The Role Of Pyroptosis In Endometriosis And COVID-19

There are a variety of physiological and pathological processes associated with regulated cell death(RCD).When the body loses control over one or more of the RCD,it will cause disease.There is a wide range of diseases related to RCD,such as development-related diseases,tumors,autoimmune diseases,neurodegenerative diseases,infection-related diseases,inflammatory bowel disease,kidney disease,and so on.Therefore,an in-depth understanding of the role and initiation mechanism of RCD under pathological conditions is of great significance for the treatment of cell death-associated diseases.Recently,with the rapid development of molecular biology,the molecular biological indicators for cell death have gradually become a research focus.Many novel forms of RCD have been identified one after another,including necroptosis,ferroptosis,pyroptosis,and so on.Among them,pyroptosis is an inflammation-dependent type of RCD,which is often mediated by inflammasomes and gasdermin(GSDMs)family proteins.The core characteristic of pyroptosis is cell membrane rupture and cell content release,so many pro-inflammatory cytokines are released during the process of pyroptosis.Many studies have shown that pyroptosis is widely involved in the initiation of various inflammatory diseases,including arteriosclerosis,enteritis,nephritis,neuritis,and infection-related inflammation.On the other hand,in addition to these inflammatory diseases that have been identified to be related to pyroptosis,there are many other inflammatory diseases that seem to be related to pyroptosis.For example,IL-1? is increased in peritoneal fluid of women with endometriosis,and severe COVID-19 had an increased LDH and IL-1? in serum.The release of LDH and IL-1? are two major outcomes of pyroptosis.These data suggest that pyroptosis may be involved in the initiation of inflammation in endometriosis and COVID-19.This study investigated whether pyroptosis is involved in the initiation of endometriosis and COVID-19-related inflammation in the ectopic endometrial microenvironment of endometriosis and the lung microenvironment of COVID-19 patients.We got the following results:Part ?:The role of pyroptosis in the pathogenesis of endometriosis1.A large number of immune cells were infiltrated in the stromal area of the ectopic endometrial microenvironmentIn order to study the pathogenesis of endometriosis,we must first understand the characteristics of the microenvironment in which the ectopic endometrium is located.we described the schematic diagram of the structure of endometriosis lesions via a series of histological assessments.Clearly understand the location of ectopic endometrial stromal cells and immune cells in it.Taking ovarian endometriosis as an example,endometriosis lesions can be divided into three parts:the outermost layer is the fibrosis area,the middle layer is the ectopic endometrial stromal area,and the innermost part of the lesion is filled with cystic fluid.The immune cells are mainly located in the ectopic endometrial area.2.T cells are the main immune cells in the ectopic endometrial microenvironmentFor further gaining insight into the local immune microenvironment of the ectopic endometrium,we analyzed and compared the immune cell composition between the ectopic endometrium and normal endometrium through Single-cell RNA sequencing(scRNA-seq),flow cytometry,and immunohistochemistry.The results showed that compared with the normal endometrium,the content of T cells in the ectopic endometrium was significantly increased,the content of nature killer cell(NK cells)was significantly reduced.There was no significant change in the content of immune cells such as macrophages and natural killer T cells(NKT cell)and B cell.Among T cells,the infiltration of CD4-positive T cell subsets is significantly increased3.Lots of active IL-16 exist in the ectopic endometrial microenvironmentBy using antibody microarray to analyze the protein components of the cyst fluid of patients with endometriosis,we found that cyst fluid contained many pro-inflammatory cytokines and chemokines.Then,we ranked the positive cytokines in the cytokine microarray according to the amount of content and found that IL-16,IL-6,and IL-8 are the top 3 abundant pro-inflammatory cytokines in cyst fluid.4.Active IL-16 can recruit and promote CD4 positive T cells to produce a series of pro-inflammatory cytokinesIL-16 is a multifunctional cytokine that exerts its effects via interacting with CD4 and CD9.IL-16 can recruit all CD4+leukocytes and is a growth factor for CD4+T cells,and this suggests that IL-16 might play a role in the accumulation and activation of CD4 positive T cells at sites of inflammation.As expected,numerous CD4 positive T cells were found to have infiltrated the ectopic endometrium.Since active IL-16 has been reported to stimulate the expression and production of various pro-inflammatory cytokines in peripheral blood monocytes,we further investigated the effect of active IL-16 on the CD4 positive T cells and found that IL-6,IL-8,TNF,and IL-1? were upregulated in the active IL-16-treated group compared with the control group.In contrast,active IL-16 exerted only a weak effect on the expression of TGF?1.These data confirmed that active IL-16 could recruit and promote the production of a variety of pro-inflammatory cytokines by ectopic endometrial CD4 positive T cells to initiate the inflammatory response in the ectopic endometrial microenvironment.5.Ectopic endometrial T cells are the main source of active IL-16In order to explore which cell type produces active