Reviewer's report
Title:Toll-like receptor-2 deficiency enhances non-alcoholic steatohepatitis
Version: 1Date:17 December 2009
Reviewer: Pranoti Mandrekar
Reviewer's report:
Comments to Reviewers
This manuscript attempts to describe the protective role and significance of
toll-like receptor 2 (TLR2) and saturated fat in the pathogenesis of non-alcoholic
steatohepatitis (NASH). The authors show that mice fed with methionine-choline
deficient diet (MCDD) enriched with 34% of calories from polyunsaturated fat
(PUFA) derived from corn oil resulted in macrovesicular steatosis and diffuse
leukocyte infiltration. However, mice administered methionine-choline deficient
diet enriched with 34% of calories from saturated fat (SAFA) derived from
coconut oil demonstrated less steatosis, inflammation and necrosis as compared
to PUFA-fed animals. Furthermore, hepatocellular injury was significantly higher
when toll-like receptor 2 (TLR2) knockout mice were fed the MCDD diet enriched
with PUFA or SAFA compared to wild-type controls fed the same diet. These
findings suggest that saturated fat and TLR2 likely plays a protective role in
NASH. The studies performed here are well designed, clearly written and to
determine the role of TLRs in NASH is physiological relevant and further
supports the role for innate immune responses in NASH. However, the lack of
elucidation of protective intracellular mechanisms in the liver by saturated fat or
TLR2 dampens the enthusiasm of this study. Further, the relevance of studies
presented here on the role of saturated fat and TLR2 together is not clear. <ore
specific concerns are as follows:
Major revisions:
1) The authors show throughout the studies the effect MCD diet enriched with
PUFA or SAFA on wild-type or TLR2 KO mice. Previous studies (Szabo et. al.)
have shown deleterious effects of MCD diet alone on TLR2 KO mice. These
controls must be shown in these studies to compare the effects of PUFA and
SAFA on TLR2 KO mice.
2) Figure 2 shows decreased TLR4 and CD14 mRNA expression in total livers of
MCD-SAFA fed mice. What is the cell type in the liver involved? Does SAFA
have an effect on macrophages or hepatocytes? In vitro studies of exposure of
macrophages cells to SAFA may further support the in vivo data.
3) In figure 4 the authors show that TLR2 protein expression in total livers is
decreased in MCD-PUFA fed mice but is still high in MCD-SAFA fed mice. Were
the TLR2 mRNA levels affected as well?
4) The authors show that proinflammatory responses such as TNF# production
and expression of innate immune receptors TLR4 and CD14 mRNA levels are
reduced in MCD-SAFA fed mice while TNF#, TLR4 mRNA and CD14mRNA are
all increased in TLR2 KO mice compared to wild-type contributing to liver injury.
The lack of protective mechanisms such as levels of anti-inflammatory cytokines
for instance IL-10 or adiponectin should be performed to clearly understand the
role of TLR2. The authors show increased expression of TLR4 and CD14 in
TLR2KO mice. Does this up-regulation occur in TLR2 KO mice or is this an effect
of the MCD diet?
5) Finally the authors show increased collagen in TLR2 KO mice fed with
MCD-PUFA and MCD-SAFA diet. Was fibrosis observed in histological sections?
This is not clear in the histological data provided. Also how does absence of
TLR2 enhance collagen expression leading to fibrosis in NASH must be further
elucidated.
Minor revisions:
1) The composition of the MCD diet enriched with PUFA or SAFA must be
described clearly in the Methods section.
2) The scoring system used to provide histological score must be detailed in the
Methods. Was any computer software used?
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare I have no competing interests.
Reviewer's report
Title:Toll-like receptor-2 deficiency enhances non-alcoholic steatohepatitis
Version: 1Date:17 December 2009
Reviewer: Pranoti Mandrekar
Reviewer's report:
Comments to Reviewers
This manuscript attempts to describe the protective role and significance of
toll-like receptor 2 (TLR2) and saturated fat in the pathogenesis of non-alcoholic
steatohepatitis (NASH). The authors show that mice fed with methionine-choline
deficient diet (MCDD) enriched with 34% of calories from polyunsaturated fat
(PUFA) derived from corn oil resulted in macrovesicular steatosis and diffuse
leukocyte infiltration. However, mice administered methionine-choline deficient
diet enriched with 34% of calories from saturated fat (SAFA) derived from
coconut oil demonstrated less steatosis, inflammation and necrosis as compared
to PUFA-fed animals. Furthermore, hepatocellular injury was significantly higher
when toll-like receptor 2 (TLR2) knockout mice were fed the MCDD diet enriched
with PUFA or SAFA compared to wild-type controls fed the same diet. These
findings suggest that saturated fat and TLR2 likely plays a protective role in
NASH. The studies performed here are well designed, clearly written and to
determine the role of TLRs in NASH is physiological relevant and further
supports the role for innate immune responses in NASH. However, the lack of
elucidation of protective intracellular mechanisms in the liver by saturated fat or
TLR2 dampens the enthusiasm of this study. Further, the relevance of studies
presented here on the role of saturated fat and TLR2 together is not clear. <ore
specific concerns are as follows:
Major revisions:
1) The authors show throughout the studies the effect MCD diet enriched with
PUFA or SAFA on wild-type or TLR2 KO mice. Previous studies (Szabo et. al.)
have shown deleterious effects of MCD diet alone on TLR2 KO mice. These
controls must be shown in these studies to compare the effects of PUFA and
SAFA on TLR2 KO mice.
