The health status alters the pituitary function and reproduction of mice in a Cxcr2-dependent manner

This study explores the effects of microbiota on reproductive function of Cxcr2 knockout animals. Cxcr2 is involved in the control of pituitary action and the subsequent development of mammary gland, uterus and ovary.

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Full guidelines are available on our Instructions for Authors page, http://www.lifescience-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/Excelfile 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.*** 1. This manuscript makes the novel discovery that the microbiota differentially affects expression of genes in mice with global loss of CXCR2 as compared to those without loss of CXCR2. Of specific interest are alterations in expression of pituitary hormones that regulate the reproductive tract and mammary gland function of female mice. While it has been known for some time by those investigators working with CXCR2 null mice that it is challenging to breed CXCR2 null mice in conventional animal housing conditions, no one has ever done the work of characterizing the reasons for this defect. This paper clearly shows that the defect is linked to the combined effect of the microbiota and loss of CXCR2 and when the CXCR2 null ovary or mammary tissue is transplanted to WT mice, under standard housing conditions, the development of the ovary and mammary gland is normal. Thus it is the effect of loss of CXCR2 on the microbiome that creates this effect leading to changes in pituitary secretion of FSH, LH, PRL and GH that bestows the phenotype of the CXCR2 null mice when housed under standard housing conditions. In SOPF conditions, the phenotype of the CXCR2-WT and CXCR2-null mice are equivocal.
2. This paper also investigates the global changes in gene expression in the CXCR2-WT vs CXCR2-null mice under SOPF versus standard housing conditions. They show that in the absence of CXCR2, bystander infections affect leukocyte migration, adhesion and function, as well as ion transport and synaptic function behavior. The data are well documented with PCR analysis in follow-up of the GO-analysis of the transcriptomes of these mice.
3. Of particular interest CXCR2-null mice in standard conditions exhibit downregulation of lactoferrin, GATA3, cyclin D1, Erbb2, Elf5, Epcam, Prlr, Krt15, Epcam, Wnt4, and Wnt5b. Of note Prlr, cyclin D1, Erbb2 and Elf5 are crucial for mammary gland development as shown by other groups. Moreover Wnt signaling activates ERapositive luminal cells--that then are stimulated by ER to release Wnt ligands that act on the myoepithelial compartment.
4. The pituitary of CXCR2 KO mice exhibited T lymphocyte enrichment and to a lesser extend B lymphocyte, macrophage and neutrophil enrichment. This type of inflammatory environment is similar to that of autoimmune hypophysitis that can led to hypo-pituitarism and atrophy of the pituitary. This finding is discussed thoroughly in the discussion and adds to the significance of the study. Importantly it has implications for the use of CXCR2 antagonist side effects with long term use--though this point is not made.
5. Another important point is the relevance of studies of mice in SOPF conditions. Based upon the findings reported here, it is clear that there will be many differences in gene expression that limit the use of studies done in SOPF conditions--since humans are never in SOPF conditions. 6. Some edits may be needed in the wording of the manuscript--but this is a minor concern.
Reviewer #2 (Comments to the Authors (Required)): The study by Timaxian et al. characterizes the pituitary function and reproductive abilities of SOPF and conventionally raised (CONV) female mice, especially in the context of a genetic invalidation of Cxcr2 chemokine. The results of the study are the following: a. Under CONV, but not SOPF conditions, Cxcr2-null female mice are unable to cycle normally, affecting their reproductive capability b. Under CONV, but not SOPF conditions, the function and histology of uterus and mammary gland of Cxcr2-null females show abnormalities c. Transplanting the ovary or mammary gland of KO mice into WT mice under CONV conditions recapitulates the phenotype observed in KO mice d. Pituitary-gonadal axis signaling is altered in Cxcr2-null females under CONV, but not SOPF, conditions. Overall, the conclusions of the study are well supported. These are very novel findings, which come in a timely manner where more and more studies aim to understand the impact of health status on the physiology of laboratory mice. I especially appreciated the transplantation experiments, which show a clear phenotype linked to the ovary of mammary gland of KO mice. I do have a few comments, however.

MAJOR:
1. The title of the manuscript should be changed, along with most references to "microbiota". While we can infer that the observed phenotypes are the consequence of the microbial environment, this does not necessarily mean that it is by the microbiota. I would replace "microbiome" in the title by "health status". If the authors want to claim that the microbiota is the culprit, I would suggest two things: a. a. Switch the bedding of SOPF and CONV mice (and vice-versa). As I understand that CONV bedding cannot be brought into the SOPF room, I'd suggest that SOPF mice be brought to the CONV room and bedding be switched between CONV and SOPF mice. b. Performing fecal microbiota transplantation from CONV to SOPF and vice-versa. Then the role of the role of the microbiota will be clear.
