Transcriptomic Establishment of Pig Macrophage Polarization Signatures

Round 1

Reviewer 1 Report

The manuscript submitted by Li et al. entitled "Transcriptomic establishment of pig macrophage polarization signatures" aims to propose new biomarkers for diagnosis in diverse clinical settings including porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), Toxoplasma gondii (T. gondii), porcine circovirus type 2 (PCV2), Haemophilus parasuis serovar 4 (HPS4), Mycoplasma hyopneumoniae (Mhp), Streptococcus suis serotype 2 (SS2), and LPS from Salmonella enterica. In this study, the authors obtained two M1 macrophages (M1_IFNγ+LPS, and M1_GM-CSF) and two M2 macrophages populations (M2_IL4+IL10, and M2_M-CSF), and compared the transcriptomic profiles between and within macrophage phenotype.Then, they identified 730 DEGs by comparing M1 (merging M1_IFNγ+LPS and M1_GM-CSF) with M2 (mering M2_IL4+IL10 and M2_M-CSF).

In general, the manuscript is well-written, with an Introduction section that clearly introduces the objectives of the work. The M&M section allows other researchers to repeat the experiments. Regarding the results, the are novel and have interest to the field. Indeed, the results reported reveal several new aspects, that bring new data and also allow the validation of the knowledge published about the human and mouse macrophages.

The main criticisms that this reviewer have about this work are the follows:

1) The authors should present and discuss the major limitations of the study e.g. the use of 7 day old Duroc × (Landrace × Yorkshire) hybrid pigs to infere the general behavior of the pig macrophages;

2) Discuss the number of MHC haplotypes in the macrophage functions;

3) underline the use of non-SPF animals to obtain the macrophages;

Author Response

Dear Editor,

We are delighted to receive the positive response from you and the reviewers. We have closely reviewed the reviewers’ comments and carefully revised the manuscript to address all concerns. All comments with our point-by-point responses are listed below: Detailed responses to Reviewers

Below, all critiques and suggestions provided by reviewers are cited in gray italics, and our responses are in black. Moreover, all revisions in the manuscript are marked in red.

Reviewer 1

Comment 1-1:

The authors should present and discuss the major limitations of the study e.g. the use of 7 day old Duroc × (Landrace × Yorkshire) hybrid pigs to infere the general behavior of the pig macrophages.

Response 1-1:

The main limitation of this paper is we built the correlation using the downloaded public transcriptome data and it worthy for ous further investigation in the future, and we added descriptions as “Nevertheless, we can never ignore the bias using public downloaded transcriptomic data.” In lines 264-265.

Talking about the animal used, indeed, at the very beginning, we tried using the adult pig to run the experiment, but hindered by several limitations, for example, adult pig is huge and the bone is hard to broken in lab, great efforts are needed to handle the material; meanwhile, the stem cells from adult pig didn’t poloraized very well thus we followed previous reported protocol using yound piglet, as well as the cost reason [1–3]. We also added limitation declare as “Of course, considering the operability of the laboratory and the better differentiation effect of young piglets, we only used the 7-day-old DLY, which has certain limitations.”.

Comment 1-2:

Discuss the number of MHC haplotypes in the macrophage functions.

Response 1-2:

Thanks for the valuable suggestion on the crucial role of MHC haplotypes in immune cells. We checked the overall expression of the MHC gene family and we  added the description as “It is worth mentioning that four MHC haplotype genes were detected expressing in porcine bone marrow-derived macrophages， such as CIITA, HLA-DOB, HLA-DRA, SLA-DMB, and two of them, HLA-DRA and SLA-DMB, are differentially expressed, comparing M1 versus M2 [39].”

Comment 1-3:

underline the use of non-SPF animals to obtain the macrophage.

Response 1-3:

Yes, non-SPF animals may have been immuned by microbial pathogens and further affecting the polarization of macrophage. However, we did not directly collect the macrophages from the pig, instead, we obtained the macrophage difference from the hematopoietic stem cells isolated from bone marrow, which suppose to have few concerns in the aspect of pre-immune. Also, plenty of works have been reported to establish macrophage from BMDM using non-SPF animals [4,5].

