Review Reports - Histamine Deficiency Inhibits Lymphocyte Infiltration in the Lacrimal Gland of Aged Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Respected authors,

Thank you for this interesting and valuable initiative to explore the role of histamine in lacrimal-gland inflammaging through measurement of relative expression of HDC mRNA, H1, H2, and H4 mRNA, lymphocytic infiltration, and increased TNFα/IL1b/IL6 and ICAM-1 in wild-type and HDC KO mice lacrimal glands.

Nevertheless, there are some major and minor issues that should be addressed before publication:

Major issues:

Each genotype×age group had 10 males and 10 females, but analysis by sex has not been performed. Lacrimal function is hormone-sensitive, so that sex differences could have an impact on results. At least include sex as a factor and report any interactions.
Identification of infiltrating cells and ICAM-1 in the lacrimal glands of aged wild-type mice: Histology and IHC lack quantification. At minimum, provide blinded, ROI-based cell density (cells/mm²) for CD3/CD4/CD8/B220/CD11c/F4/80 and AOD for ICAM-1. That simple >50-cells/5-sections threshold isn’t enough to back mechanistic claims.
Statistical analysis states that the t-test was used for paired samples. For qPCR analyses, multiple t-tests could be replaced with a two-way ANOVA, which would include age, genotype, and sex if possible.
Acknowledge study limitations- semiquantitative nature of qPCR for HDC, H1-4, and ICAM-1 and propose future directions.

Minor issues:

Abstract (Methods): Please state the exact number of specimens per group (n per genotype/age, and per sex if applicable).
Introduction: Overall structure is solid. Some improvements could be incorporated: Line 48: Clarify “reciprocal stimulation,” e.g., “Histamine promotes inflammation via bidirectional crosstalk with immune cells: histamine activates H1/H4 receptors on immune cells, inducing cytokines (e.g., TNFα, IL-1β) that, in turn, upregulate histamine synthesis and receptor expression.”
Introduction: When you cite your own work, add independent citations to provide a field-level synthesis. For tone, prefer “previous studies, including ours”.
Results (terminology): When reporting HDC and H1–H4 receptor data, use “relative mRNA expression (RT-qPCR transcript levels)” rather than “expression” to avoid implying protein quantification.
Results – Figure 1 (legend/text): The legend states results for WT and HDC-KO; confirm whether all panels include both genotypes or only WT and revise accordingly. Also correct wording to: (B) “Relative mRNA expression of H1 (Hrh1) and other histamine receptor subtypes in the lacrimal gland.” (C) “Relative mRNA expression of H2 (Hrh2) in the lacrimal gland.”
Results (line 97): The phrase “and HDC knockout (HDC-KO)” is redundant after the first definition.
Results – Figure 1B (lines 167–170): Make the histology caption diagnostic and specific, e.g., “Lacrimal gland from a 12-month-old wild-type mouse showing pronounced lymphocytic infiltration.”
Results – Table 1 (legend): Clarify variables and statistics. Example: “Prevalence of lymphocytic infiltration in lacrimal glands by age and genotype. Data are n/N (%). Infiltration defined as ≥50 mononuclear cells across five HPFs. p-values from χ² test (df stated in footnote).”
Materials and Methods (lines 287–288): Move “The data obtained were analyzed using a chi-square test” to the Statistical analysis subsection (if additional analysis with quantification of IHC results is not possible).
Materials and Methods (line 299): If the study analyzed lacrimal (not parotid/submandibular/sublingual) glands, correct to: “Total RNA was isolated from lacrimal glands …” (and ensure consistency throughout).

Recommendation: Major revision

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

We appreciate your constructive comments and insightful suggestions, which have greatly contributed to improving our manuscript. Considering all your comments, we have thoroughly revised the manuscript, incorporating the necessary changes, and in line with these changes, we have also revised the Abstract. The changes made to the figures and tables are as follows: Please find our responses to the individual comments and suggestions below. Our manuscript was edited in English by Editage Language Edit prior to the submission of the revised version. We have marked all changes made between the first submission and re-submission in red in the revised manuscript.

Figure 1: We have added sex-specific HDC mRNA expression data to panel B.