2. Since Cxcr2-null mice are used, I think that it would be very useful to have the levels of the chemokine in WT CONV and SOPF mice. What is the rationale to focus on that chemokine specifically?
3. What is the rationale to focus solely on female mice? I understand that adding male mice to the analysis doubles the amount of work, but there should be an explanation to it. Did the authors perform semen analysis of male mice? 4. Why is SOPF data absent in several figures (such as Figure 2A)? That would be extremely important.
5. Authors should quantify the data when that is possible (e.g. branching in mammary gland in Figure 6D and lutheal bodies in Figure 6C). In the same manner, I'd suggest merging the quantification data from supplementary Figure 2 into Figure 3. If qualitative data (images) can be quantified, it should be done.
6. In page 6, the authors claim that "rederivating conventional animals to remove pathogens led to animals with full reproduction ability". The data needs to be shown. Figures 4 and 7, a multilinear model would be very useful. Adding a PCoA plot would add to the understanding of the study, if the plot shows clear segregation in CONV but not SOPF conditions. In Fig. 7E, two-way ANOVA should be used to include the two variables: health status and genotype (with adjusted p-values). Chi-2 or Fisher exact test should be done in Figure 6C.

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8. In transplanted animals, at least hormone levels should be measured. 9. Can the authors imagine that stimulating KO mice in CONV conditions with LH or FSH would restore the WT phenotype? If this cannot be done, at least it should be discussed.
MINOR COMMENTS: 1. Nomenclature of genes should be respected for the species. In mice, the nomenclature of a gene is Cxcr2, with only the first letter being capital.
2. The authors should include in the discussion the paper by Rosshart et al. (Science, 2019), showing that laboratory mice born to wild mothers retain certain immunological abilities.
3. In order for the figures to appear easier to read, I'd suggest adding CONV or SOPF as headers whenever those mice are used. GO  Reviewer #1 (Comments to the Authors (Required)): Answer: There is no specific point raised by the reviewer except in the wording, that we have done our best to improve.
Reviewer #2 (Comments to the Authors (Required)): 1. The title of the manuscript should be changed, along with most references to "microbiota". While we can infer that the observed phenotypes are the consequence of the microbial environment, this does not necessarily mean that it is by the microbiota. I would replace "microbiome" in the title by "health status". If the authors want to claim that the microbiota is the culprit, I would suggest two things: a. a. Switch the bedding of SOPF and CONV mice (and vice-versa). As I understand that CONV bedding cannot be brought into the SOPF room, I'd suggest that SOPF mice be brought to the CONV room and bedding be switched between CONV and SOPF mice. b. Performing fecal microbiota transplantation from CONV to SOPF and viceversa. Then the role of the role of the microbiota will be clear.
Answer: We agree with the reviewer comment that it could be a bit confusing. Indeed, SOPF animals were brought to a conventional room and to favor the rapid acquisition of microbiota from the facility, the bedding of animals already in conventional conditions was added to the one of transferred SOPF animals during the first weeks. We have added this to the materials and methods. We have not done fecal transplantations, but the added bedding is close to that. The reviewer is also right, conventional animals cannot go to a SOPF facility. So, we have replaced in the title microbiome by heath status and in the manuscript the term microbiome by microbiota.
2. Since Cxcr2-null mice are used, I think that it would be very useful to have the levels of the chemokine in WT CONV and SOPF mice. What is the rationale to focus on that chemokine specifically?
Answer: Our work starts with the serendipitous discovery that we observed specifically on CXCR2 KO animals the loss of fertility when SOPF animals were transferred to a conventional facility. To our knowledge, no one has reported this for any other chemokine or chemokine receptor KO animal. The question of the reviewer about chemokines is interesting but is also relatively broad. We looked at the levels of CXCR2 ligands in the mammary gland and pituitary of WT animals in conventional and SOPF conditions based on RNAseq analysis. From this analysis, we did not find any significantly regulated CXCR2 ligands between WT animals in the mammary gland of conventional and SOPF conditions. The same was true for WT pituitaries of conventional and SOPF animals. We have mentioned it in the discussion of the manuscript. Regarding other chemokines, only the chemokines CCL6 and CCL8 are down-regulated in the mammary gland of WT SOPF animals compared to CONV animals.
3. What is the rationale to focus solely on female mice? I understand that adding male mice to the analysis doubles the amount of work, but there should be an explanation to it. Did the authors perform semen analysis of male mice?
Answer: The reviewer is right, looking at males animals would require an entire study by itself and may dilute the message of this paper. What we can say is that CXCR2 KO males are also infertile. They also show a reduced size of prostate and seminal glands. We plan to study this more in details in the future.
