Experimental Schistosoma japonicum-induced pulmonary hypertension

Background Schistosomiasis, a major cause of pulmonary arterial hypertension (PAH) worldwide, is most clearly described complicating infection by one species, Schistosoma mansoni. Controlled exposure of mice can be used to induce Type 2 inflammation-dependent S. mansoni pulmonary hypertension (PH). We sought to determine if another common species, S. japonicum, can also cause experimental PH. Methods Schistosome eggs were obtained from infected mice, and administered by intraperitoneal sensitization followed by intravenous challenge to experimental mice, which underwent right heart catheterization and tissue analysis. Results S. japonicum sensitized and challenged mice developed PH, which was milder than that following S. mansoni sensitization and challenge. The degree of pulmonary vascular remodeling and Type 2 inflammation in the lungs was similarly proportionate. Cross-sensitization revealed that antigens from either species are sufficient to sensitize for intravenous challenge with either egg, and the degree of PH severity depended on primarily the species used for intravenous challenge. Compared to a relatively uniform distribution of S. mansoni eggs, S. japonicum eggs were observed in clusters in the lungs. Conclusions S. japonicum can induce experimental PH, which is milder than that resulting from comparable S. mansoni exposure. This difference may result from the distribution of eggs in the lungs, and is independent of which species is used for sensitization. This result is consistent with the clearer association between S. mansoni infection and the development of schistosomiasis-associated PAH in humans.

Rebuttal: Thank you for the comment. The results section text has been revised to remove descriptions of methods, and associated references; where appropriate the methods section has been revised to incorporate this text and citations.
--------------------Conclusions -Are the conclusions supported by the data presented?-Are the limitations of analysis clearly described?-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?-Is public health relevance addressed?
Reviewer #1: Conclusions are well based by the results and bring up the perspective of the findings Rebuttal: Thank you for the positive comments.
Reviewer #2: Yes the conclusions arise from the results.
The limitations are described correctly.
Rebuttal: Thank you for the positive comments.
--------------------Editorial and Data Presentation Modifications? Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend "Minor Revision" or "Accept".
Reviewer #1: Some points would benefit of clarification / exploration if the data are available.
-the authors explored the quantity of eggs per tissue and also the thickness of the granuloma peri-egg. I wonder if the authors have quantified the number of granulomas per slide -as another form to support that S. japonicum might have a different immunogenicity Rebuttal: Thank you for the comment. We have performed the requested analysis by quantifying the number of granulomas per slide, divided by the area (mm2) of tissue analyzed, and have included this as revised Figure 4B (and associated text in the Results section). The result was no significant difference between the 4 groups (Sm/Sm; Sj/Sj; Sj/Sm; Sm/Sj). Please note that per stereological principles, the number of observed 3-dimensional objects captured in a representative 2-dimensional plane is the mathematical product of the number of objects present and the average size of the objects (eg, see Chapter 5 in: Howard and Reed. Unbiased Stereology 2 nd Edition. 2005, Garland Science/BIOS Scientific Publishers, NY, NY). For the specific example of comparing Sm/Sm to Sj/Sj, we interpret the result of no difference in granuloma number visualized as arising from the mathematical product of a non-significant trend towards more eggs ( Figure 4A) and a significant decrease in granuloma volume ( Figure 3A), respectively, in the Sj/Sj group as compared to the Sm/Sm group.
-in sensitized mice, there was no difference in IL-4. Although the authors raised the potential limitation caused by the number of animals in each group, another potential reason could be a more limited role of IL4 it self in the inflammatory cascade triggered by schistosoma. The discussion would benefit of a paragraph trying to better explain the role of each one of the ILs (4 and 13) and the plausibility of the different roles according to the different species Rebuttal: Thank you for the comment, indeed there are important differences between IL-4 and IL-13 which we had not directly addressed. The potential discrete roles of IL-4 and IL-13 have been discussed in a new paragraph in the discussion section.
-The difference in sensitized and non sensitized was an elegant way to reinforce the concept that this model is immunologically driven. It would be very nice if the authors have any data showing translation of these immunological phenomena to endothelial function. This could support potential differences between patients continuously exposed to schistosoma as compared to those already outside endemic regions minor comment -in the author summary, japonicum is mentioned twice in the same sentence, where mansoni should be the comparator.

Rebuttal. Thank you for the comment, which raises an interesting link between host immunity and phenotype of non-immune cells, such as endothelial cells. We have not previously assessed endothelial cell phenotype in Schistosoma-PH. Others have investigated endothelial cell phenotype in
Rebuttal: Thank you catching this typo, which has been corrected.
Reviewer #2: Remove text for methods and remove discussion from the results section.
Rebuttal: Thank you for the comment, which we appreciate. Text describing methods or discussion of results has been moved from the results section to more appropriate places in the manuscript.
--------------------Summary and General Comments. Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.