Previous Reviewers Comments:
Re: JACI-D-13-01373 "Human rhinovirus infection causes different DNA
methylation changes in nasal epithelial cells from healthy and asthmatic
subjects"
Dear Dr. McErlean:
Your manuscript, referenced above, has been reviewed. On the basis of the
recommendations of the Reviewers and the Editorial Office review, it has not
been accepted for publication. We asked the Reviewers to consider the quality of
the study design, the novelty of the conclusions, and the relevance of the
manuscript to the field of allergy and immunology. Both their confidential
comments to the Editors and their comments to the authors were taken into
account, along with the Journal's editorial policies and the current needs of the
JACI and its readers.
The significant increase in the number of manuscripts submitted to the Journal
has resulted in intense competition for available space. Currently, the Journal
accepts less than 20% of the manuscripts received. Editors and Reviewers have
therefore had to raise the bar in defining the criteria for acceptability for
publication. As a result, many well-conducted studies and interesting reports
that would previously have been acceptable to the JACI must now, regrettably, be
declined.
Please note that this decision means that this manuscript can no longer be
considered for publication in this journal, even in revised form.
COMMENTS FROM REVIEWER #1:
The objective of this study was to investigate whether epigenetic mechanisms
could be involved in virus-induced asthma exacerbations. The authors profiled
DNA methylation in asthmatic and healthy nasal epithelial cells during HRV
infection in vitro. The main findings were evidence of specific methylation
patterns associated with asthma and with HRV infection. There were a few
pathways that were different in HRV infection from the asthma vs. normal group.
Data relating changes in methylation of SNORA12 to RV infection and lung
function were of interest.
Major Comments
1. The characteristics of the study populations need to be described in the text.
What is the difference between population 1 and population 2? Were they
recruited at different times? Were the same criteria used for both? Differences in
the asthma vs. non-asthma populations should be noted. The ethnicity of the
asthma and non-asthma subjects in "Population 1" is quite different, which could
have influenced the results. It would be hard to separate differences in asthma
from differences in ethnicity.
2. Figures 1 and 2 seem to present discordant information. In Figure 1, the RVrelated changes in methylation were only present in the asthma group, while in
Figure 2, the PCA showed that RV caused changes in methylation patterns in the
normal cells but not in the asthma cells. Please explain this apparent
discrepancy.
3. For the methylation analysis of individual genes, 3 normal and 6 asthma
subjects were used. Were these from population one or two? Three seems like a
low number of subjects for this analysis, in which 500 loci were considered. The
low number of subjects may be the reason that "the presence of virus infection
did not influence either the 208 loci or patterns of methylation characterizing
Healthy and Asthma NECs"---perhaps the power of the analysis was too low
(type I statistical error).
4. The meaning of the following sentence was not clear to me (lines 210-211):
"Ranking of negative loadings of PC2 and PC4 identified unique loci whose
methylation changed after HRV infection in NECs from healthy and asthmatic
subjects respectively". Please explain in the text "ranking of negative loading".
5. One major limitation of the study is that effects of atopy and asthma cannot be
separated. This is acknowledged by the authors, but it limits interpretation of the
findings.
Minor Comments
1. Please briefly describe in the Methods section how the nasal epithelial cells
were obtained.
2. FEV1/FVC ratio in the table would be best represented as the calculated
number instead of percent predicted.
COMMENTS FROM REVIEWER #2:
In this study, primary nasal epithelial cells from asthmatics and controls were
studied for global and loci specific methylation pattern and its change upon
HRV16 infection.
My Comments
1. Exclusion criteria on the recruitment to the study should be stated. Were all of
the study participants non-smokers, free of asthma exacerbation and/or upper
respiratory infection within 4-6 weeks period preceding nasal epithelium
sampling? Demographic data (Table 1) or Methods should categorize asthma
severity, presence of allergic rhinitis/rhinosinusitis.
