Nasal Administration of Lipopolysaccharide Exacerbates Allergic Rhinitis through Th2 Cytokine Production from Mast Cells

Round 1

Reviewer 1 Report

In the present manuscript, the authors setup the mouse allergic rhinitis model to investigate the effect of lipopolysaccharide (LPS) on allergic rhinitis. An interesting work is presented here and results are shown. There are some weak points as described below and need to clarify

1. Abstract: the content is a little too long, it need be concise.
2. Statistical analysis should be describe detailed.
3. In all Figure, (1) please add quantification chart for cytokines level by western blotting; (2) please add color arrow to sign where the eosinophil in each HE staining field.
4. In Figure 4 B and C, please sign *p value between which two groups and statistically analyze by ANOVA.
5. Please add scale bar in the Fig. 1B, Fig. 2B and Fig. 3A
6. Author should add some results for evaluation of mast cell using Toluidine blue staining.
7. There are some grammatical errors in the sentences. Suggest authors that please go through the entire manuscript carefully.

Author Response

Thank you for your detailed peer review.

We have corrected as much as possible regarding the points you pointed out.

Of the many reports of lower respiratory tract allergies or mast cells and LPS, we consider this to be the first report of LPS and nasal allergies.

We apologize for the inconvenience, but please review it.

1) Abstract: the content is a little too long, it need be concise.

As you pointed out, we have shortened Abstract.

2) Statistical analysis should be describe detailed.

We have examined the statistical method, performed statistical processing again, and described it. For comparison of sneezing, after evaluating with ANOVA, each group was compared. Mast cell degranulation and cytokine production were evaluated by ANOVA and then Dunnet's test was performed. Therefore, it is not stated that ANOVA was used because F-test is not required.

3）In all Figure,

(1) please add quantification chart for cytokines level by western blotting;

This time, Western blotting does not perform quantitative analysis. In all experiments, proteins are collected from the same amount of tissue by performing IP using the primary antibody and protein G beads under the same conditions.

(2) please add color arrow to sign where the eosinophil in each HE staining field.

As you pointed out, the tissue section is not cut well and eosinophils are difficult to understand. Showed an arrowhead on eosinophils.

4)In Figure 4 B and C, please sign *p value between which two groups and statistically analyze by ANOVA.

We conducted an ANOVA study and conducted a Dunnet's test on Figures 4A, B, and C. Figure 4B is compared with negative control, and 4C is compared with positive control, so horizontal lines have been added for clarity.

5)Please add scale bar in the Fig. 1B, Fig. 2B and Fig. 3A

As you pointed out, we added a 10µm bar to Figure 1B, reviewed the images and made all the images the same scale.

6)Author should add some results for evaluation of mast cell using Toluidine blue staining.

Thank you for your valuable feedback. We are working on confirming BMMC with Truidin Blue, but we did not leave a photo because we were doing it for the purpose of confirming that it is a mast cell. Until now, β-hexosaminidase release assay has been performed for degranulation of mast cells, but the degranulation rate was 25 to 30% even with positive control. The remaining granules are thought to remain within the mast cells. Therefore, it may be difficult to evaluate the amount of granules remaining in the cells by evaluating toluidine blue to determine whether or not the cells have been degranulated using this experimental system. In addition, as added in the text from Reference 17, it has been reported that even if LPS derived from Escherichia coli is added to BMMC, it does not degranulate, and we believe that our experimental results are correct.

7)There are some grammatical errors in the sentences.

We requested a native speaker to proofread the paper.

Reviewer 2 Report

The aim of the present research paper is of utmost importance both for the clinician and for the scientist -  the exacerbation of nasal congestion when infectious rhinitis develops in patients with allergic rhinitis. Although the phenomenon is widely acknowledged by the clinician , the underlying  mechanisms are unknown. Thus, the authors present a very interesting experimental study, assessing the effect of lipopolysaccharide (LPS) on  allergic rhinitis using a mouse allergic rhinitis model. Although research papers have been published addressing the lower respiratory tract, this is the first to study the relationship between infection and allergy in nasal mucosa. The study is well conducted, the methods are clearly explained. The use of appropriate immunological assays, has enabled the authors to clearly demonstrate that the nasal administration of LPS together with the antigen exacerbate nasal allergy via 125 TLR4 of mast cells. Besides, the study also demonstrated that  LPS does not exacerbate mast cell degranulation but promotes TH 2 production from 146 mast cells via expression of GATA-3 gene. The conclusion is pertinent. The paper is a must for everyone interested in the management of allergic patients. 

Author Response

Thank you for your detailed peer review.

Of the many reports of lower respiratory tract allergies or mast cells and LPS, we consider this to be the first report of LPS and nasal allergies.

We have corrected the part pointed out by another reviewer and proofread by a native speaker.

We apologize for the inconvenience, but please review it.

Round 2

Reviewer 1 Report

Authors have been responded point by point. 

The manuscript can be accepted.