Simultaneous Assessment of mTORC1, JAK/STAT, and NLRP3 Inflammasome Activation Pathways in Patients with Sarcoidosis

Round 1

Reviewer 1 Report

Raisa Kraaijvanger and co-authors present a quality and well-written experimental manuscript focused on simultaneous assessment of mTORC1, JAK/STAT and NLRP3 inflammasome activation pathways in patients with sarcoidosis.

Authors aimed to explore the potential co-expression of these three inflammatory pathways in patients with sarcoidosis and see whether possible differences are related to disease outcome. They hypothesized that there will be differences between activation of these three pathways in individual patients. Furthermore, they assessed whether presence or differences in these inflammatory pathways could be linked to disease outcome. 

Authors used tissue of 60 patients with sarcoidosis to determine activity of these three signaling pathways by immunohistochemistry. They found that activation of NLRP3 was present in 85% of all patients and activation of mTORC1 and JAK/STAT was present in 49% and 50% of patients, respectively. Furthermore, presence of NLRP3 activation at diagnosis was associated with a chronic disease course of sarcoidosis.

Finally, authors conclude that their findings of different new conceptual inflammatory tissue phenotypes in sarcoidosis could possibly guide future treatment studies using available inhibitors of either NLRP3, JAK/STAT and mTORC1 inhibitors in a more personalized medicine approach.

Overall, the manuscript is highly valuable for the scientific community and should be accepted for publication after the corrections are made.

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Other comments:

1) Please check for typos throughout the manuscript.

2) Please update/improve figures and tables where appropriate.

3) Authors are kindly encouraged to cite the following article that describes the structural and functional aspects of one of the primary downstream targets of JAK/STAT pathway. DOI: 10.1371/journal.pone.0131218

Author Response

We thank the reviewer for taking the time to read our manuscript and for the different suggestions to improve the article. We have corrected multiple typos throughout the manuscript. Considering the figures and tables in the manuscript, we have looked at all the content and have decided to change nothing to the figures/tables.
The suggested article shows conformational dynamics of DMSO-treated SOCS2:EloC:EloB, indicating which parts of the complex form a rigid unit in relation to one another. The resulting structure provides a means for solving future structures and conformational changes in this structure reveal flexibility within SOCS2 that match those postulated by previous molecular dynamics simulations.  Since SOCS2 is a negative regulator of the JAK/STAT pathway there is some overlap between this article and our paper.  Although very interesting, the scope of our article was to study different regulators of the three investigated pathways and therefore we decided to not cite it in our paper.

Reviewer 2 Report

The manuscript provides a potentially novel insight into the pathogenesis of sarcoidosis by focusing on three hitherto unexplored pathways in combination with one another. The problem is of course, that these three are not working independently from a series of other mechanisms, thus immediately limiting the usability of the described pathways. Ideally, we would want to see a more thorough genetic makeup of each patient, in order to properly put these things into context (albeit, this would be a daunting task to perform, despite a theoretical advantage). The approach sounds reasonable, although I am slightly worried if the 60 patients present enough homogeneity and enough variance to be sufficient to make a solid conclusion (the balancing problem is that insufficient homogeneity will not yield significant results, while insufficient variance will not yield usable result). This is further complicated by the fact that the tissue type overlap is not complete, thus residing in a fair share of conjecture while synthesising all the evidence. The clinical status/grading of the disease is inexistent, thus further limiting the context. It would be much better if there was a thorough clinical assessment in the analysis, which might provide some dose effect signal detection. Also, early vs. Later onset of the disease might yield more relevant information. When this is coupled with varying treatment schemes and duration, one must wonder if the sample is homogenous enough. Sadly, there is no way to verify this, but only list this as a possible limitation toward the end of the Discussion. Some modest confounding effects are explored, but only suggesting more metabolically active pathways in younger patients. Do not treat P of 0.070 as insignificant, but please suggest this was a marginally insignificant result. 

Fine

Author Response

We thank the reviewer for taking the time to read our manuscript. We agree with the reviewer that it would be interesting to see the genetic makeup of each patient because of the complexity of sarcoidosis and the variance between patients.  We will include the following notes to our limitations:

• Clinical outcome status (COS) was divided into two groups: resolved (COS 1-6) and persistent (COS 7-9). This grading system limits the context regarding clinical assessment in the analysis, but was performed due to limited sample size
• Biopsy staining only gives information regarding one time point of the disease; therefore, of the following disease course cannot be visualized by this approach. Gathering at different time points during disease course would yield more relevant information on the role or the signaling pathways in the pathogenesis of sarcoidosis.