International Medical Conference Endometriosis 2025:
Endometriosis 2025: Your Mother Should Know, Your Doctor Should Know Better!
Immunology of Endometriosis-Hugh Taylor, MD
My name is Danielle Luciano, and I'll be introducing Dr. Hugh Taylor, who is the Anita O'Keefe young professor of Obstetrics and Gynecology and Reproductive Sciences at Yale School of Medicine and the chief of Obstetrics and Gynecology at Yale New Haven Hospital. He is a renowned researcher and clinician. Dr. Taylor has made groundbreaking contributions to the understanding and treatment of endometriosis, infertility, and reproductive health. Dr. Taylor,
Well, thank you very much. Appreciate it. Glad to be here with you. I want to update you with this talk and some of the work we've done in immunology. That's not something I've really focused on for most of my career, but the last couple of years we've been doing more and more, and I've got a few people in my laboratory group are here joining today, have done a lot of this work and want to thank them for being here. But I think we'll all agree that the immune system plays a large role in endometriosis, and it is a disease of inflammation. I think you know the pathogenesis of endometriosis, there's this retrograde menstruation then that causes an inflammation that leads to altered adhesion, altered invasion, increased angiogenesis, and then this immune system activation where we get a lot of inflammatory cytokines made. But the real paradox for me has always been the idea that there is also an evasion of the normal immune response. A lot of times, a lot of these cells are cleared largely by cytotoxic T cells and others. They're cleared from the peritoneal cavity, but endometriosis has this ability to evade that immune response. So there's a one hand immune activation and inflammation, and on the other hand, this immune evasion and decreased immune function.
So again, we get the endometriosis forming from the retrograde menstruation. It starts this inflammation, and there are changes in the immune complement that come into this lesion. But the lesion has its own immune profile, its own complement of immune cells, which as you'll see as we go forward, behave differently when they're part of that immune complex. Again, this immune complex in endometriosis, we believe very much in some ways resembles the inflammatory profile that we see in tumors, and the tumors have their own unique inflammatory environment. Well, one of the things we did was looked at first where these cells come from, how many are actually carried out with the endometrium in the retrograde menstruation, and how many come from bone marrow derived cells? This is some single cell seek work we've did that. We're in the process of publishing now and the green cells here, we use GFP and we transplanted GFP endometrium into wild type mice. And then we did the single cell C. And you can see that green cells, the identity of them are the fibroblasts, the epithelial cells, the endothelial cells, endothelial progenitors, but pretty much all of the immune cells are not green. In other words, they come from the host bone marrow. They join the lesion and that are transformed in the lesion rather than being some inherent immune deficiency in the retrograde menstruated endometrium.
The other thing we know in normal endometrium, again, there's a compliment of immune cells, and as the endometriosis lesions form and progress, there is a change in that immune compliment. Initially, it is a more of a inflammatory type or more of the M two macrophage, which is a wound healing phenotype that may allow for creation of the lesion, establishment of the lesion. But later it matures to the more M1 macrophage type, the more immunogenic, more inflammatory macrophage. Not only that, but once you get this inflammatory environment in the lesion itself, it impacts all of the surrounding organs. We know that the bladder, the ovary, the uterus, the bowel, are all affected by this peritoneal inflammation that originates from that endometriosis lesion. And some of the work I talked to you about before is the idea that it's not just the peritoneal cavity, but this signal from the endometriosis is really systemic.
It goes throughout the whole body. And one of the things that transmits this signal is not just these inflammatory cytokines which do enter the circulation from the endometriosis, but also these microRNAs that are these small non-coding RNAs that are made by the endometriosis lesions. And then normally these small RNAs influence gene expression in the lesions, which they do. And I'll show you that here we looked at the expression differences in some of these microRNAs in endometriosis, let seven family of microRNAs are all decreased and even more decreased in the more severe endometriosis. And they're playing a role that I won't go into here today in the endometriosis lesions themselves. But they also are packaged released into the circulation either as naked microRNAs or in microvesicles exosomes where they can travel great distances in the circulation and influence other organs. And we published some time ago now some work looking at differences in abundance of microRNAs in the circulation of women with endometriosis.
And we found that some are greatly increased, some are greatly decreased, but there are clearly differences in these microRNAs in the circulation I and from the tumor biology world, we know that these microRNAs can be taken up by other cells and influence gene expression in other cells remote from where they're produced. So they could come from the tumor. In this case endometriosis travel through the circulation to a remote organ. Today I'm going to focus just on their effects on inflammation and the immune system, but we've shown they have an effect on multiple different organ systems, including brain and liver and adipose tissue and many others. But what we looked at and what I'll show you here are some data we had looking at the effect of these microRNAs on macrophages in the circulation. So we looked at macrophages, we treated them with the micro RNA levels to reproduce the microRNA levels that are seen in endometriosis, and that's the solid black bars there.
And when we increased 1 25, which is elevated in the circulation of women with endometriosis, or when we decreased let seven using an inhibitor, which is in the decrease in the circulation of women with endometriosis, we see these macrophages took on a more inflammatory phenotype. They make more TNF alpha, they make more IL one beta more IL six more IL eight. So again, these circulating macrophages or rather circulating microRNAs are activating macrophages throughout the body creating this inflammatory environment. And when we looked in women and correlated the amount of each of these microRNAs in the circulation to the level of these inflammatory cytokines, there was a close correlation, at least for TNF alpha IL one beta and IL eight rather than IL six, but not IL eight. So there clearly a correlation, the amount of microRNA in the circulation and the amount of inflammatory cytokine production from macrophages.
