PREreview of Maternal adaptations in mouse lactation are vulnerable to diet-induced excess adiposity

This review reflects comments and contributions by Aude Angelini, Marina Schernthanner, Shaunak Deota, Pablo Ranea-Robles, Luciana Gallo and Femi Arogundade. Synthesized by Pablo Ranea-Robles.

This study explores how a high-calorie diet-induced increase in body fat impacts the way mothers' bodies adapt during lactation in a mouse model. The results revealed that this excess body fat caused changes in the structure of the intestines, the types of immune cells present, and how well the intestinal barrier worked. Another focus of this study was whether these changes persist after lactation and how a HC-diet affects them. The study highlights the implications of excess adiposity for maternal health during the periconceptual/perinatal period and emphasizes the need for further research to understand its effects on postpartum health. Overall, the crowd reviewers found that the conclusions of this manuscript are well-supported by the data, and that this is a relevant topic of research that deserves more attention. Below, we provide our major and minor comments for this study.

Major comments:

• The study addresses an important and relevant topic by examining the impact of excess adiposity during lactation, a period often understudied, on maternal health. This has significant implications for understanding postpartum health and potential interventions. This paper is very informative in terms of characterizing changes in tissue-resident and circulating immune cell populations, intestinal tissue morphology, metabolism, intestinal permeability etc. in lactating females when exposed to a high-calorie diet. The use of a mouse model allows for controlled experiments and observations that can help elucidate complex interactions between diet, adiposity, and maternal adaptations. The study employs a longitudinal approach by examining the effects of excess adiposity both during lactation and up to two months post-lactation. This contributes to a better understanding of the persistence and resolution of maternal adaptations.

• The authors make experimental data available on a public platform (figshare), allowing other researchers to verify the results and perform further analyses, promoting transparency and reproducibility.

• In Fig. 9, the authors should compare the HC-diet lactation and post-lactation groups with a HC-diet fed non-pregnant female group to assess the differential effect of diet vs lactation. We think this is a crucial aspect that should be included in this manuscript. In Fig. 9d, the authors compare to a control diet-non-pregnant female group, which is not ideal. The diet by itself can cause most of the dysregulation and may have an overwhelming effect, especially considering that the authors have reported that HC diet itself increases intestinal permeability in a recent publication.

• Following up on Figure 9, the effects observed in the post-lactation group in Fig. 9d seems caused by an apparent outlier in that group. Have the authors evaluated that possibility? If so, this should be accounted for in the discussion of the results. 

Minor comments:

• We think the introduction is overall okay, but perhaps is missing a bit of focus on the difference between physiological adaptations to pregnancy and pathophysiological adaptations to pregnancy when fed a high-caloric diet. Authors are experts in this topic, but we found this aspect was only well-described in the discussion. A specific mention to the changes in intestinal permeability during physiological pregnancy-lactation in the introduction could improve the understanding of the topic while reading the introduction, especially to differentiate the results of this paper to the ones found in the recent Plos one from the same authors, where they studied these changes during gestation.

• Authors may want to include details regarding the live/dead gate in their gating strategy to gate out dead cells.

• The increase in body weight is clear in Figure 2a. However, when looking at the body weight over time in Supp Fig 2, BW during lactation does not seem to change between the two diet groups. This can be confusing for the reader. It could probably be related to the different type of statistical analysis, but, we think, at least, that authors could include the p-value at day p21 in the Supp 2a body weight graph.

• The expression of several tight junction proteins such as occludin (Ocln), claudins (Cldn-1, -2, -3, -4, -5, -7, -8) and zonula occludens (Z0)/tight junction proteins (Tjp1) are downregulated in the gut during metabolic disorders and inflammation. Assessing their expression can be an additional proof that the gut barrier integrity is affected.

• The change in select macrophage populations in intestinal tissue is interesting - did the authors check if those populations localize differently (relative to the crypt) in mice fed a control versus a high calorie diet?

• Did the authors observe general changes (increase?) in gut-homing signals such as a4b7 integrin, CCR9 on immune cells or CCL25 on intestinal epithelial cells upon high-calorie diet? We were curious about changes in gut-homing signals, since they observed changes in the number of certain immune subsets (f.e. the change in monocyte-derived macrophages) between their two diets. Which could be due to changes of immune cell proliferation in the tissue or a change in recruitment of immune cells to the tissue. Or decreased exit of immune cells through lymphatics (but that would be mostly for dendritic cells and T cells, for which they don't see such drastic changes in number). Technically a4b7 integrin (on immune cells) is less of a gut-homing signal and rather facilitates exit of immune cells out of the blood vessel and into the tissue, so we think checking for chemokines (f.e. CCL25 expressed by the epithelium, which would recruit immune cells into the tissue) would make more sense.

• The authors observed a change in mesenteric fat weight and later on in selected immune cell populations. Did they check for differences in the number and composition of lymphoid structures (f.ex. Peyer's patches in the small intestine) or composition and size of the draining mesenteric lymph nodes?

• For all of their immune cell isolations from intestinal tissue - were those done after removing secondary lymphoid structures such as Peyer's patches?

• Given the reported differences in crypt length - did the authors observe differences in proliferation (EdU pulse, Ki67 staining etc.) and the number of intestinal stem cells as assessed via canonical stem cell markers such as Lgr5 (for both, small and large intestine) or Olfm4 (only in the small intestine)?

Comments on reporting:

• We agree with the authors choice of the dam as biological replicate, not the offspring, for analysis

• We missed more details regarding euthanasia methods, both for the pups and the adult animal at the end-point of the study, as well as regarding sample size for each experiment in material and methods / figure legends.

• Even though Figure 1 was quite informative regarding the experimental design, we think it would be useful to indicate the age of the dams at the point when they were allocated to one of the diets, to facilitate the understanding of the experimental design.

• Figure legend of fig. 2 would be more clear if authors add the information on the lactation endpoint used here, i.e P21-23 as explained in the text

• We think that Supp figure 2b does not add anything informative to what is already present in Supp 2a.

• It is crucial to include a dedicated section within the preprint that thoroughly discusses the limitations of the study. This comprehensive discussion of limitations is essential to provide a well-rounded and balanced interpretation of the results presented.

Suggestions for future studies

• It remains unclear how exposure to a high-fat diet during pregnancy might change the susceptibility of those female mice to f.ex. intestinal infections, acute inflammatory insults etc. A follow-up challenge model in previously pregnant mice would have been interesting to see and would have perhaps helped to shed more light on long-lasting immune cell phenotypes following exposure to a high-fat diet. Alternatively, similar characterization studies of intestinal tissue morphology, function and immune landscape, as done for lactating and post-lactating mice in this study, would have been interesting to see in the mice's offspring upon reaching adulthood. Body weight of the offspring coming from these dams was already increased at p21, suggesting transgenerational changes happen in this model.

• Following up on future studies on offspring, authors may want to assess if the high-caloric diet also affects the cytokine levels or bacterial products in breast milk, as this could have a major effect on offspring

• We found intriguing the possibility that the changes in immune cells may have an effect on the adipose tissue of these dams, could be a nice addition for future experiments.

• The fact that the cecal weight is different might suggest an altered microbial composition following a high calorie diet. Did the authors look into this? Alternatively, it could be related to differences in fiber content of the diets, which vary quite a bit in carbohydrate amounts etc. Is it known whether the source and composition of fibers is comparable between the high calorie and control diets used in this study?

Competing interests

The author declares that they have no competing interests.