Dear Dr Stijlemans:

Thank you very much for submitting your manuscript "Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection." (PPATHOGENS-D-19-01509) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

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If you have any questions or concerns while you make these revisions, please let us know.

Sincerely,

Christian R. Engwerda

Section Editor

PLOS Pathogens

David Sacks

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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Reviewer 1 has raised several points that we would like you to address without further experimentation. Please address all their comments and let us know how you have changed the manuscript. Thanks.

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Here, Stijlemans et al. use IL-10 reporter mice to analyze potential unexpected sources of IL-10 during T. congolense infection of mice. These experiments show that hepatocytes from infected mice express IL-10, both in vitro and in vivo. The authors also show that either parasite or parasite products can stimulate hepatocytes to express IL-10 in vitro. The authors explore the biological relevance of hepatocyte-derived IL-10 using a genetic system (Albumin-Cre.IL-10fl/fl). In these in vivo studies, various cellular, inflammatory, and physiologic parameters are compared among T. congolense-infected TgAlbCre-IL-10-/- mice (but not appropriately matched genetic controls that include either mice with WT alleles of IL-10 or IL-10 floxed mice lacking expression of Cre) and either WT and LysM-Cre.IL-10 fl/fl mice. The rationale for focusing on myeloid-derived IL-10, but not T cell-derived IL-10 is not provided. The experiments show that mice lacking hepatocyte-derived IL-10 expression exhibit elevated parasite burdens and accelerated and exacerbated morbidity and mortality, compared to either WT mice or mice lacking myeloid-derived IL-10.

The paper is generally well written, easy to follow and understandable. The key strength of the manuscript is the unexpected phenotype in T. congolense-infected TgAlbCre-IL-10-/- mice. However, key weaknesses include the substantial volumes of correlative data that appear to mainly derive form a time point when the bulk of animals in the TgAlbCre-IL-10-/- cohort have succumbed to infection. Another weakness relates to studies shown in Figure 12. The studies in Figure 12 both lack comprehensive experimental control groups (e.g. anti-MIF-treated WT mice and irrelevant/non-specific Nb reagents) and stand in direct opposition to the primary parasitemia phenotype reported in Figure 1A.

Other questions and concerns are noted in Parts II and III below.

Reviewer #2: T congolense infection is affecting the livestock making it to a serious problem. Protection is associated with an early and strong Th1 response which has to be tightly regulated to prevent immune pathology by hyper inflammation. An already known and very important factor is IL-10. In IL-10 KO mice are suffering from disease symptoms which are similar to affected cattle.What the authors have convincingly shown is that the important source of IL-10 are not immune cells but hepatocytes.This hepatocyte-derived IL-10 can be triggered by trypanosome lysate or soluble VSG.

The manuscript is in general well written and methods are very good described. Several control were undertaken to really verify the key finding that IL-10 is made by hepatocytes and the importance of this IL-10 was known using tissue specific IL-10 ko mice.The consequences of the lack of IL-10 are clearly described. One important factor that papered to be regulated by IL-10 is MIF. Neutralizing MIF can prevent over inflammation in this model.

In conclusion the manuscript addresses an important disease and provide new insides in immune regulation which might also operate in other diseases.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1) The last sentence of the introduction is an overstatement. The data do support that hepatocyte-derived IL-10 is essential, but the authors have not shown that this is the “only” source that is critical. T cell-specific IL-10 KO may very likely result in the same susceptibility phenotype, and the authors have not explored this. This is also relevant to the authors’ lack of clearly defined rationale for experimentally focusing on myeloid-derived IL-10, but not T cell-derived IL-10. That myeloid cells could produce IL-10 is not much of an argument for ignoring T cell-derived IL-10. Have others already used T cell-specific IL-10 deficient genetic systems to show that T cells are (or are not) a biologically relevant source of IL-10 during T. congolense infection? If those experiments have been published, the reference(s) should be provided. More rationale should be provided.

2) Figure 4: All of the "significant" cellular and inflammatory perturbations occur after parasite control is lost in TgAlbCre-IL-10-/- mice (after day 18, Figure 1A) and after at least half the TgAlbCre-IL-10-/- mice are dead (day 45, Figure 1B). The authors should attempt to reconcile these data. Why do TgAlbCre-IL-10-/- mice lose control of the infection after the second week? Why are the TgAlbCre-IL-10-/- mice dying? These analyses of cellular and inflammatory perturbations, which are quite superficial, precede neither the loss of parasite control nor the death of the mice so it is not at all surprising that very sick mice with very high parasite burdens exhibit these perturbations.

