PREreview of Irgm proteins attenuate inflammatory disease in mouse models of genitalChlamydiainfection

Review of: Dockterman, et al. July 9, 2022. “Irgm proteins attenuate inflammatory disease in mouse models of genital Chlamydia infection.” BioRxiv. DOI: https://doi.org/10.1101/2022.07.06.499085

This review was written by an undergraduate student at Mount Holyoke College (MA, USA) who is a member of the Lijek Lab which also studies Chlamydia trachomatis and immunopathology in mouse models. The methodology of this review was carried out following ‘A Rubric to Evaluate Preprint Peer Reviews’ (https://doi.org/10.5281/zenodo.6471333) which was created collaboratively by undergraduates at Mount Holyoke College as an assignment in a course taught by Dr. Rebeccah S. Lijek.

COI: The review author declares no conflict of interest and has no personal or financial relationship with the study's author.

Summary

The manuscript investigates the role of Irgm proteins in murine Chlamydia infection. Murine Irgm proteins have been shown to be important for host defense against intracellular pathogens. The authors use C. trachomatis in primary mouse embryonic fibroblasts (MEFs) and both C. trachomatis and C. muridarum in their mouse models. The authors measured bacterial burden and pathology to show that Irgm knockouts display increased immunopathology with limited changes to burden. I believe that this research is important and the manuscript can be strengthened by incorporating the suggestions herein.

Strengths

• Studying gross pathology on day six gives that data more weight. Because the difference in bacterial burden at this time is not significant, the pathology seen can be separated and the role of Irgm is better understood. 

• Infecting the mice with C. muridarum transcervically rather than intravaginally allows for a better comparison of the effects of Irgm in Chlamydia infection.

• Using both primary MEFs and mice allows for both in vivo and in vitro assessment which provides an increased likelihood of reproducibility and translatability. 

• I appreciate the thoroughness put into the evaluation throughout the narrative of the manuscript. The flow between experiments and experimental groups felt like a logical progression. 

Major Critiques

• In addition to the wild-type control, I believe that showing a mock-infected Pan-Irgm mouse serves as a helpful comparator group. Specifically, within the pathology results, this would allow us to separate the inflammatory effects of Irgm knockout from the response to Chlamydia. It would also be helpful to understand how often changes such as granulomatous formations occur in uninfected Pan-Irgm mice. 

• I found the findings regarding inflammation and pathology confusing. If the audience for this paper is pathologists, I believe the current explanations would be sufficient. I believe that additional clarification on how these characteristics were defined and seen would be beneficial. A few examples are as follows: 

  • In Figure 2B, the neutrophils and edema are clear but I am not confident in what the arrowhead in the middle denotes. 

  • Additional elaboration as to how histiocytic inflammation was noticed and defined would help to better understand these findings. 

  • How is the difference between edema and dilation defined?

• Some of the figures have issues with pieces being missing or mislabeled. I believe that adjusting this would help with the comprehension of the manuscript. A few examples are as follows:

  • The figure legend for Figure 1A states, “letters denote statistical significance,” but does not provide a key to indicate what the significance is.

  • For Figure 6, the figure legend does not match the labeling of the images. 

  • Figure 2G shows flow cytometry data not described in the manuscript. The quantification of neutrophils adds to the characterization of pathology, but it would be helpful to see specific data and methodology. 

Minor Critiques

• What is the speculated difference between the changes seen in Irgm1/3 and Pan-Irgm knockouts? If Irgm2 is believed to be ineffectual, then Irgm1/3 and Pan-Irgm should not carry much of a meaningful difference. Providing additional information or rationale here would be helpful.

• Where and how is ‘tissue destruction’ being measured or defined? If it isn’t, please take this term out of the results section or include justification. 

• Is there Chlamydia within the granuloma seen in Figure 3? Knowing this could help to separate the role of Irgm in general from its role in Chlamydia infection. 

• It could be helpful to define some abstract qualities that are being looked at such as host fitness vs host resistance vs disease tolerance. This could create specific parameters for the reader to follow. Alternatively, the authors could instead name the qualities looked at, such as pathology or cell-autonomous immunity, to clarify.

• In Figure 1A, I would appreciate being able to see the undivided data for the bacterial burden in MEFs. This would allow the reader to evaluate the different conditions and IFNɣ treatment for themselves.

• The cystic formations seen in the wild-type uterus of Figure 6 would benefit from some additional explanation. I’m curious if this is a coincidental finding or somehow connected to the experimental question.

• The conclusions in lines 20-2 of the discussion section (pages 15-16) of the PDF are overly strong without better statistical data, as it stands, it reads as an overinterpretation.

Competing interests

The author declares that they have no competing interests.