Review for 10.1186/s12876-022-02208-x (2022)

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:
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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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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
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Yes
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Yes
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from apparent manipulation?
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