Dear Authors,

The submitted manuscript reviews the current literature on equine macrophages in different organ systems (bone marrow, intestines, lungs) as well as known equine macrophage subtypes and related diseases. The manuscript also directly compares what is known in the horse to the established macrophage biology in humans and mice. Please see below my review comments on your manuscript.

MAJOR COMMENTS
1. The title of this review states that this is a discussion of equine monocytes and macrophages, but only goes into detail describing equine macrophages. Due to the fact that monocytes are also a significant component of the MPS, it is also necessary to add an additional section on blood monocytes. This is important considering that blood monocytes are readily available in the horse (compared to bone marrow precursors) and are often used to generate macrophages in vitro. Please add a blood monocyte section, including known monocyte subtypes and a comparison to human/murine blood monocytes, before discussing macrophages and macrophage subtypes.

1. The figures do not clearly describe the MPS of the horse. Instead, there should be a figure similar to Fig 2 but illustrating what is specifically known about the horse MPS. A lot of the manuscript focuses on LPS and TLR signaling and the differences between species. Include another figure summarizing the signaling pathways and genetic variants of TLRs between species.

2. In the description of LPS and TLR activation of equine monocytes (lines 143-165), please add more connection between LPS signaling (lines 143-158) and TLR activation (lines 160-165). Does equine TLR2 form a heterodimer with TLR1 or 6 as in other species? Lines 385-400 discuss TLR4 ligand information that would fit nicely in this earlier section.

3. Not all references in the text are also cited in the reference list. For example, line 290 cites “Lee et al (2017)”, line 292 cites “Lee, Kiupel et al (2017)”, and line 537 cites “Burns and Roberts, 2018”, but there are no citation numbers and no citation lines in the reference list for these publications.

MINOR COMMENTS
1. Cytokine nomenclature includes a dash between “IL” and the number. This needs to be corrected throughout (ex: IL-4, not IL4). Interferon gamma is abbreviated IFN-γ, not INFG (line 81). Macrophage colony-stimulating factor 1 is also commonly referred to as M-CSF, and granulocyte-macrophage colony-stimulating factor 2 is also commonly referred to as GM-CSF. Please mention these additional abbreviations, even if you do not use them.

1. For clarity, please refer to macrophages or monocytes in different species with the species name (ex: equine macrophage, murine macrophage, human macrophage) as much as possible. Especially in the first half of the manuscript, it is often unclear which species is being discussed.

2. On line 201 (and lines after), when describing surface proteins that identify muscularis macrophages (MM), “ve” is added after MHCII+, CD11b+ and CD169+. This either needs to be clarified as this is uncommon nomenclature, or fixed if it is a typo.

3. On line 271, the “(“ needs to be removed from the reference citation.

4. There is inconsistency with in-text references. Lines 290, 292-293, 310-317, 428-429, 442-443, and 542 do not use [#], while the rest of the manuscript does.

5. On line 340, please correct the spelling of “phagocytose”.

6. Please be consistent with the order when listing different molecules. For example, line 347 reads “TLR9 and TLR4” and line 352 reads “TLR4 and TLR9”. I would recommend the latter order.

Dear Authors,

The revised manuscript compares each macrophage type in horses and ultimately argues that horses serve as a better biological model for human innate immunity than mice. This revision includes new figures that very nicely illustrate the equine MPS cell types with their tissue locations and distinguishing features, TLR signaling, and homology of relevant genes between humans, horses, mice and rats. Thank you for adding these additional illustrations, as well as a description of equine monocytes and monocyte derived macrophages at the beginning. Please pay special attention to the “major comments” which are regarding cytokine nomenclature and the inclusion of monocytes in the review.

