Equine alveolar macrophages and monocyte-derived ...

19 Jan 2023

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Editorial Response to Initial Review

ACADEMIC EDITOR: The statistical analysis was inadequate or absent. Many parts of the methods were unclear. Furthermore, comparing two different cell types—alveolar macrophages (AMs) and monocyte-derived macrophages (MDMs)—raises concerns about data contamination between these types. Improved data interpretation and visualization needed.

In response: A new statistical analysis using a one-factor factorial in a randomized complete block design was performed to account for inter-horse variability. For details, see the updated statistical analysis section.

We acknowledge the presence of monocytes in the BALF-derived macrophage pool and have added this discussion to the manuscript.

Journal Requirements for Revision

1. Ensure manuscript meets PLOS ONE's style requirements, including file naming. Templates available at:

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

File names have been modified accordingly.

2. The phrase “data not shown” does not meet PLOS data sharing requirements. Authors must provide all relevant data within the manuscript, in supplementary files, or in a public repository. Citations or data access URLs, DOIs, or accession numbers should be provided.

We have included all original data in the resubmission. "Data not shown" was replaced with "S2 File".

Reviewer Questions and Responses

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Partly

Addressed as noted above.

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

Addressed as noted above.

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #2: No

Addressed as noted above.

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Review Comments to Author

Reviewer #1: The study focuses on severe equine asthma associated with exposure to A. fumigatus spores, gram-negative bacteria LPS, and silica microspheres, investigating the expression of molecules like CD163 and CD206 and cytokine secretion. Some aspects were intriguing:

Line 159: TrypLE select enzyme used to detach cells prior to immunostaining and flow cytometry might affect CD163/206 antigen presence. Have the authors investigated this? We used TrypLE select enzyme, preserving cell-surface epitope expression as proven with other proteins like CD24 (Panchision et al. 2007).

Line 171: Six-hour experiment length—why not 12, 24, or 48 hours? Previous gene expression curves from our lab justify 6 hours (Odemuyiwa, 2012). In vitro environment and viable spore germination beyond 10 hours made longer incubation impractical.

Flow cytometry: Anti-human CD163 and CD206 PE antibodies need validation. Supplementary Figure S1 is updated with scatter plots to display gating strategy. Clones were validated in equine studies (Kang et al., 2022).

Figure Recommendations:

Figure 1: Remove as it's redundant with Figure 2. Updated accordingly.

Figure 2: Non-significant results addressed with updated statistical analysis, reformatted into dot plots representing individual horses.

Line 246: Removed non-significant cytokine mentions, revised text appropriately.

Discussion: Address potential reasons for unexpected flow cytometry results—detachment technique, sample size, or incubation time. Also measured CD163 and CD206 in soluble forms, added comprehensive references including Tsuchiya et al. (2019).

Key Findings and Insights:

Provided detailed responses to all of Reviewer #2's suggestions including incorporating individual horse data, relevant citations (Sage et al., 2022; Berghaus et al., 2014), and clarification on methodologies.

Popular Perspectives and Additional FAQs

1. What are the primary differences between equine alveolar macrophages (AMs) and monocyte-derived macrophages (MDMs)?

AMs primarily reside in the alveoli under homeostatic conditions, whereas MDMs are derived from monocytes recruited to the lungs in response to inflammation.

2. How do these macrophages respond differently during an inflammatory stimulus?

The study shows differential expression of surface markers and cytokine secretions, with MDMs exhibiting a slightly reduced expression of CD206 post FLS challenge.

3. What are the limitations of using anti-human antibodies in equine studies?

While validated for certain equine studies, the specificity and affinity might vary, introducing potential discrepancies in observed data.

4. What future directions can amplify findings in such studies?

Using a larger sample size, extended incubation times, and incorporating in vivo conditions can provide more comprehensive insights. Also, exploring additional cytokines including IL-4 and soluble forms of key markers can offer a broader understanding.

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