PONE-D-20-26981

Blocking IL-10 signaling with soluble IL-10 receptor restores specific lymphoproliferative response in dogs with leishmaniasis caused by Leishmania infantum

PLOS ONE

Dear Dr. Oliveira,

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PLOS ONE

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3. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere:

'Some data on lymphocyte proliferation has been previously published, however, detailed information on cloning and in vitro evaluation of soluble canine IL-10 receptor was no included in the previous publication.'

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors identified and produced a recombinant canine sIL-10R1 and used that protein to block IL-10 signaling in vitro. Blocking of IL-10 signaling was done in vitro in PBMC obtained from infected and healthy / uninfected dogs. The work is interesting and can be considered for publication in Plosone, however some points must be clarified.

• Please modify the title to make it clear that this is an in vitro study. As it is, it makes us think that some treatment was done in vivo on the animals. In this sense, also modify the discussion and conclusion sections.

• In the abstract, define the abbreviation CanL as canine leishmaniasis, as it should be written (line 25).

• In relation to healthy animals, the authors should consider that they cannot exclude the possibility of Leishmania infection only by the result of blood qPCR. Some authors have shown in the literature that the load / determination of the parasite in the blood of infected animals can fluctuate during infection, sometimes showing negative results. Authors should associate the method used to identify parasites in the blood with serological methods such as DPP Leshmaniose or Elisa as currently described in the literature.

• In lines 77-78, the authors described that “Dogs that develop the symptomatic form of leishmaniasis exhibit higher IL-10 and lower IFN-γ concentrations in the blood, while the inverse it true in asymptomatic dogs [34, 35]”. However, the authors must consider that these results are controversial in the literature. Some authors have demonstrated a reduction in the expression of pro and anti-inflammatory cytokines in symptomatic dogs, including IL-10.

• The figure 1 caption looks confused. Please indicate only the letters A and B as shown in the figure.

• How was the statistical analysis of the data represented in figure 4?

• Figure 4 shows the effect of blocking IL-10 signaling in the presence of recombinant IL-10 and IL-4 on mast cell proliferation. Why wasn't the test also performed using only recombinant IL-10 and casIL-10R? Blocking this signaling appears to have had little effect on mast cell proliferation.

• In the caption of figure 5, please indicate that the recovery of the lymphoproliferative response was detected in vitro using cells obtained from dogs. Thus, it does not appear that any treatment has been done on animals in vivo.

• In figure 5, include the letters A and B for the upper graph and the lower graph, respectively.

• The authors wrote in lines 68-70 that “IL-10 also inhibits some T lymphocyte functions indirectly through decreased antigen presentation [24] and directly by inhibiting CD4 T cell proliferation and cytokine production (IL-2 and IFN-y) [25, 26]”, and in lines 277-280 that “Dogs with leishmaniasis exhibit limited specific-cellular immune response and increase in IL-10 production [34, 47], to determine if blocking IL-10 signaling would revert Leishmania- 9 specific lymphoproliferative unresponsiveness, CFSE labeled-PBMCs from healthy or infected dogs were cultured together with, or without, rcasIL-10R1, and with or without the addition of SLA, or in the presence of PHA alone for five days.” In this context, how could the authors explain that cells from healthy donors stimulated with PHA proliferated less than cells from sick dogs? As shown in figure 5.

• In relation to the above comment, it would be interesting to measure the production of IL-10 from the culture supernatant and dog plasma. It would also be interesting to measure the production of IFN-g and TNF after blocking IL-10 signaling.

Reviewer #2: It is an interesting paper, which propose the obtaining of a recombinant canine soluble IL-10R1 receptor using bacmid expression in insect cells and that this recombinant protein could restore in vitro lymphocytes proliferation in response to antigen from dogs naturally infected with Leishmania. However some points should be revised by the authors:

Line 40 – aggression replace by damage

Line 74 lower case for chagasi

Line 230. The authors could provide the comparison of amino acid sequence of human and canine IL-10 receptor R1

Line 266-272 and Figure 4 legends - The results description should be rewritten considering the percentage of inhibition as well as the authors could show the data as % of inhibition. It is better visualized.

Other point is about the experiments: what was the n of experiments ? one experiemtn in triplicates, two ??? and the statistical analysis ? Is there any statistical test applied ? It is not described in the graphic and legend.

Figure 5- Please, identify the graphics as A and B . It is quite confused this data , the graph below seems to show the proliferation of lymphocytes from disease dogs, and PHA induce cells a significant profliferation compared to medium alone, since a reduction on CFSE MFI is observed. Therefore the lines 289-287 describe “In diseased dogs, although CFSE-labeled lymphocytes cultured with PHA showed reductions in MFI, these were not statistically significant (Fig 1B).

Line 287 - Fig1B should be Fig 5B

Line 288 - These data should be better explained : “ Lymphocytes from diseased dogs showed proliferative response when cultured with rcasIL-10R1, regardless of the addition of SLA to cultures (without SLA addition, rcasIL-10R1: 2.9, 2.0, and 10.2 vs medium: 128, 117, and 205; with SLA addition, rcasIL-10R1: 3.8; 1.3, and 12.1 vs medium: 121, 91 87, and 176) (Fig 5B).”

Sincelery.

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Reviewer #1: Yes: Fernanda Morgado

Reviewer #2: No

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Blocking IL-10 signaling with soluble IL-10 receptor restores specific lymphoproliferative response in dogs with leishmaniasis caused by Leishmania infantum

PONE-D-20-26981R1

Dear Dr. Oliveira,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Yara M. Traub-Csekö

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

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Reviewer #1: No