Figure 2: Toluidine blue staining and quantitative data on mast cell counts have been included.

Figure 3: This figure corresponds to Figure 2 in the original manuscript (HE staining data). We have added quantitative data on cellular infiltration per unit area to panels C and D. Table 1 has been removed from the manuscript.

Figure 4: This figure corresponds to Figure 3 in the original manuscript (immunostaining results).

Figure 5: This figure corresponds to Figure 4 in the original manuscript (cytokine PCR data).

Figure 6: This figure corresponds to Figure 5 in the original manuscript (ICAM-1 data).

Comment

Major issues:

Each genotype×age group had 10 males and 10 females, but analysis by sex has not been performed. Lacrimal function is hormone-sensitive, so that sex differences could have an impact on results. At least include sex as a factor and report any interactions.

Answer:

We have added data comparing male and female wild-type mice for HDC expression and histological quantification (Figure 1B, Figure 3F) and incorporated these additions into the Abstract, Methods and Discussion sections. Consequently, no significant differences were observed between the male and female groups.

Identification of infiltrating cells and ICAM-1 in the lacrimal glands of aged wild-type mice: Histology and IHC lack quantification. At minimum, provide blinded, ROI-based cell density (cells/mm²) for CD3/CD4/CD8/B220/CD11c/F4/80 and AOD for ICAM-1. That simple >50-cells/5-sections threshold isn’t enough to back mechanistic claims.

Answer:

For the histological data on cellular infiltration, we added quantitative analyses per unit area (Figure 3E and F). In contrast, immunohistochemistry was performed to qualitatively identify infiltrating cells in aged wild-type mice; therefore, it was not subjected to a quantitative analysis in the present study.

Statistical analysis states that the t-test was used for paired samples. For qPCR analyses, multiple t-tests could be replaced with a two-way ANOVA, which would include age, genotype, and sex if possible.

Answer:

In this study, our aim was not to determine which group was more likely to develop significant changes but rather to compare the differences between two distinct conditions, such as young versus aged and wild type versus HDC-KO. Therefore, we performed analyses using a t-test.

Acknowledge study limitations- semiquantitative nature of qPCR for HDC, H1-4, and ICAM-1 and propose future directions.

Answer:

We have added this point to the Discussion section (lines 251–253) as a limitation of our study, and to highlight possible future research directions..

Minor issues:

Abstract (Methods): Please state the exact number of specimens per group (n per genotype/age, and per sex if applicable).

Answer:

We improved Abstract.

Introduction: Overall structure is solid. Some improvements could be incorporated: Line 48: Clarify “reciprocal stimulation,” e.g., “Histamine promotes inflammation via bidirectional crosstalk with immune cells: histamine activates H1/H4 receptors on immune cells, inducing cytokines (e.g., TNFα, IL-1β) that, in turn, upregulate histamine synthesis and receptor expression.”

Answer:

The wording has been revised for clarity, and we believe that it now better reflects the intended meaning.

Introduction: When you cite your own work, add independent citations to provide a field-level synthesis. For tone, prefer “previous studies, including ours”.

Answer:

The wording has been revised for clarity, and we believe that it now better reflects the intended meaning.

Results (terminology): When reporting HDC and H1–H4 receptor data, use “relative mRNA expression (RT-qPCR transcript levels)” rather than “expression” to avoid implying protein quantification.

Answer:

The notations in the figures have been revised accordingly. In the main text and figure legends, the term “expression” was retained; however, “mRNA” was explicitly specified to prevent confusion with protein expression.

Results – Figure 1 (legend/text): The legend states results for WT and HDC-KO; confirm whether all panels include both genotypes or only WT and revise accordingly. Also correct wording to: (B) “Relative mRNA expression of H1 (Hrh1) and other histamine receptor subtypes in the lacrimal gland.” (C) “Relative mRNA expression of H2 (Hrh2) in the lacrimal gland.”

Answer:

The wording has been revised for clarity, and we believe that it now better reflects the intended meaning.

Results (line 97): The phrase “and HDC knockout (HDC-KO)” is redundant after the first definition.

Answer:

The wording has been revised for clarity, and we believe that it now better reflects the intended meaning.

