Studies On The Mechanisms Of Peritoneal Macrophages Of Different Origins In The Pathogenesis Of Endometriosis

Background:The incidence of endometriosis(EM)in women at the reproductive stage is approximately 10%to 15%and tends to increase yearly.The main symptoms of EM include infertility and various pelvic visceral pains,which seriously affect the daily life and work of the patients.In addition,the high recurrence rate after EM treatment causes great pain to the patient's body and mind.However,the pathogenesis of EM remains unclear.The existing hypothesis of EM pathogenesis includes retrograde menstruation theory,endometrial stem cell implantation theory,coelomic metaplasia theory,and Mullerian residue theory.At present,retrograde menstruation theory is widely recognized by scholars but cannot explain these two main phenomena:1)as many as 90%of women have menstrual reflux,but only a minority of them suffers from EM;and 2)the reflux of endometrial debris began to appear in women after menarche,but the age at onset of patients with EM is 25-45 years.This theory cannot explain the 10 years delay between the onset of endometrial reflux and the disease.The latest research shows that menstrual reflux may be the only possible incentive,and immune dysfunction plays an important role in EM planting,adhesion,and growth.Understanding of EM pathogenesis is still at its infancy,and the unclear pathogenesis leads to the lack of specific etiological therapy.Based on existing EM etiology,the pathogenesis of this disease must be further elucidated.Macrophage is an important component of the innate immune system and exhibits multiple functions,including antigen presentation,phagocytosis,and cytokine synthesis and release;peritoneal macrophages play a decisive role in the occurrence and progression of endometriosis(EM).After the endometrial tissue flows counter-currently into the peritoneal cavity,peritoneal macrophages are activated,enriched,and infiltrated into the ectopic tissue.The activated macrophages secrete a large number of cytokines to participate in the formation of neovascularization and nerve fibers in ectopic lesions.Clinical studies reported abnormalities in the function and quantity of peritoneal macrophages and in the expression of related cytokines[interleukin(IL)-1?,IL-6,IL-8,and IL-10]in patients with EM.Experiments using animal models of EM revealed the following:1)drug elimination of peritoneal macrophages can suppress the growth of ectopic lesions;2)lipopolysaccharide-induced conversion of peritoneal macrophages into Ml inhibits the growth of ectopic foci;and 3)M2 polarization of macrophages induced by IL-4 can promote the growth of ectopic foci.Therefore,the functional status of peritoneal macrophages could likely determine the occurrence and development of EM.However,contradicting results were reported by studies on the polarization and functional status of peritoneal macrophages in the course of EM.1)Bacci et al.found that the expression levels of CD 163 and CD206 in macrophages increased in the ectopic lesions of patients with EM and in mouse models of EM.This finding indicates that CD 163 and CD206 were converted into M2.By contrast,Takebayashi et al.conducted immunohistochemistry analysis and found that the CD206 expression in patients with EM was lower than that in the control group.2)In ex-vivo experiments,the expression of representative M1 and M2 markers increased in macrophage culture supernatants after stimulation of THP-1 induced macrophages with conditioned medium derived from endometrial cells.Beste et al.isolated,purified,and cultured peritoneal macrophages from patients with EM in vitro.A high-throughput multifactor detection technology was used to determine cytokine secretion pattern;results indicated that Ml-specific cytokines(IL-1?,IL-12)and M2-specific cytokines(IL-10)were upregulated in peritoneal macrophages derived from patients with EM.In summary,the functional status of peritoneal macrophages in the pathogenesis of EM and the specific mechanism remain unclear.Macrophages can be divided depending on their origins into tissue-specific macrophages(e.g.,microglia in the brain and Kupffer cells in the liver)and macrophages derived from peripheral monocytes.These macrophages possess different phenotypes and functions,that is,different subpopulations of macrophages exhibit different functional polarization states after receiving the same peripheral stimuli.The two sources of macrophages in the peritoneal cavity are large peritoneal macrophage(LPM)derived from the yolk sac and small peritoneal macrophage(SPM)derived from hematopoietic stem cell monocytes.LPM and SPM also have different phenotypes and functional properties.Previous studies on macrophages in EM neglected and were confused with regard to the heterogeneity of the two macrophages,leading to inconsistent results and conclusions.The imperfection of macrophage function evaluation system also leads to inconsistent research results.Macrophages are sensitive to their external stimulatory factors and undergo large-scale transcriptional regulation changes after activation.The functional status of macrophages is simply classified as M1 or M2-type macrophages.However,their metergasis is extremely complicated.M1-M2 polarization is a continuous process of functional change.Therefore,most research results cannot accurately reflect the functional characteristics of peritoneal macrophages during the pathogenesis of EM.In this regard,high-throughput experimental methods must be developed to screen comprehensive indicators of the immediate functional status of macrophages.Objective:In this study,we focus on the difficulties encountered and the contradicting findings of previous works.We will use peritoneal macrophages as the core of our research and explore the following aspects.1)High-throughput techniques were used to comprehensively assess changes in the functional status of peritoneal macrophage subpopulations in the course of EM and improve our understanding on the mechanism of peritoneal macrophages in EM.2)We will assess the effect of EM on the body from multiple layers by further examination of peritoneal macrophage-associated T cells and gut microbiota.3)Finally,we will explore new approaches of EM therapy by regulating the function of macrophages.Methods:A mouse model of EM was used in this study.1)Flow cytometry was used to detect changes in peritoneal macrophage subpopulations.Trace amounts of LPM and SPM were obtained by flow sorting technology,and their functional status was detected by mRNA sequencing analysis.2)Flow cytometry was also used to detect changes in peritoneal macrophage-associated T cells.16S rRNA sequencing was conducted to analyze intestinal microbiological abundance and compositional changes