4. Why is SOPF data absent in several figures (such as Figure 2A)? That would be extremely important.
Answer: SOPF data are present in most of the figures and as the differences between WT and animals were only observed in conventional conditions, we did not include this in figure 2. We did not see any alteration of reproduction nor of ovary or uterus histology of SOPF animals as shown in figure 2B and 3A. We have now added SOPF smears to the manuscript. To limit the size of the figure 2, we have added the SOPF smears as a supplementary figure 2.
5. Authors should quantify the data when that is possible (e.g. branching in mammary gland in Figure 6D and lutheal bodies in Figure 6C). In the same manner, I'd suggest merging the quantification data from supplementary Figure 2 into Figure 3. If qualitative data (images) can be quantified, it should be done. 6. In page 6, the authors claim that "rederivating conventional animals to remove pathogens led to animals with full reproduction ability". The data needs to be shown.
Answer: We have effectively rederivated conventional animals to transfer them into a SOPF facility. We have never been able to have pups from homozygous animals in conventional facility. On the contrary, all homozygous CXCR2 KO animals which were rederived from conventional animals that we have mated in SOPF conditions were fertile. This is what is shown in Fig. 1A, these are animals in SOPF conditions after having been rederivated from conventional animals.
7. In Figures 4 and 7, a multilinear model would be very useful. Adding a PCoA plot would add to the understanding of the study, if the plot shows clear segregation in CONV but not SOPF conditions. In Fig. 7E, two-way ANOVA should be used to include the two variables: health status and genotype (with adjusted p-values). Chi-2 or Fisher exact test should be done in Figure 6C.
Answer: We have added a supplementary figure 4 that shows the PCA for both mammary gland and pituitary RNAseq. This shows that the transcriptome of WT and KO animals are much more different in conventional conditions than in SOPF conditions both in the mammary gland and the pituitary. Pituitary and mammary gland samples are clearly different as expected. Finally, without taking in account the genotype of the animals (WT or KO), the transcriptomes of conventional animals are also different from the one of SOPF animals both in the pituitary and the mammary gland.
In figure 7E (now labelled 7F), we have re-run statistical and analysis and we have used Kruskall-Wallis test was used followed by Dunn's multiple comparisons test to compare all plots between each other. This confirms that the main differences are between WT and KO animals in CONV conditions. In addition, statistical differences are seen for some genes between KO from CONV and SOPF conditions, but not between WT animals. We have performed a Fisher exact test on Fig. 6C, and added it to the figure legend, this shows no significant difference in successful breeding between WT and KO donors (p= 0.5147).
8. In transplanted animals, at least hormone levels should be measured.
Answer: The main goal of doing ovarian transplantations was to show that the WT females transplanted with KO ovary were fertile and to show that the histology of transplanted KO ovaries was normal in WT environment. For these reasons, transplanted females were mated after transplantation and when they gave birth, the females were sacrificed to check the ovary histology. We did not collect blood samples for these animals as they had been pregnant (and had thus obviously a very particular status) and could not be compared with any of the animals of this study assessed before which were all virgins.
9. Can the authors imagine that stimulating KO mice in CONV conditions with LH or FSH would restore the WT phenotype? If this cannot be done, at least it should be discussed.
Answer: Treating mice with LH or FSH is a good suggestion but these hormones are acting in a cyclic manner. So to be effective, this would require to have some kind of pulsatile injections at correctly chosen times according to the estrus cycle. The problem is that these animals have a weird estrus cycle, so, there is no way to know when these animals should be treated synchronously. Moreover, progesterone and estradiol levels are altered and the histology of the uterus and ovary severely impaired, it might not work. We have now discussed this in the paper.
MINOR COMMENTS: 1. Nomenclature of genes should be respected for the species. In mice, the nomenclature of a gene is Cxcr2, with only the first letter being capital. 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. Dear Dr Leibfried, Please find attached a revised version of our revised manuscript #LSA-2019-00599-T entitled "The health status alters the pituitary function and reproduction of mice in a Cxcr2-dependent manner", which we submit to Life Science Alliance for consideration for publication.
Here are the novel modifications: -We provide also a rebuttal letter with the answers to the reviewers.
-We have deposit the RNA-seq data in GEO and have provided the accession number in the methods section of your manuscript (GSE144231).
-We have added the statistical tests in the figure legends.
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-We have added callouts in the manuscript text to Fig 8 (now named Fig 10) and S1 supplemental figure.
- Thank you for submitting your Research Article entitled "The health status alters the pituitary function and reproduction of mice in a Cxcr2-dependent manner". 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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