2. A difference in the global Alu repeats methylation between asthmatics and
controls was very small---i.e., 0.5%. What exactly was the method used to
discriminate between methylated and unmethylated genomic Alu templates. Did
the authors use parallel sequencing (ref. 20 use is inappropriate---the method is
cited in the referenced paper as ref. 24 [Xie et al. Nucleic Acid Res. 2009;
37:3371]), or methylation-specific PCR was used, or a microarray suggested in
the Discussion, line 226. Many methods have standard deviations as much as 2%
(1.6% for Alu pyrosequencing).
3. Percentage of 5mC found in NEC from this study (44%) is very different from
the one reported by Baccarelli (24%). The main study difference is non-cultured
nasal brushing vs. cultured one. If in vitro culture can increase global
methylation, comparable primary culture time should be stated.
4. Were experimental HRV16 infections compared for similar efficiency in
asthmatics and controls? This might be easily done by qRT-PCR from cell lysates.
5. Results: Please put into the table a list of top-10 genes discriminative between
asthmatics and controls; Fig. 2 B and C require indexes at abscissa to identify
paired samples (e.g., M1-RV1).
6. Lines 208-207: "Remarkably however, the presence of virus infection did not
influence either the loci or patterns of methylation..."---this does not make sense,
because these loci were picked up by PCA as having constant loading on casescontrols despite HRV infection.
7. A promising correlation between SNORA12 expression and FEV1% is in
contrast with lack of any impact of the gene methylation. This discrepancy
should be adequately discussed. What is known about regulatory elements of
this gene serving the source for RNA guide during uridine to pseudoridine
isomerisation?
COMMENTS FROM REVIEWER #3:
This manuscript details a study looking at DNA methylation status in nasal
epithelial cells from healthy and asthmatic subjects before and after HRV
infection (in vitro).
General Comments
The authors document rather modest effects on DNA methylation with HRV
inoculation, and really only in cells from asthmatic subjects. However, in
principal component analysis there appears to be more of difference in the
normal subjects than in the asthmatics. These differences raise concerns over the
reproducibility of these data. Further, the lack of any real mechanistic insight
greatly lessens enthusiasm for this report.
Specific Comments
1. For the HRV infection experiments, it would make sense to use a TLR agonist
(such as UV inactivated HRV) to determine if the changes seen are related to
viral replication or just TLR agonism.
2. Was population 2 (Table 1) the group used for the microarray? This is
unclear. Also, were any of the subjects on intranasal or inhaled corticosteroids
(or antihistamines, antileukotrienes, etc.)? From the table it appears that
albuterol was the only medication being used. Also, were any of the demographic
data in Table 1 significantly different?
3. The relationship between nasal epithelial cell DNA methylation and airway
hyper reactivity is unclear, and this is the only significant association in Table 2.
4. Figure 5B showed no difference in cells from asthmatics, even though the
initial screen did show an effect. Also, Figure 5C is redundant (essentially the
same as 5B---also, there is a fold of 0, which probably supposed to be a 1).
Similarly, the SNORA12 data (Figure 6) is very modest and 6C is redundant
(same as 6C---also, same issue with a fold of 0). While the authors try to provide
some understanding of this in terms of a mechanism, it is quite unclear. Why is
this one gene important rather than this being more a case of variability in an
assay, and this just happened to be an association? The lack of any real
mechanistic studies just obscures the importance of these findings.
5. A minor issue: In Figure 3A, are the differences between the asthmatic mock
versus HRV PC4 statistically different? Also, the labels in 3B and 3C seem
incorrect (could they be "mock" and "HRV" rather than "Healthy" and "Asthma"
for each one?)---as B is supposed to be from Healthy and C from Asthma subjects.
We hope that you can use the comments provided to improve your manuscript
and enhance your work for other uses.
Thank you for inviting us to consider your paper.
Sincerely,
Joshua A. Boyce, MD
Associate Editor
Dear Dr. McErlean:
I write you in regards to manuscript # thoraxjnl-2013-204940 entitled "Human
rhinovirus infection causes different DNA methylation changes in nasal epithelial
cells from healthy and asthmatic subjects." which you submitted to Thorax.
