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Title: Origins and phenotypic heterogeneity of macrophages in endometriosis
Author: Hogg, Chloe Elizabeth
ISNI:       0000 0004 9349 4539
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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Endometriosis is a chronic inflammatory disorder associated with pelvic pain and infertility that affects ~176 million reproductive age women worldwide. It is defined by the presence of endometrial-like tissue outside the uterus (lesions). The aetiology of endometriosis is not fully understood however we know that menstrual effluent is refluxed into the peritoneal cavity during menstruation in 90% of women, and it is hypothesised that in some women this tissue can adhere to form lesions (Sampsons theory). Current management of endometriosis is unsatisfactory and there is an unmet need for new treatments. Macrophages play a key role in the growth, vascularisation and innervation of endometriosis lesions. We know from other diseases such as cancer that macrophages with differential origins and phenotypes play distinct roles in disease development, however our current knowledge of macrophage origins, recruitment pathways and phenotype in endometriosis is limited. In this thesis I aimed to define the origin of lesion-resident macrophages, the recruitment pathways active in disease and assess lesion macrophage phenotypic heterogeneity using the Edinburgh mouse model of endometriosis. This mouse model mimics the retrograde reflux of menses material by stimulating an artificial menstruation-like event in mice, whereby this tissue is collected and injected into the peritoneal cavity of recipient mice. I hypothesised that lesion resident macrophages have multiple origins, that CCL2 is an important chemokine for macrophage recruitment and that multiple macrophage populations exist within endometriosis lesions that have differential phenotypic profiles. I used Csf1r-EGFP mice (MacGreen; macrophages express green fluorescent protein) to characterise the origins of lesion resident macrophages. Endometriosis was induced in WT mice using donor endometrial tissue from Csf1r-EGFP mice. 16% of lesion macrophages were GFP+ endometrial-derived macrophages and the remaining 84% were GFP- infiltrating macrophages of recipient origin. To assess infiltration of tissue-resident large peritoneal macrophages (LPM) and monocytederived small peritoneal macrophages (SPM) into lesions, I adoptively transferred LPM or SPM (isolated from Csf1r-EGFP mice) into the peritoneal cavity of endometriosis mice. GFP+ LPM infiltrated lesions however GFP+ SPM were not identified within lesion tissue. Using Ly6C, F4/80 dual immunofluorescence I identified both Ly6C+ monocytes and Ly6C+ F4/80+ monocyte-derived macrophages in lesions. Mice with endometriosis exhibited a ~3 fold increase of peritoneal macrophages expressing CCR2 (receptor for chemokine (C-C) motif ligand 2 (CCL2)) compared to sham controls and 53% of lesion resident macrophages expressed CCR2. To assess the importance of CCR2 in the development of endometriosis lesions, endometriosis was induced in CCR2 knockout mice compared to WT. CCR2 knockout mice developed a similar number of endometriosis lesions to WT mice and exhibited an influx of Ly6C+ monocytes into the peritoneal cavity, which also infiltrated into lesions. Notably, endometriosis lesions from CCR2 knockout animals had fewer F4/80+ macrophages. CCL2 knockout mice developed more endometriosis lesions than WT and demonstrated similar infiltration of Ly6C+ monocytes and F4/80+ macrophages into lesion tissue compared to WT. Importantly, CCL2 knockout mice had an increase in CD45+ leukocytes within the peritoneal cavity which could have attributed to the difference observed in lesion number. Initially, I performed flow cytometry analysis on lesion tissue to assess phenotypic heterogeneity and demonstrated that multiple macrophage populations were present, based on differential expression of CCR2, MHC II, CX3CR1 and CD86. I also observed that endometrial macrophage phenotypic heterogeneity was lost upon incorporation into lesions, suggesting macrophage reprogramming upon incorporation into the lesion microenvironment. To gain further granularity into the phenotypic heterogeneity of lesion macrophages, using single-cell RNA-sequencing, I performed unbiased transcriptional profiling of CD45+ cells in lesions (6006 cells), donor endometrial tissue (1306 cells) and peritoneal lavage cells from sham (5645 cells) and endometriosis mice (6720 cells). Clustering analysis identified 5 lesionresident macrophage populations that had differential transcriptomic profiles. Comparison of lesion-resident macrophage populations with macrophage clusters from endometrial tissue and the peritoneum confirmed that lesions contained macrophages from different origins. Collectively, these results indicate that lesion resident macrophages are derived from multiple origins and that 5 different macrophage populations exist within lesions that have differential transcriptomic profiles. Macrophage/monocyte recruitment may be in part mediated by the CCL2/CCR2 signalling pathway but when this pathway is disrupted in our model of endometriosis there appears to be compensation such that monocytes are still recruited to the peritoneal cavity and lesions. Herein, I provide an important insight into macrophage biology in endometriosis and provide a platform for further studies that aim to identify disease-specific macrophage populations that could potentially be targeted for clinical benefit.
Supervisor: Greaves, Erin ; Pollard, Jeffrey ; Horne, Andrew Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: endometriosis ; macrophages ; mouse model ; CCR2 ; CCL2