MAJOR:

1. Regarding the nomenclature for cytokines, the HUGO gene nomenclature describes gene and gene sequence nomenclature, not protein nomenclature. Throughout the manuscript, most cytokine references are referring to assays that measure protein levels and therefore should be referred to following the standardized protein nomenclature, which refers to Interleukin 4 as IL-4, etc. Please refer to the Journal of Immunology’s standard abbreviation list where they state that interferons are abbreviated “IFN-ɣ” and interleukins are abbreviated “IL-2”.
   https://www.jimmunol.org/info/abbreviations

Chemokine nomenclature is also standardized (J Leukoc Bio. 2001 Sep;70(3):465-6.) and includes an example of chemokines that have also been given interleukin names with this correct nomenclature (Interleukin 8 = CXCL8 = IL-8).

1. In the section on monocytes, the focus is primarily on differentiating monocytes into macrophages and dendritic cells. Please also add a more specific summary of the functional role of blood monocytes to begin this section (line 174). This is described in part earlier in the manuscript (for example lines 84-85, 113-114) but needs to be expanded in this section because of the focus on blood monocytes. Please also describe that there are different monocyte subtypes and the functional relevance of each (i.e. classical, intermediate and nonclassical monocytes). In table 1, CD14+ CD16+ monocytes are stated to be detected in the horse. CD16 expression on some equine monocytes (reference 16) should also be added to the section describing monocyte surface protein expression (line 213).

2. Line 512 – add “/kg” dose to human lethal dose of LPS to make the comparison with horses more comparable. These seem like significantly different lethal doses between humans and horses, which is not what is stated in the sentence prior (line 510 says humans and horses have similar LPS sensitivity). Please clarify.

MINOR:

1. Format of references to other review articles uses different brackets in different places throughout the manuscript (ex: compare references on line 86 and line 128).

2. Line 175-176 – “monocytes derived from PBMC” makes it sound like monocytes have to be differentiated in vitro. Replace “derived” with “isolated” or “purified”.

3. Line 176 and also throughout – PBMC is the common abbreviation for the plural “peripheral blood mononuclear cells” and does not need an “s” at the end.

4. Line 191-192 – fix parentheses for RANTES. I think it should say “and secreted RANTES (also known as CCL5) induction”.

5. Line 235 – I am not sure what happened to the text on this line. It is a larger font, bolded and looks like multiple lines of text on top of each other making it illegible.

6. Line 301 – I recommend changing the double negative to “it is not surprising”. The double negative could easily be misleading or confusing.

7. Line 480 – add statement with surface proteins/gene expression that distinguishes and identifies PIMs. Differences in TLR expression on PIMs is shown in Figure 4 but absent from the text. Please update the text to describe what is also shown in the figure.

8. Section starting on line 503 on the horse as an animal model for human disease research – This paragraph feels incomplete until the rest of the “interspecies variation” section is read. I recommend incorporating the information in this first paragraph into the following section OR adding a sentence to line 515 that introduces the next subsections as a more thorough summary of the horse as a model for human immunology.

9. Line 591: add “for example, postoperative ileus is….”, or something similar in order to more clearly connect this sentence with the sentence prior.

10. Lines 611-612 : confusing difference between 450 different breeds vs. 175 defined breeds. Please clarify.

11. Lines 618-620 : sentence starting with “furthermore” is unclear and seems redundant with itself.

12. Paragraph starting on line 651 – As it is currently written, it is easy to confuse the definitions of synteny and homology making this paragraph difficult to interpret. I recommend adding something that says “while 50% of chromosomes have conserved synteny, gene homology is still highly conserved between species”.

13. In Table 1, is there an estimated number of hereditary conditions conserved between mice and humans? It would be more informative under diseases to have the first section say “number of hereditary conditions that serve as models for humans” and then to put the number estimate in each box : >100 for horses, and a number range for mice.

14. Excellent figure additions. In Figure 4, please label green circle with MD2 similar to how Lipid A or dsRNA are labelled. Consider introducing the figure from left to right. This would mean describing TLR2/MyD88 signalling (red arrow) first, then TLR 4/MyD88 (yellow) and TRIF (black dotted) second and finally TLR3 (blue arrow) last.