Microbiota regulates the turnover kinetics of gut macrophages in health and inflammation

Maintenance of the gut lamina propria–resident macrophage cell pool requires monocyte input during adulthood. Here, Ruedl and colleagues demonstrate that the intestinal microbiome positively influences the replenishment of tissue-resident macrophages under both steady-state and inflammatory conditions.

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Thank you for this interesting contribution to Life Science Alliance. We are looking forward to receiving your revised manuscript. --High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, https://www.life-science-alliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max. 200 characters including spaces). This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title and running title. It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person. Author names should not be mentioned.
--By submitting a revision, you attest that you are aware of our payment policies found here: https://www.life-sciencealliance.org/copyright-license-fee B. MANUSCRIPT ORGANIZATION AND FORMATTING: Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** Reviewer #1 (Comments to the Authors (Required)): In the current study, authors demonstrated that microbiota affects F4/80(hi) resident macrophage turnover in the intestine. Authors utilized fate-mapping mouse system which labels BM originated cells and showed that various myeloid cells are replenished by BM. To assess the cell type-specific contribution of microbiota during their turnover, mice were also housed in germ-free condition. Colonic F4/80(hi) macrophages were the most affected cell type by microbiota in terms of cell turnover under the steady-state and inflammatory settings, suggesting that microbial stimulation supports F4/80(hi) macrophage turnover. In small intestine where microbes are less populated, F4/80(hi) macrophage turnover was less prominent, especially for longlived Tim-4(+) subpopulation. Although the study provides the evidence of microbial involvement in F4/80(hi) macrophage replenishment, its detailed mechanism is still obscure. Still this approach provides useful information to the researchers in related fields.
Specific comments: 1. F4/80(hi)MHCII(+) in Figure 1C seems monocyte, which should be F4/80(hi)MHCII(-). 2. Did authors checked YFP expression of c-kit(+) cells in the BM at 2~4 months after TAM treatment? It would be important to check whether YFP(-)c-kit(+) cells are not present in TAM-treated mice at the time point when analyzed. Also, please explain why authors used different time points after TAM treatment. 3. Figures need to be labelled following order they appear in the text. Currently Figure 2E appears after Figure 3B. Alternatively, Figure 2E and 3B can be combined and presented as new set of figure. 4. Please show representative plots, at least for F4/80(hi) macrophages in Figure 3. Also, representative plot for Figure 4C would be informative. 5. Infection and injury induces influx of various cell types but number of resident macrophages are generally less affected. DSS obviously increases cell influx to large intestine and relative ratio of F4/80(hi) macrophage should be decreased. Authors need to show absolute number of F4/80(hi) cells in Figure 4A to show this population actually "lost".
We thank all the reviewer for his/her constructive comments on our work. We have tried to address as well as we can on the experimental concerns and provide detailed answers to the raised remarks. We have included a new Fig. 4 and revised the old ones based on the comments. Amendments in the manuscript have been highlighted in red.
We hope that with the described changes and responses, the paper is now suitable for publication in LSA.

In the current study, authors demonstrated that microbiota affects F4/80(hi) resident macrophage turnover in the intestine. Authors utilized fate-mapping mouse system which labels BM originated cells and showed that various myeloid cells are replenished by BM. To assess the cell type-specific contribution of microbiota during their turnover, mice were also housed in germ-free condition. Colonic F4/80(hi) macrophages were the most affected cell type by microbiota in terms of cell turnover under the steady-state and inflammatory settings, suggesting that microbial stimulation supports F4/80(hi) macrophage turnover. In small intestine where microbes are less populated, F4/80(hi) macrophage turnover was less prominent, especially for long-lived Tim-4(+) subpopulation. Although the study provides the evidence of microbial involvement in F4/80(hi) macrophage replenishment, its detailed mechanism is still obscure. Still this approach provides useful information to the researchers in related fields.
Specific comments:

Did authors checked YFP expression of c-kit(+) cells in the BM at 2~4 months after TAM treatment? It would be important to check whether YFP(-)c-kit(+) cells are not present in TAMtreated mice at the time point when analyzed. Also, please explain why authors used different time points after TAM treatment.
Since HSC are expressing c-kit ( also known as stem cell factor receptor), a 5 day treatment with TAM will maintain the YFP signal in the BM over time, hence there will be a continous seeding of YFP + BM cells into the periphery. Depending on their turnover rates distinct lymphoid and myeloid cells will be labelled at different kinetics. In particular, some specific intestinal macrophage subpopulations (e.g. Tim-4 + ) are known to be replaced slowly over time instead some other subpopulations are swiftly refilled (Tim-4 -). Therefore to capture more efficiently the refilling kinetics at steady-state, we have chosen two different time-points (2 [ Fig. 2 B, C and Fig. 3] and 4 months [Fig. 2D]). On the other hand, under inflammatory conditions (e.g. DSS treatment) we opted for a shorter window (1 month) since we expected a faster monocytemediated replenishment process. Figure 2E appears after Figure 3B. Alternatively, Figure 2E and 3B can be combined and presented as new set of figure.

Figures need to be labelled following order they appear in the text. Currently
As suggested, we have included a new figure 4 which included the cytokine and chemokine qPCR analysis of colon and small intestine. Figure 3. Also, representative plot for Figure 4C would be informative.

Please show representative plots, at least for F4/80(hi) macrophages in
As suggested, representative flow cytometry plots were included in both Fig. 3 and new Fig. 5C. Figure 4A to show this population actually "lost".

Infection and injury induces influx of various cell types but number of resident macrophages are generally less affected. DSS obviously increases cell influx to large intestine and relative ratio of F4/80(hi) macrophage should be decreased. Authors need to show absolute number of F4/80(hi) cells in
We agree with the reviewer that sometimes the frequency does not reflect a reduction or increase in particular if there is a massive infiltration of another cell population. We have added changed the % in absolute numbers (New Fig. 5A). 1 st Revision -Editorial Decision Thank you for submitting your revised manuscript entitled "Microbiota regulates the turnover kinetics of intestinal macrophages". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
Along with points mentioned below, please tend to the following: -As a one of main myeloid cell population, Ly6C+MHCII+ cells need to be included in Figures 2,3,5. Also, please update text accordingly -please add the Twitter handle of your host institute/organization as well as your own or/and one of the authors in our system -please add Abstract in our system -please note that titles in the system and the manuscript file must match -please consult our manuscript preparation guidelines https://www.life-science-alliance.org/manuscript-prep and make sure your manuscript sections are in the correct order and labeled correctly -please use the [10 author names, et al.] format in your references (i.e. limit the author names to the first 10) -please label Figure 5 correctly -please provide the link to access to original flow cytometry data in your data availability section If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. Authors addressed comments raised by this reviewer.
Minor point: As a one of main myeloid cell population, Ly6C+MHCII+ cells need to be included in Figures 2,3 Thank you for submitting your Research Article entitled "Microbiota regulates the turnover kinetics of gut macrophages in health and inflammation". It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance. Congratulations on this interesting work.
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You can contact the journal office with any questions, contact@life-science-alliance.org Again, congratulations on a very nice paper. I hope you found the review process to be constructive and are pleased with how the manuscript was handled editorially. We look forward to future exciting submissions from your lab.
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