2) Figure 2 shows decreased TLR4 and CD14 mRNA expression in total livers of
MCD-SAFA fed mice. What is the cell type in the liver involved? Does SAFA
have an effect on macrophages or hepatocytes? In vitro studies of exposure of
macrophages cells to SAFA may further support the in vivo data.
3) In figure 4 the authors show that TLR2 protein expression in total livers is
decreased in MCD-PUFA fed mice but is still high in MCD-SAFA fed mice. Were
the TLR2 mRNA levels affected as well?
4) The authors show that proinflammatory responses such as TNF# production
and expression of innate immune receptors TLR4 and CD14 mRNA levels are
reduced in MCD-SAFA fed mice while TNF#, TLR4 mRNA and CD14mRNA are
all increased in TLR2 KO mice compared to wild-type contributing to liver injury.
The lack of protective mechanisms such as levels of anti-inflammatory cytokines
for instance IL-10 or adiponectin should be performed to clearly understand the
role of TLR2. The authors show increased expression of TLR4 and CD14 in
TLR2KO mice. Does this up-regulation occur in TLR2 KO mice or is this an effect
of the MCD diet?
5) Finally the authors show increased collagen in TLR2 KO mice fed with
MCD-PUFA and MCD-SAFA diet. Was fibrosis observed in histological sections?
This is not clear in the histological data provided. Also how does absence of
TLR2 enhance collagen expression leading to fibrosis in NASH must be further
elucidated.
Minor revisions:
1) The composition of the MCD diet enriched with PUFA or SAFA must be
described clearly in the Methods section.
2) The scoring system used to provide histological score must be detailed in the
Methods. Was any computer software used?
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Toll-like receptor-2 deficiency enhances non-alcoholic steatohepatitis
Version: 1Date:17 December 2009
Reviewer: Pranoti Mandrekar
Reviewer's report:
Comments to Reviewers
This manuscript attempts to describe the protective role and significance of
toll-like receptor 2 (TLR2) and saturated fat in the pathogenesis of non-alcoholic
steatohepatitis (NASH). The authors show that mice fed with methionine-choline
deficient diet (MCDD) enriched with 34% of calories from polyunsaturated fat
(PUFA) derived from corn oil resulted in macrovesicular steatosis and diffuse
leukocyte infiltration. However, mice administered methionine-choline deficient
diet enriched with 34% of calories from saturated fat (SAFA) derived from
coconut oil demonstrated less steatosis, inflammation and necrosis as compared
to PUFA-fed animals. Furthermore, hepatocellular injury was significantly higher
when toll-like receptor 2 (TLR2) knockout mice were fed the MCDD diet enriched
with PUFA or SAFA compared to wild-type controls fed the same diet. These
findings suggest that saturated fat and TLR2 likely plays a protective role in
NASH. The studies performed here are well designed, clearly written and to
determine the role of TLRs in NASH is physiological relevant and further
supports the role for innate immune responses in NASH. However, the lack of
elucidation of protective intracellular mechanisms in the liver by saturated fat or
TLR2 dampens the enthusiasm of this study. Further, the relevance of studies
presented here on the role of saturated fat and TLR2 together is not clear. <ore
specific concerns are as follows:
Major revisions:
1) The authors show throughout the studies the effect MCD diet enriched with
PUFA or SAFA on wild-type or TLR2 KO mice. Previous studies (Szabo et. al.)
have shown deleterious effects of MCD diet alone on TLR2 KO mice. These
controls must be shown in these studies to compare the effects of PUFA and
SAFA on TLR2 KO mice.
2) Figure 2 shows decreased TLR4 and CD14 mRNA expression in total livers of
MCD-SAFA fed mice. What is the cell type in the liver involved? Does SAFA
have an effect on macrophages or hepatocytes? In vitro studies of exposure of
macrophages cells to SAFA may further support the in vivo data.
3) In figure 4 the authors show that TLR2 protein expression in total livers is
decreased in MCD-PUFA fed mice but is still high in MCD-SAFA fed mice. Were
the TLR2 mRNA levels affected as well?
4) The authors show that proinflammatory responses such as TNF# production
and expression of innate immune receptors TLR4 and CD14 mRNA levels are
reduced in MCD-SAFA fed mice while TNF#, TLR4 mRNA and CD14mRNA are
all increased in TLR2 KO mice compared to wild-type contributing to liver injury.
The lack of protective mechanisms such as levels of anti-inflammatory cytokines
for instance IL-10 or adiponectin should be performed to clearly understand the
role of TLR2. The authors show increased expression of TLR4 and CD14 in
TLR2KO mice. Does this up-regulation occur in TLR2 KO mice or is this an effect
of the MCD diet?
5) Finally the authors show increased collagen in TLR2 KO mice fed with
MCD-PUFA and MCD-SAFA diet. Was fibrosis observed in histological sections?
This is not clear in the histological data provided. Also how does absence of
TLR2 enhance collagen expression leading to fibrosis in NASH must be further
elucidated.
Minor revisions:
1) The composition of the MCD diet enriched with PUFA or SAFA must be
described clearly in the Methods section.
2) The scoring system used to provide histological score must be detailed in the
Methods. Was any computer software used?
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare I have no competing interests.
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