IL-16 in the ectopic endometrial microenvironment.We first analyzed the expression of IL-16 mRNA in various immune cell subsets through single-cell transcriptome sequencing and found that almost all immune cells in the ectopic endometrium express IL-16 mRNA.Combined with the data that the proportion of T cells in the ectopic endometrium is higher than 80%,we believe that the T cells in the ectopic endometrium are the main source of active IL-16.Through Western blot experiments,we proved that ectopic endometrial T cells are the main source of active IL-16.6.The iron of cystic fluid promotes ectopic endometrial T cells to produce active IL-16In order to investigate the reason for the production of active IL-16 by ectopic endometrial T cells,we reviewed the results of antibody microarray and want to find the trigger.The results of the antibody microarray revealed that the cystic fluid at the focus of ovarian endometriosis contains a large amount of ferritin and transferrin.Therefore,we speculate that the iron in the cystic fluid induces the release of active IL-16 by inducing iron overload of ectopic endometrial T cells.In order to prove this hypothesis,we first proved that the T cells in the ectopic endometrium are in an iron overload state through western blot and immunofluorescence experiments.Then it was confirmed by western blot that the production of active IL-16 depends on the iron overload pathway.7.Ectopic endometrial T cells release active IL-16 through the iron overload-caspase-3-GSDME pathwayActive IL-16 is a non-classical cytokine without signal peptide,so it cannot be secreted directly outside the cell through the cell membrane.Therefore,we hypothesized that pyroptosis or other lytic cell death forms may be involved in the release of activated IL-16.By flow cytometry and western blot,we demonstrate the existence of activated caspase-3 and GSDME in ectopic endometrial T cells,block the activation of GSDME or caspase-3 could inhibit the production of active IL-16 in iron overload T cells.The results indicate that ectopic endometrial T cells release active IL-16 through the iron overload-caspase-3-GSDME axis.Part ?:The role of pyroptosis in the pathogenesis of COVID-191.A variety of cellular 'danger' signals accumulate in the lungs of COVID-19 patientsIn order to study the initiation mechanism of S ARS-CoV-2 related cytokine storms,we collected six lung tissues of COVID-19 patients and three para-carcinoma tissues of lung adenocarcinoma patients as the patient group and control group for RNA-seq transcriptome analysis.Functional analysis of the upregulated genes revealed enrichment in functions related to the Transmission of cellular 'danger' signals.Previous studies have suggested that cellular 'danger' signals are reported to activate of NLRP3 inflammasome signaling pathway.Here,through RNA-seq transcriptome analysis we found many genes associated with cellular danger' signals are upregulated in the lungs of severe COVID-19 patients.2.The SARS-CoV-2 infection causes more inflammatory macrophages to accumulated in the lungs of severe COVID-19In the process of analyzing the transcriptome sequencing of patients with severe COVID-19,we observed chemokines recruiting monocytes in the lungs of COVID-19 patients were significantly up-regulated compared to control.Through immune infiltration analysis of transcriptome and immunohistochemical staining of tissue sections,we confirmed a large number of inflammatory macrophages(CD14+CD16+CD163+)infiltrated in the lungs of COVID-19 patients.3.Inflammatory macrophages are the main producer of pro-inflammatory cytokinesTo determine whether these infiltrating pro-inflammatory macrophages are involved in the SARS-CoV-2-associated cytokine storm,a series of experiments was performed.First,we measured three cytokines including pro-inflammatory IL-1?,IL-6,and IL-18,which were included in the SARS-CoV-2 associated cytokine storm,in the lung tissues of severe COVID-19 patients and control donors.Second,we co-stain these pro-inflammatory cytokines with macrophages,As expected,we confirmed that pulmonary macrophages of COVID-19 could release a number of proinflammatory factors including IL-1?,IL-6,and IL-18,suggesting that pulmonary macrophages are the main source of pro-inflammatory cytokines and involved in the SARS-CoV-2-associated cytokine storm.4.Inflammatory macrophages ignite the SARS-CoV2 associated cytokine storm through the pyroptosis pathwayIn our previous results,we found that a series of genes related to cellular 'danger'signals,which could active the NLRP3 inflammasome pathway,was upregulated in the lungs of COVID-19 patients,so we speculate that the process of pyroptosis may be involved in the initiation of SASR-CoV-2 related cytokine storms.By observing the pathological characteristics of the lungs of patients with COVID-19,we found that the pulmonary macrophages of patients with COVID-19 have the characteristics of swelling and edema,which is a typical morphological feature when the cells undergo pyroptosis.Then through in vitro experiments,we proved that SARS-CoV-2 can indeed induce pyroptosis.Cleaved GSDMD is a classic indicator for detecting cell pyrosis.In order to further prove that the pulmonary macrophages of COVID-19 patients did undergo pyroptosis,we tested the expression of cleaved GSDMD in the pulmonary macrophages of COVID-19 patients.The results show that there is a large amount of cleaved GSDMD in the macrophages of COVID-19 patients.These results indicate that inflammatory macrophages ignite the SARS-CoV2 associated cytokine storm through the pyroptosis pathway.