Results – Figure 1B (lines 167–170): Make the histology caption diagnostic and specific, e.g., “Lacrimal gland from a 12-month-old wild-type mouse showing pronounced lymphocytic infiltration.”

Answer:

Following the recommendation of the proofreading service, the information presented in the Results section has not been repeated in the figure legends in this manuscript.

Results – Table 1 (legend): Clarify variables and statistics. Example: “Prevalence of lymphocytic infiltration in lacrimal glands by age and genotype. Data are n/N (%). Infiltration defined as ≥50 mononuclear cells across five HPFs. p-values from χ² test (df stated in footnote).”

Answer:

Table 1 has been removed, and the corresponding quantitative data on cellular infiltration have been incorporated into Figure 3 for a clearer presentation.

Materials and Methods (lines 287–288): Move “The data obtained were analyzed using a chi-square test” to the Statistical analysis subsection (if additional analysis with quantification of IHC results is not possible).

Answer:

It has been relocated to the “4.5 Histological quantification and statistical analysis” section.

Materials and Methods (line 299): If the study analyzed lacrimal (not parotid/submandibular/sublingual) glands, correct to: “Total RNA was isolated from lacrimal glands …” (and ensure consistency throughout).

Answer:

We have corrected the manuscript accordingly to ensure consistency.

Reviewer 2 Report

Comments and Suggestions for Authors

A interesting study, and quite well written - at first. The Introduction and methodology sections are fine.

Results and discussion are a mixed bag.

Results - This needs to be FAR MORE concise (too chatty). Just state the results, no interpretations or explanations for why techniques are used (that belongs in Discussion and methods sections respectively).

e.g.: 2.2 - delete lines 94-97 + 99-100 (rework the results into the reformed sentences: methods) and lines 106-108 (discussion).

e.g.: 2.3. delete line 111 (methods) and lines 120-122 (discussion). delete "(data not shown)"

e.g.: 2.4. delete lines 124-126 (methods) and 140-141 (discussion)

e.g.: 2.5 delete lines 143-146 (methods) and lines 153-155 (discussion).

Please make sure that what remains makes sense after the deletions.

Discussion - please rewrite to explain your data and how that supports/ contrasts to published studies.

There were 5 interpretations offered in the results - 1 per section. That is where you begin.

e.g.: 2.1: Histamine levels and #H1 receptor expressing cells increase with age in wild type. Is this new, unusual, other publications? Significance?

e.g.: 2.2: HDC deficiency tends to suppress age-associated lymphatic infiltration in the LG. Is this new, unusual, other publications? Significance?

e.g.: 2.4: age-associated upregulation of inflammatory cytokines observed in wild-type mice tend to be suppressed in HDC-KO mice. Is this new, unusual, other publications? Significance?

e.g.: 2.5: aging indices ICAM-1 upregulation in LG, histamine may contribute to this increase. Is this new, unusual, other publications? Significance?

Conclusion - too short - only refers to the last finding. What is the overarching theme and what needs to be done next?

Author Response

Dear Reviewer,

Figure 1: We have added sex-specific HDC mRNA expression data to panel B.
Figure 2: We have included toluidine blue staining as well as quantitative data on mast cell counts.
Figure 3: This figure corresponds to Figure 2 in the original manuscript (HE staining data). We have added quantitative data on cellular infiltration per unit area to panels C and D. Table 1 has been removed from the manuscript.
Figure 4: This figure corresponds to Figure 3 in the original manuscript (immunostaining results).
Figure 5: This figure corresponds to Figure 4 in the original manuscript (cytokine PCR data).
Figure 6: This figure corresponds to Figure 5 in the original manuscript (ICAM-1 data).