during the course of EM progression.3)Two intervention strategies based on peritoneal macrophage(CD47 antibody blockade and retinoic acid-induced intraperitoneal macrophage subpopulation transformation)were explored for treatment of EM.Results and conclusions:1)Experimental results showed that the proportion of LPM in total peritoneal macrophages considerably decreased in the early stage of EM and gradually increased in the later period.By contrast,the proportion of SPM in the total peritoneal macrophages increased significantly in the early EM period and showed a decreasing trend in the later period.After 2 weeks of EM modeling,bipolar polarization occurred in total peritoneal macrophages,and the proportions of NOS2-and CD206-positive cells increased.Further analysis indicated that LPM cells were the main source of M1 macrophages,and SPM cells were the source of M2 macrophages.These findings indicate that LPM and SPM have different functional differentiation directions in EM,leading to biphasic functional differentiation of total peritoneal macrophages.Flow sorting technology and mRNA sequencing of trace amounts of LPM and SPM were performed to comprehensively analyze and compare their functional differences.The results showed large-scale changes in the gene expression of LPM and SPM in both EM and control mice.LPM and SPM exhibited different functions in EM mice.Analysis of the KEGG pathway suggested that the metabolic pathways of LPM and SPM considerably changed.Alteration in the metabolic state of macrophages could be related to their functional differentiation/polarization.Remodeling of the metabolic state provided the required energy to transform the function of macrophages.Directly modulating the key signal pathways of cell metabolism can affect the functional differentiation of macrophages.This finding further indicates that the two macrophages exhibit different levels of functional differentiation and thus play different roles in the occurrence and development of EM.Interventional regulation of key cellular metabolic pathways can excavate new therapeutic targets in EM.2)The peritoneal macrophages of mice consist of LPM and SPM with different sources,phenotypes,and functions.LPM plays a major role in eliminating apoptotic cells in the tissue environment,and SPM is mainly involved in inflammation caused by abdominal infection.LPM tends to differentiate into M1 macrophages during EM progression,thereby promoting inflammation.SPM differentiates into M2 macrophages,which inhibit inflammation and promote the repair of damaged tissues.Therefore,LPM may play a major role in inhibiting the growth of endometrial cells in lesions by strong direct phagocytosis and indirect M1 macrophage differentiation during EM progression.Based on the flow cytometry data,the phagocytic capacity of LPM was found to be twice that of SPM.Retinoic acid plays an important role in LPM development and can induce the transformation of SPM into LPM.After administration of retinoic acid to EM mice,the markers of SPM were transformed into LPM,and retinoic acid-induced SPM exhibited strong phagocytic capacity.The CD47-SIRPa pathway is an important self-protection mechanism in innate immunity.Macrophages express SIRPa,which after binding to CD47 on the target cell surface,will inhibit macrophages from phagocytosing the target cell.The blockade of the CD47-SIRPa signaling pathway has contributed to development of macrophage immunotherapy for cancers.The effect of blocking CD47 on the survival of ectopic endometrial cells was also investigated in this study.First,tissue immunofluorescence staining was conducted.The CD47 expression in glandular epithelial cells in the ectopic endometrium was higher than that in the eutopic endometrium.Flow cytometry and live cell labeling techniques were then conducted.After blocking the CD47 molecule on the surface of endometrial cells,the ability of macrophages to engulf endometrial cells was enhanced,and the apoptosis of endometrial cells was increased.3)The peritoneal immune environment is very complex and includes a variety of immune cells,such as macrophages,T cells,and B cells.Peritoneal macrophages are key factors in the pathogenesis of EM.Once activated,macrophages can affect the differentiation of T-helper cells(Th)and T-regulatory cells(Treg)through direct or indirect interactions with other immune cells.LPM can regulate the synthesis of IgA in the intestinal mucosa-associated lymphoid tissue by regulating B1 cells in the peritoneal cavity,thereby affecting the microorganisms in the intestinal tract.Flow cytometry was performed at multiple time points to detect changes in the proportion of helper Th and Treg cells during EM pathogenesis.The results suggest that after EM modeling,the proportion of Thl and Treg cells increased in peritoneal cavity in the 6th week,the proportion of Th17 cells increased in the 4th week and continued until the 6th week,but Th2 cells ratio did not change significantly.Wherein Thl and Thl7 cells are proinflammatory immune cells,but Treg cells are anti-inflammatory immune cells.The ratio of both types of cells is increased,which is consistent with the bipolar polarization of total peritoneal macrophages and may be related to the different directions of LPM and SPM functional differentiation.This study is the first to apply high-throughput 16S rRNA sequencing technology for multiple time point assessments of EM-related intestinal microbial changes.The experimental results suggested that within 6 weeks after EM modeling,EM did not cause body weight changes and intestinal inflammation in mice.Analysis of a diversity showed no remarkable difference between the EM models and control mice within 6 weeks.However,? diversity analysis showed that the composition of the intestinal microflora in mice changed significantly in the 6th week after EM modeling.Furthermore,UPGMA and PCOA analyses at the phylum classification level revealed that the microbial composition in the intestine of the EM mice significantly differed from that in the other groups in the 6th week.LEFse analysis showed that the change of Firmicutes and Bacteroidetes was the main factor that altered the microbial flora in EM mice.The Firicutes/Bacteroidetes ratio,which is an important reference for measuring the dysbacteriosis of intestinal flora,in EM mice was found to be twice that of the control group.Hence,EM mice developed dysbacteriosis.This work improved our understanding on the quantitative changes and functional status of peritoneal macrophages derived from different sources in the pathogenesis of EM.We also initially explored new strategies for EM treatment by intervening macrophages.Overall,the results clarified the effect of EM on the body from the perspective of intestinal microbiota.