Microbe-Immune Crosstalk: Evidence That T Cells Influence the Development of the Brain Metabolome

Round 1

Reviewer 1 Report

Major Comments:

In this paper the authors describe the relationship of microbiota, T cells and brain metabolites. The describe the correlation between presence of microbiota in the feces/cecum and presence of T cells using mice models. They then characterize the brain metabolites present in the two mouse models (B6 and TCR-) and show the differences that exists. The authors here have not proven the molecular mechanism of the association. This has been mentioned in the discussion. However, one of the major issues is that the paper only provides correlation. I would strongly urge the authors to provide evidence that if T cells are replaced in early stage of life, the microbiota returns to B6-like phenotype. They can also show that such a replacement early in life also associates with brain metabolites being more similar to B6-like. Even though in the discussion section, the authors have tried to link their findings with previously published work, I feel unless they show it this current manuscript, it would be difficult to convince bidirectional communication.

 

Minor comments:

Fig 1A and B – Please mention p-values for each comparison pairs to delineate which ones are significant vs which ones aren’t.

The result section “2.5” number has been repeated. Please rectify.

Result section 2.4 – the paragraph describing about the difference in metabolites (line 195 onwards) is wordy. It might be beneficial to put some of the differences into context here in results rather than discussion.

 

 

Author Response

Major comment:

We appreciate the reviewer’s time and comments. We agree that an important step is to demonstrate that replacement of T cells in early life or other approach restores or partially restores the phenotype of wild type mice. This is outside the scope of the current study and the use of knock out mice provides novel insight on its own. This study is comparable to numerous published studies using immunocompromised mice or germ-free mice that did not include replacing immune cells or conventionalizing germ-free mice. Importantly, the current work demonstrated that the impact of loss of T cells occurs during the postnatal period with long lasting changes in the brain into adulthood. There are several methodological and analytical strengths in the current paper: the inclusion of a comprehensive panel of parallel measures in fecal, colon, and brain samples for metabolomics, as well as 16S rRNA sequencing in both cecal and fecal samples; the use of microbiome analytics that considered the compositional nature of the microbiota; mulitvariate analysis of metabolomics; and the DIABLO integrated analysis of the connection between microbes and metabolites that is superior to simple correlative analysis.

Minor comments:

Fig 1A and B – Please mention p-values for each comparison pairs to delineate which ones are significant vs which ones aren’t.

Reply: Details have been added to the figure legend.

The result section “2.5” number has been repeated. Please rectify.

Reply: corrected

Result section 2.4 – the paragraph describing about the difference in metabolites (line 195 onwards) is wordy. It might be beneficial to put some of the differences into context here in results rather than discussion.

Reply: Result section 2.4 has been reorganzied and interpretive statements have been added.

Reviewer 2 Report

The authors used mice lacking β and δ receptors on T cells to analyze bacterial flora differences between Cecal and Fecal, as well as metabolomics, in comparison to B6 counterparts. The analysis of metabolites included the large intestine and also examined changes in age. We also compared metaboites in the cortex, hippocampus, and hypothalamus of the brain.

As a result of each, there are some items that make a significant difference, and they are expressed. However, it has not been clarified whether the results obtained in each of the immunity-intestine-brain axes, which the authors paid particular attention to, are directly linked.

In the last paragraph of the discussion, the authors describe the experiments they need to do in the future. However, it is still unclear whether it is possible to verify the axis of immunity-intestine-brain, and a more specific description is required. 

Author Response

As a result of each, there are some items that make a significant difference, and they are expressed. However, it has not been clarified whether the results obtained in each of the immunity-intestine-brain axes, which the authors paid particular attention to, are directly linked.

We thank the reviewer for their time and comments.

Reply: this comment was addressed in the reply to reviewer 1 and is reproduced here for convenience.

In the last paragraph of the discussion, the authors describe the experiments they need to do in the future. However, it is still unclear whether it is possible to verify the axis of immunity-intestine-brain, and a more specific description is required. 

We agree that an important step is to demonstrate that replacement of T cells in early life or other approach restores or partially restores the phenotype of wild type mice. This is outside the scope of the current study and the use of knock out mice provides novel insight on its own. This study is comparable to numerous published studies using immunocompromised mice or germ-free mice that did not include replacing immune cells or conventionalizing germ-free mice. Importantly, the current work demonstrated that the impact of loss of T cells occurs during the postnatal period with long lasting changes in the brain into adulthood. There are several methodological and analytical strengths in the current paper: the inclusion of a comprehensive panel of parallel measures in fecal, colon, and brain samples for metabolomics, as well as 16S rRNA sequencing in both cecal and fecal samples; the use of microbiome analytics that considered the compositional nature of the microbiota; mulitvariate analysis of metabolomics; and the DIABLO integrated analysis of the connection between microbes and metabolites that is superior to simple correlative analysis.

We have edited the final paragraph of the discussion as well.

Round 2

Reviewer 1 Report

To prove a bidirectional association, a causation study is necessary. Without the replacement of T cells experiment, it is not scientifically appropriate to have the claims depicted in the manuscript.