Comment
A interesting study, and quite well written - at first. The Introduction and methodology sections are fine.
Results and discussion are a mixed bag.
Results - This needs to be FAR MORE concise (too chatty). Just state the results, no interpretations or explanations for why techniques are used (that belongs in Discussion and methods sections respectively).
Answer:
In response to the reviewers’ comments, we have carefully revised the manuscript, including the sections indicated. The Results section now exclusively presents the results, while content more appropriately belonging to the Materials and Methods or Discussion sections has been relocated to those sections.

e.g.: 2.1 - remove lines 81-83 (methods) and 91-92 (discussion) and condense the remaining part to state 1) There is a significant increase in HCD and H1 expression...6 week - 12 months... and 2) there is an insignificant increase in H2 and H4 ....6 weeks to 12 months.
e.g.: 2.2 - delete lines 94-97 + 99-100 (rework the results into the reformed sentences: methods) and lines 106-108 (discussion).
e.g.: 2.3. delete line 111 (methods) and lines 120-122 (discussion). delete "(data not shown)"
e.g.: 2.4. delete lines 124-126 (methods) and 140-141 (discussion)
e.g.: 2.5 delete lines 143-146 (methods) and lines 153-155 (discussion).
Please make sure that what remains makes sense after the deletions.

Discussion - please rewrite to explain your data and how that supports/ contrasts to published studies.
There were 5 interpretations offered in the results - 1 per section. That is where you begin.
e.g.: 2.1: Histamine levels and #H1 receptor expressing cells increase with age in wild type. Is this new, unusual, other publications? Significance?
Answer:
To our knowledge, this is the first report describing the age-related expression of HDC and H1 in the lacrimal gland. We have added this point to the Discussion section (lines 210–219) in the revised manuscript.

e.g.: 2.2: HDC deficiency tends to suppress age-associated lymphatic infiltration in the LG. Is this new, unusual, other publications? Significance?
Answer:
Apart from our previous study in the submandibular gland, which demonstrated that HDC deficiency attenuates age-related inflammation, no other studies have reported an association between HDC and age-related inflammation. To the best of our knowledge, the present study is the first to demonstrate such an association in the lacrimal gland. We have added this point to the Conclusion (lines 333-338).

e.g.: 2.3: Infiltrating cells in aged LG (wildtype) were predominantly diffuse lymphocytes (not follicular clusters. How does this compare to other LG or other occular glands (Harderian gland has such clusters too in alligators, birds and lizards). Is this new, unusual, other publications? Significance?
Answer:
Although lymphoid tissues such as CALT and TALT are known to exist in the conjunctiva and nasolacrimal duct, even in normal mice, they do not infiltrate the functional glandular tissue, as observed in this study. In Sjögren’s syndrome model mice, an increase in lymphocytes within the lacrimal gland and a reduction in tear secretion have been reported, suggesting that the present findings may also lead to functional impairment.

e.g.: 2.4: age-associated upregulation of inflammatory cytokines observed in wild-type mice tend to be suppressed in HDC-KO mice. Is this new, unusual, other publications? Significance?
Answer:
To date, age-associated alterations in the histamine and cytokine levels within the lacrimal gland have not been reported. We have added to the Discussion that inflammatory cytokines were attenuated in HDC-KO mice in association with the upregulated expression of PPARγ (line 238-241).

e.g.: 2.5: aging indices ICAM-1 upregulation in LG, histamine may contribute to this increase. Is this new, unusual, other publications? Significance?
Answer:
Since it has been reported that an H1 antagonist suppresses ICAM-1 expression (reference No.25), the increased HDC in the lacrimal gland and suppression of ICAM-1 expression observed in HDC-KO mice in the present study may be related. As ICAM-1 is also involved in T-cell migration (reference No.27), it is highly likely that ICAM-1 is associated with inflammation. We have added this point regarding ICAM-1 to the Discussion (line 244-248)

Conclusion - too short - only refers to the last finding. What is the overarching theme and what needs to be done next?
Answer:
We have revised and expanded the Conclusions section to provide a broader perspective, summarizing the overarching themes of our findings and outlining potential directions for future research.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Respected Authors,

Thank you for the revised version of your manuscript. The responses are thorough, and the paper has improved considerably. The discussion is balanced, clearly relates the findings to inflammaging and Sjögren’s syndrome, and the conclusions are concise. It would strengthen the paper to add one or two sentences on future perspectives in the Discussion, as this is mentioned but could be emphasized more.

Overall, the paper is well written, and I recommend acceptance after minor revision.

Author Response

Dear Reviewer

Thank you for your constructive comment. In accordance with your suggestion, we have added a paragraph on future perspectives in the Discussion section to further emphasize the potential implications of our findings (line 246-252).

Sincerely.