitors in autoimmune diseases including
inflammatory bowel disease. Have there been any clinical trials inhibiting
necroptosis and targeting RIP3 pathways? Have RIP3 and related pathways been
previously targeted in terms of drug development?
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?
Yes
Are the methods sufficiently described to allow the study to be repeated?
Yes
Is the use of statistics and treatment of uncertainties appropriate?
Yes
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