The white-footed deermouse, an infection tolerant reservoir for several zoonotic agents, tempers interferon responses to endotoxin in comparison to the mouse and rat

Reviewer #1 (Public Review):

Summary: A well-executed series of experiments that will likely be of immense interest to (a) vector-borne disease researchers and (b) gram-negative sepsis/bacteremia researchers. The study uses comparative transcriptomics to begin probing what makes Peromyscus leucopus a unique host for numerous pathogens. Most issues with the paper are trivial, relating to descriptions of statistical cutoffs. While the paper does not provide mechanistic insight into how P. leucopus restrains its immune response to LPS or other microbial invaders, it is likely that this paper will be frequently consulted by researchers trying to understand that phenomenon.

Strengths:

o Use of outbred M. musculus is a commendable choice for the studies here.
o Excellent decision by the authors to use their published dataset (with appropriate statistical normalization) to improve their statistical power to examine sex-biased gene expression. Is it possible to go one step further and briefly incorporate their prior BALB/c data to see how the BALB/c compare to the outbred mice. This could perhaps be just a PCA plot to see if they cluster with the outbred mice and/or Peromyscus, or are separate.
o The correlations and ratios used to try to understand immune cell dynamics are clever and likely reflect interesting biology, but caution should be used when interpreting these indirect measures. As there are no tools for cell separation in P. leucopus, the authors should continue to include these data to stimulate ideas in the field, but readers should understand the "conclusions" are hypotheses due to the nature of the bulk RNAseq.

Weaknesses:

o Supplemental Table 1 only lists genes that passed the authors statistical thresholds. The full list of genes detected in their analysis should be included with read counts, statistics, etc. as supplemental information
o While P. leucopus is a critical reservoir for B. burgdorferi, caution should be taken in directly connecting the data presented here and the Lyme disease spirochete. While it's possible that P. leucopus have a universal mechanism for limiting inflammation in response to PAMPs, B. burgdorferi lack LPS and so it is also possible the mechanisms that enable LPS tolerance and B. burgdorferi tolerance may be highly divergent.
o Statistical significance is binary and p-values should not be used as the primary comparator of groups (e.g. once a p-value crosses the deigned threshold for significance, the magnitude of that p-value no longer provides biological information). For instance, in comparing GO-terms, the reason for using of high p-value cutoffs ("None of these were up-regulated gene GO terms with p values < 1011 for M. musculus.") to compare species is unclear. If the authors wish to compare effect sizes, comparing enrichment between terms that pass a cutoff would likely be the better choice. Similarly, comparing DEG expression by p-value cutoff and effect size is more meaningful than analyses based on exclusively on p-value: "Of the top 100 DEGs for each species by ascending FDR p value." Description in later figures (e.g. Figure 4) is favored.
o The ability to use of CD45 to normalize data is unclear. Authors should elaborate both on the use of the method and provide some data how the data change when they are normalized. For instance, do correlations between untreated Mus and Peromyscus gene expression improve? The authors seem to imply this should be a standard for interspecies comparison and so it would be helpful to either provide data to support that or, if applicable, use of the technique in literature should be referenced.
o Regarding the ISG data-is a possible conclusion not that Peromyscus don't upregulate the antiviral response because it's already so high in untreated rodents? It seems untreated Peromyscus have ISG expression roughly equivalent to the LPS mice for some of the genes. This could be compared more clearly if genes were displayed as bar plots/box and whisker plots rather than in scatter plots. It is unclear why the linear regression is the key point here rather than normalized differences in expression.
o Some sections of the discussion are under supported:
 The claim that low inflammation contributes to increased lifespan is stated both in the introduction and discussion. Is there justification to support this? Do aged pathogen-free mice show more inflammation than aged Peromyscus?
 The claim that reduced Peromyscus responsiveness could lead to increased susceptibility to infection is prominently proposed but not supported by any of the literature cited.
 References to B. burgdorferi, which do not have LPS, in the discussion need to ensure that the reader understands this and the potential that responses could be very different.

Reviewer #2 (Public Review):

Milovic, Duong, and Barbour investigate the inflammatory response of three species of small mammals (P. leucopus, M. musculus, and R. norvegicus) to endotoxin lipopolysaccharide (LPS) injection via genome-wide transcriptomics from blood samples. Understanding the inflammation response of P. leucopus is of importance as they are a reservoir for several pathogens. The study is a thorough, controlled, well researched analysis that will be valuable for designing and interpreting future studies. The authors discuss the limitations of the data and the potential directions. Clearly P. leucopus respond differently to the LPS exposure which is very interesting and opens the door for numerous other comparative studies.

The conclusions of the manuscript are thoughtful and mostly supported by the data, but there are a couple of points for clarification.

1. How were the number of animals for each experiment selected? Was a power analysis conducted?

2. The authors conducted a cursory evaluation of sex differences of P. leucopus and reported no difference in response except for Il6 and Il10 expression being higher in the males than the females in the exposed group. The data was not presented in the manuscript. Nor was sex considered for the other two species. A further discussion of the role that sex could play and future studies would be appreciated.

3. The ratio of Nos2 and Arg1 copies for LPS treated and control P. leucopus and M.musculus in Table 3 show that in P. leucopus there is not a significant difference but in M.musculus there is an increase in Nos2 copies with LPS treatment. The authors then used a targeted RNA-seq analysis to show that in P. leucopus the number of Arg1 reads after LPS treatment is significantly higher than the controls. These results are over oversimplified in the text as an inverse relationship for Nos2/Arg1 in the two species.