We receive many more manuscripts than we can publish. In view of the
comments of the reviewer(s) found at the bottom of this letter, your manuscript
has not reached a high enough priority for publication in Thorax. I am sorry to
disappoint you, and I hope that nonetheless the comments are of value to you as
you move forward with the work.
The reviewers and editors felt that the study is too preliminary without
confirmation in another cohort and some functional analysis of the locus
indicated.
Thank you for considering Thorax for the publication of your research. I hope
the outcome of this specific submission will not discourage you from the
submission of future manuscripts.
Sincerely,
Professor Andrew Bush
Editor-in-Chief, Thorax
Professor Ian Pavord
Editor-in-Chief, Thorax
Reviewer(s)' Comments to Author:
Reviewer: 1
Comments to the Author
The authors here present data on methylation changes in normal and asthmatic
subjects, studying changes in nasal epithelial cells ex vivo. I recognise that the
contribution of methylation-based regulation to HRV induced inflammation has
not been well studied before. The data shown also must have taken quite a lot of
work: there's some significant arrays and bioinformatics analysis here. I also
note that I am not someone who usually works with arrays (though I've done
some), and I'm not experienced in bioinformatics. I therefore confine some of my
comments to issues around the cells, clinical populations, conclusions and
significance. The authors are welcome to argue that I've missed key points, but
here are my views.
Whilst I think these data are tantalising and have some interest, I also find that
there are many reasons to consider the overall conclusions still fairly
preliminary. In part as acknowledged by the authors, there are many areas
requiring clarification. I have the following comments.
1. The inclusion/exclusion criteria are not that well explained. I take it to mean
that subjects have mild asthma not requiring any nasal or pulmonary
topical/inhaled steroids, and are controlled with SABAs only. Subjects also have
not had recent exacerbations. These are therefore at the mildest end of the
disease spectrum. Whilst asthma exacerbations can present in patients with mild
disease, it might be argued that these subjects are not particularly at risk of
severe exacerbations, and it would have been interesting to see if the data
pattern was born out at some level, even if just for SNORA12, with patients with
slightly more severe disease. Patients on ICS might have confounders from the
effects of steroids, but might also have more or smaller changes in gene
methylation and expression that would add value to the data set here. This is
also pertinent for the idea of correlating data with lung function etc, as these
relatively mild asthmatics (assuming I've interpreted the methods section
clearly) would only be expected to have minor changes in their PFTs, so it would
be slightly surprising to see big correlations with potentially modest gene
expression changes and modest changes in FEV1.
2. The study of nasal epithelial cells as a model of the lung has validity, but
published work confirms that these are not always the same as bronchial
epithelial cells. Whilst some studies have shown that these cells may be useful
models of lower airway cells (eg. McDougall CM, Blaylock MG, Douglas JG,
Brooker RJ, Helms PJ, Walsh GM. Nasal epithelial cells as surrogates for bronchial
epithelial cells in airway inflammation studies. Am J Respir Cell Mol Biol. 2008
Nov;39(5):560–8. ), others show these cells are not equivalent (Lopez-Souza N,
Favoreto S, Wong H, Ward T, Yagi S, Schnurr D, et al. In vitro susceptibility to
rhinovirus infection is greater for bronchial than for nasal airway epithelial cells
in human subjects. J All Clin Immunol. United States; 2009 Jun;123(6):1384–
90.e2.). I would therefore have liked to have seen some data examining the key
gene phenotype in some primary bronchial epithelial cells, even just
commercially purchased normal cells.
3. I struggle with data in figure 5 and 6; changes in methylation or gene
expression may be statistically significant, but it is hard to know how biologically
significant they are. When presented isolated from other stimuli, I don't know if
these are big changes or small changes. Some parallel controls looking to see if
obvious proinflammatory pathway stimuli (poly(I:C), IL-1, IFNbeta) cause
similar or larger changes would have helped greatly to get a feel for the overall
importance of these findings.
Overall, I think there are some interesting ideas and leads here, but not enough
to make me sure that this is a key biological finding.
Reviewer: 2
Comments to the Author
In this study, McErlean and colleagues investigate DNA methylation changes in
response to human rhinovirus infection (HRV) of nasal epithelial cells (NECs).