Given the critical role of secreted antibody in controlling this parasite, an obvious question relates to whether humoral immunity derailed in the absence of hepatocyte-derived IL-10. Measuring total cells in the blood does not reveal whether critical circuits/networks such as germinal center reactions, plasma cells, B cell activation, antibody secretion, CD4+ T follicular helper cell numbers and function, or Th1 cells are impaired. Minimally, some discussion is warranted. Relevant to this point, Page 19 lines 13-14: there are no data to support that reduced B cell numbers could explain the elevated parasitemia, given that B cell numbers remain identical through day 28 to 38 p.i., which is well after the TgAlbCre-IL-10-/- mice have lost control of the parasite (which occurs on 18 p.i., day according to Figure 1A).

3) Figure 12: These data represent a very interesting and exciting attempt to understand the inflammatory, parasitological, morbidity and mortality phenotypes in observed in TgAlbCre-IL-10-/- mice, compared to WT mice. As described, one cohort of TgAlbCre-IL-10-/- mice was treated on day 30 p.i. with regular injections of a nanobody targeting host MIF. The data in Figure 12 show that neutralizing MIF abrogates the exacerbated anemia phenotype observed in T congolense-infected TgAlbCre-IL-10-/- mice, but has no effect on modulating parasite burdens. Based on these data the authors argue that MIF signaling mediated the phenotype observed in TgAlbCre-IL-10-/- mice. The authors should specify which phenotype.

This lack of precision aside, there are concerns regarding the parasitemia data shown in Figure 12A. According to these results, there are no differences in parasite burden in untreated WT and untreated TgAlbCre-IL-10-/- mice on either day 35, 40, or 45 p.i. (days 5, 10, 15 post-treatment, respectively, as shown on the graph). However, Figure 1A shows that by either day 30, 35, 40, or 45 p.i., the TgAlbCre-IL-10-/- mice should exhibit burdens that exceed the WT mice by 80% or more (TgAlbCre-IL-10-/- mice have substantially elevated parasite burdens by days 18, 34, 40 and 48 as shown in Figure 1A). Thus, the data in Figure 12 stand in clear opposition to the data shown in Figure 1A and undermine the robustness of the model and data sets.

Reviewer #2: No further experiments required

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 1) Typographical error, Page 7 line 5. Extend should be “extent.”

2) Page 8 line 5: Either data should be shown or a reference should be provided to support the statement that, “levels of IL-10 progressively increase during the course of infection…”

3) The authors argue that IL-10 functions in an autocrine manner to potentiate IL-10 production in hepatocytes. This should be shown by either culturing hepatocytes with recombinant IL-10 or blocking IL-19R signaling in the cultures treated with either lysate, parasites, or VSG.

4) The legend for Figure S1 states that TgAlbCre-IL-10-/- mice did not show any detectable IL-10 protein levels. However, the graph shows substantial proteins levels from leukocyte pools from the TgAlbCre-IL-10-/- mice. Please clarify.

5) In Figure 3, the dashed red line should be defined. Do these lines denote the limit of detection of the assay, or levels measures in naïve mouse sera?

6) Why are the mice infected with T. brucei dying? It appears to have nothing to do with parasitemia (Fig. S2A), which therefore would leave inflammatory immunopathology as the likely cause, which similar to the T. congolense model, would be expectedly counteracted by IL-10. Why might hepatocyte-derived IL-10 not matter after day 25-20 in the T. brucei experimental system?

7) Figure 5D: It is not clear that the IFN-g, TNF, MIF and IL-6 serum data collected on day 45 p.i. represent fundamentally new information given that the relative cytokine levels and relationships were established on day 48 as part of Figure 3.

Reviewer #2: The authors showed that also IL-6 is very differently regulated (indeed more than MIF). Some information should be given why they concentrate on MIF and not IL-6. Indeed new therapeutic strategies to modify IL-6 in humans are already near to clinical application.

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Reviewer #2: No

Dear Dr Stijlemans,

We are pleased to inform that your manuscript, "Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection.", has been editorially accepted for publication at PLOS Pathogens. 

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Best regards,

Christian R. Engwerda

Section Editor

PLOS Pathogens

David Sacks

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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Reviewer Comments (if any, and for reference):