References

1. Kapetanovic, R.; Fairbairn, L.; Beraldi, D.; Sester, D.P.; Archibald, A.L.; Tuggle, C.K.; Hume, D.A. Pig Bone Marrow-Derived Macrophages Resemble Human Macrophages in Their Response to Bacterial Lipopolysaccharide. J. Immunol. 2012, 188, 3382–3394, doi:10.4049/jimmunol.1102649.
2. Kapetanovic, R.; Fairbairn, L.; Downing, A.; Beraldi, D.; Sester, D.P.; Freeman, T.C.; Tuggle, C.K.; Archibald, A.L.; Hume, D.A. The Impact of Breed and Tissue Compartment on the Response of Pig Macrophages to Lipopolysaccharide. BMC Genomics 2013, 14, 1, doi:10.1186/1471-2164-14-581.
3. Song, Y.; Song, L.; Wan, X.; Shen, B.; Fang, R.; Hu, M.; Zhao, J.; Zhou, Y. A Comparison of Transcriptional Diversity of Swine Macrophages Infected With TgHB1 Strain of Toxoplasma Gondii Isolated in China. Front. Cell. Infect. Microbiol. 2020, 10, 1–16, doi:10.3389/fcimb.2020.526876.
4. Trouplin, V.; Boucherit, N.; Gorvel, L.; Conti, F.; Mottola, G.; Ghigo, E. Bone Marrow-Derived Macrophage Production. J. Vis. Exp. 2013, 1–6, doi:10.3791/50966.
5. Moreau, I.; Andreoni, C.; Caux, C.; Saeland, S.; Rigal, D. Modification of Human Long‐term Bone Marrow Cultures: Establishment of a Functional Stromal Microenvironment Devoid of Myeloid Progenitors. Eur. J. Haematol. 1992, 49, 29–35, doi:10.1111/j.1600-0609.1992.tb00910.x.

Author Response File: Author Response.docx

Reviewer 2 Report

This paper seems very interesting, look at these points to improve it:

- Lines 65-75: "The ability to diagnose or cure a variety of..." It is not clear what is the purpose of this paper. Please state it better in this point.

- Lines 93-96: "To verify the reliability of polarized macrophages... markers for M1 and M2, respectively" Authors should add more details about this result.

- Lines 231-234. How does this study translate to humans? Some missing references should be added: --- PMCID: PMC9169371 -- PMCID: PMC9223226 --  PMID: 35841593

- Lines 266-268: "At the same time, we used the four established gene characteristics to identify various pig infectious diseases with prognosis and predictive values." Where was this discussed in the results?

- Table 2 is too long. Try to reduce it.

Author Response

Dear Editor,

We are delighted to receive the positive response from you and the reviewers. We have closely reviewed the reviewers’ comments and carefully revised the manuscript to address all concerns. All comments with our point-by-point responses are listed below: Detailed responses to Reviewers

Below, all critiques and suggestions provided by reviewers are cited in gray italics, and our responses are in black. Moreover, all revisions in the manuscript are marked in red.

Reviewer 2:

Comment 2-1:

Lines 65-75: "The ability to diagnose or cure a variety of..." It is not clear what is the purpose of this paper. Please state it better in this point.

Response 2-1:

To better declare the purpose of this paper, we rewrite this part as “Since different macrophage phenotypes are profoundly involved in the the development and outcome of many microbial infected diseases and are key cells in controlling mormal physiological processed, here we question whether a restricted set of gene signatures could be applied to define a particular functional phenotype encountered in the context of mcrobial infected diseases” to displace Lines 65-75: "The ability to diagnose or cure a variety of...".

Comment 2-2:

Lines 93-96: "To verify the reliability of polarized macrophages... markers for M1 and M2, respectively" Authors should add more details about this result.

Response 2-2:

We changed the description as “To verify the reliability of polarized macrophages, 49 classical macrophage marker genes were retrieved from the previous literature, containing 29 and 20 macrophage markers for M1 and M2, respectively (Supplementary S2 Table). Consistently, all 29 classical M1 and 20 M2 markers are highly expressed in M1 and M2, respectively (Fig.1C, Additional file1)”.

Comment 2-3:

Lines 231-234. How does this study translate to humans? Some missing references should be added: --- PMCID: PMC9169371 -- PMCID: PMC9223226--PMID: 35841593.

Response 2-3:

Thanks for the rigorous point, PPARG, NTRK1, ADIPOQ, FLT1, HMOX1, IL6, CD4, IGF1, IL10, PTGS2, FOS, PPARG, are all those genes are highly conserved ortholog genes across humans, pig as well as mouse. And we added the citation of the papers as suggested.

Comment 2-4:

Lines 266-268: "At the same time, we used the four established gene characteristics to identify various pig infectious diseases with prognosis and predictive values." Where was this discussed in the results?

Response 2-4:

Lines 154-178, Part 2.3, we described the correlation analysis between the four types of polarized macrophages and macrophages infected with different microorganisms using downloaed transcriptomic data. Based on this, four macrophage models were constructed to identify various porcine infectious diseases with prognostic and predictive value.

Comment 2-5:

Table 2 is too long. Try to reduce it.

Response 2-5:

We adapted the table as suggested.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors answered positively to all requests. Thus, in the opinion of this reviewer, the manuscript reaches a suitable form to be published.

Reviewer 2 Report

Good.