The study demonstrates changes in DNA methylation that are both disease
specific (differences between asthma and healthy) or infection specific (induced
by HRV infection). The focus then moves to SNORA 12 a small nucleolar RNA that
exhibited increased methylation upon HRV infection, in healthys but not
asthmatics. Expression analysis shows that asthmatics have increased SNORA 12
expression upon infection, which correlations generally suggesting that DNA
methylation prevents transcription. The manuscript is well written with well
planned and executed experiments. The study is the first to investigate DNA
methylation in relation to HRV infection, and is therefore of importance to the
field. The role of epigenetics in asthma is an under reported area of study.
Major Comments.
1.
The authors examine NECs (rather than lower airway cells) for epigenetic
and gene expression studies. What is the reason for this? Some justification
should be made in the introduction and expanded upon in the discussion. With
asthma being a disease of the lower airways, the comment is an obvious one yet
hasn’t been adequately addressed in the manuscript.
2.
In Figures 1, 5 and 6 all statistical analysis are with non-parametric
methods (Mann-Whitney) while data is presented as mean +/- SEM. This is
incorrect and data showed be presented as median and range if non-parametric
stats are used.
3.
Why was the initial epigenetic studies performed on only 3 controls while
the rest of the data (and overall number of patients in both groups) much
greater?
4.
Why are there two populations of asthmatics, and do they differ in any
other clinical parameters? Is the first study group (population 1) more severe?
This should be commented upon to assist the reader. It would also be useful to
include IgE levels, ACQ data and PC20 (histamine) data if possible. This is of use
and may also be helpful in performing other correlations (see below).
5.
The methylation data in HRV infected NECs while interesting and
certainly a first, identifies SNORA 12 as potentially an RNA species of interest
however the data show no function for SNOA 12 and no further studies to
elucidate this are attempted. While various correlations with clinical endpoints
are performed, I would suggest expanding on this with IgE, PC20 and ACQ if
possible. Were any virus inducible factors (cytokines chemokines etc measured?)
to also correlate SNOA 12 methylation of expression data with? HRV replication
would also be of interest. This may assist in establishing a mechanistic role for
SNORA 12 and add interest to the manuscript.
Minor Comments
1.
In Table 2, how is the atopy data correlated with the methylation data?
This reviewer was under the assumption that atopy was a qualitative
measurement (yes or no) rather than quantitative, and all the other correlation
data are with quantitative data. How this has been performed should be
explained.
2.
The study recruitment section is confusing, and as written it is unclear if
respiratory infection, stable asthma (no exacerbation in previous 4 weeks) is an
inclusion or exclusion criteria. I would suggest rewriting as what is inclusion and
what is exclusion criteria.
PONE-D-14-02696
Human rhinovirus infection causes different DNA methylation changes in nasal
epithelial cells from healthy and asthmatic subjects
PLOS ONE
Dear Dr. McErlean,
Thank you for submitting your manuscript to PLOS ONE. After careful
consideration, we feel that it has merit, but is not suitable for publication as it
currently stands. Therefore, my decision is "Major Revision."
We invite you to submit a revised version of the manuscript that addresses the
points below:
The authors suggest that human rhinovirus infection causes DNA methylation
changes in airway epithelial cells that differ between asthmatic and healthy
subjects. However, the nasal epithelial cell culture was performed by using a
single dose of HRV-16 infection and a single time point (48 hr). The authors need
to validate their data by performing a time course and dose response study.
Moreover, nasal epithelial cells were cultured under submergerd condition.
Well-differentiated cells at the air-liquid interface may be considered to confirm
the submerged cell culture data. There was no experiment done to determine the
function of SNORA12 in anti-viral and pro-inflammatory responses. Some of the
proteins or genes involved in inflammatory and anti-viral responses can be
easily measured in the collected epithelial supernatants of normal versus
asthmatic nasal epithelial cells. In summary, the conclusion of the current study
will be strengthened by performing aforementioned experiments.
We encourage you to submit your revision within 120 days of the date of this
decision.
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http://pone.edmgr.com/ and following the Submissions Needing Revision link.
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