Reviewer #2 (Public Review): Endocannabinoids and their receptors modulate endometriosis pathogenesis and immune response

Peer review process Revised: This Reviewed Preprint has been revised by the authors in response to the previous round of peer review; the eLife assessment and the public reviews have been updated where necessary by the editors and peer reviewers. Read more about eLife’s peer review process.Editors - Reviewing EditorSang Jun HanBaylor College of Medicine, Houston, United States of America - Senior EditorTadatsugu TaniguchiThe University of Tokyo, Tokyo, Japan Reviewer #1 (Public Review): Summary: The endocannabinoid system (ECS) components are dysregulated within the lesion microenvironment and systemic circulation of endometriosis patients. Using endometriosis mouse models and genetic loss of function approaches, Lingegowda et al. report that canonical ECS receptors, CNR1 and CNR2, are required for disease initiation, progression, and T-cell dysfunction. Strengths: The approach uses genetic approaches to establish in vivo causal relationships between dysregulated ECS and endometriosis pathogenesis. The experimental design incorporates both bulk and single-cell RNAseq approaches, as well as imaging mass spectrometry to characterize the mouse lesions. The identification of immune-related and T-cell-specific changes in the lesion microenvironment of CNR1 and CNR2 knockout (KO) mice represents a significant advance Weaknesses: Although the mouse phenotypic analyses involves a detailed molecular characterization of the lesion microenvironment using genomic approaches, detailed measurements of lesion size/burden and histopathology would provide a better understanding of how CNR1 or CNR2 loss contributes to endometriosis initiation and progression. The cell or tissue-specific effects of the CNR1 and CNR2 are not incorporated into the experimental design of the studies. Although this aspect of the approach is recognized as a major limitation, global CNR1 and CNR2 KO may affect normal female reproductive tract function, ovarian steroid hormone levels, decidualization response, or lead to preexisting alterations in host or donor tissues, which could affect lesion establishment and development in the surgically induced, syngeneic mouse model of endometriosis. Reviewer #2 (Public Review): Summary: The endocannabinoid system (ECS) regulates many critical functions, including reproductive function. Recent evidence indicates that dysregulated ECS contributes to endometriosis pathophysiology and microenvironment. Therefore, the authors further examined the dysregulated ECS and its mechanisms in endometriosis lesion establishment and progression using two different endometrial sources of mouse models of endometriosis with CNR1 and CNR2 knockout mice. The authors presented differential gene expressions and altered pathways, especially those related to the adaptive immune response in CNR1 and CNR2 ko lesions. Interstingly, the T-cell population was dramatically reduced in the peritoneal cavity lacking CNR2, and the loss of proliferative activity of CD4+ T helper cells. Imaging mass cytometry analysis provided spatial profiling of cell populations and potential relationships among immune cells and other cell types. This study provided fundamental knowledge of the endocannabinoid system in endometriosis pathophysiology. Strengths: Dysregulated ECS and its mechanisms in endometriosis pathogenesis were assessed using two different endometrial sources of mouse models of endometriosis with CNR1 and CNR2 knockout mice. Not only endometriotic lesions but also peritoneal exudate (and splenic) cells were analyzed to understand the specific local disease environment under the dysregulated ECS. Providing the results of transcriptional profiles and pathways, immune cell profiles, and spatial profiles of cell populations support altered immune cell population and their disrupted functions in endometriosis pathogenesis via dysregulation of ECS. L386: Role of CNR2 in T cells: Finding nearly absent CD3+ T cells in the peritoneal cavity of CNR2 ko mice is intriguing. Interpretation of the results is well-described in discussion. Weaknesses: The study was terminated and characterized 7 days after EM induction surgery without the details for selecting the time point to perform the experiments. The authors also mentioned that altered eutopic endometrium contributes to the establishment and progression of endometriosis. This reviewer agrees L324-325. If so, DEGs are likely identified between eutopic endometrium (with/without endometriosis lesion induction) and ectopic lesions. It would be nice to see the data (even though using publicly available data sets). Figure 7 CDEF. Please add the results of the statistical analyses and analyzed sample numbers. L444-450 cannot be reviewed without them. This reviewer agrees L498-500. In contrast, retrograded menstrual debris is not decidualized. The section could be modified to avoid misunderstanding. The authors addressed all my concerns. I do not have any comments.