So there's this whole body inflammation that at least in part is mediated through not just the secretion of inflammatory cytokines locally by the endometriosis, but by this activation of macrophages throughout the body. Then we looked at not only those macrophages in the circulation that are normal, but disease associated macrophages, the macrophages that take on a new phenotype in the endometriosis. And this is from a paper we published just a couple of months ago. We found, and I'll show you more detail in a minute, that macrophages and endometriosis are unique in that they express high levels of an enzyme called TET three. TET three is an epigenetic modifier. The Tet molecules, there are three of them. Tet three is particularly the one associated with endometriosis, but the Tet proteins in general catalyze the conversion of methyl cytokine to hydroxy methyl tine. So methylation of DNA is adding an extra methyl group on the cytokine base pair, and this converts that to hydroxy methylene, which eventually leads to loss of that methylation.
So it's essentially a dem methylator. Well, we looked, and I won't go through all the details in here, but this TE three is highly expressed in endometriosis associated macrophages. So you can see the TET three expression in the first, that single celle that shows that it's much higher. And then we localize that using a marker of macrophages to show that they're very high levels of that Tet three in general and endometriosis. And specifically what we show on the bottom is that TET three co localizes to these endometriosis specific macrophages. If we knock down that TE three, either I'll show you in a minute, either by using a monocyte specific knockout or by targeting it with irna. And you see here the Tet three when we use that targeting micro or rather targeting irna, the TET three goes down and it's specific to TET three, TET two doesn't go down. You can see then the protein goes down in the western and B and in C, you can see that that leads to an increase in apoptosis. This is a tunnel assay and you can see that it's greatly increased. And looking at that in two different cell lines here, you can see both that the knockdown of TET three leads to increase apoptosis.
Here we look at this again, I'll show you that both the knockdown and the knockout, the monocyte specific knockout that knocks this gene out just in macrophages. You can see here that we see a decrease when we knock that down. We see a decrease in the Tet protein, but we also see a decrease in all of these inflammatory cytokines that are associated with endometriosis IL eight IL one, beta IL six. And we show that both in the RNA on the left and the protein in the right, the top is the S irna knockdown. On the bottom is the monocyte specific knockout. When we look in the monocyte specific knockout in these animals, there's less of all of these inflammatory cytokines. So this TET three seems to be very important for inflammation and preventing proptosis. When we look at the TET three knockouts and we try and create endometriosis, we see that in the Tet three knockout, you get much smaller lesion size.
It leads to inability to create large lesions or shrinkage of the lesions when we transplant them in. You can see both for when we look at total volume or epithelial surface area in the Tet three knockout. Again, this is the monocyte myeloid specific knockout. They don't form large lesions. And you can see again in the bottom, we co localize a lot of the expression of these inflammatory cytokines to these disease associated macrophages. The macrophages that are specifically in the endometriosis when we knock in the knockout, those inflammatory cytokines are all dramatically decreased and again, localized to those macrophages. We also not only looked at the knockout, but there's a small molecule inhibitor of TE three called bobcat that blocks the enzymatic activity of TE three, and it's something that has a very good safety profile, possibly could be a drug. And when we treat animals with endometriosis with this small molecule inhibitor of Tet three, again, the lesions shrink. So we get a very nice effect and we also see a reduction in all of those inflammatory cytokines. And again, co localizing that expression to the macrophage monocytes that specifically decreased expression there.
So here's a summary of our model. These macrophages normally produce various molecules, but in the disease associated macrophages, there's a change in these molecules that are expressed more inflammatory cytokines. When we knock Tet three down, it blocks through Lett seven, the production of several of these inflammatory cytokines and also leads to apoptosis. So TET seven rather Tet three is really clearly a very important regulator of these disease associated macrophages, not so much in macrophages from other areas, but specifically dramatically elevated in the disease associated macrophage. I'm going to now talk a little bit about the role of the T cells. So that's how one of the ways that these macrophages are causing the inflammation, but it's also a defect in T-cell IMMUNOSURVEILLANCE T cells. The cytotoxic T cells normally would clear endometriosis lesions, and that's not happening adequately in endometriosis. So you get this, again, inflammatory endometriosis nodule where many of the cells are behaving differently.
I've already shown you how the macrophages behaved differently, but also there are dysfunctional T cells within the lesion itself. The T cell induction and tolerance involves molecule PD one and PD L one. The ligand PD one is the receptor on the T cells and PD one, L one and L two are the ligands for that receptor. And we'll show you in a minute. In the T cells associated with endometriosis, we see defects in this signaling. This is a signaling pathway that's probably best known for its rolling tumors that tumor cells cancers make the ligand, which blocks the receptor on the T cells and prevents immune surveillance that clears cancers. Here's a PD one on the antigen presenting cell. The macrophage makes that ligand PD L one. The PD one receptor on the T cell then is activated when presented with PD L one and prevents that from that cytotoxic activity from happening with immune clearance of a tumor or in this case endometriosis. Well, others have shown this is in our work, but others have shown that the PD one, PD L one is expressed at higher levels in endometriosis. So clearly we suspect there's a role there.
This is a little small and hard to see, but what it's basically showing you is that the macrophages here, the disease associated macrophages are making that PD one or rather PD L one, the ligand and making it very high levels. And we show that it co localizes again to those disease associated macrophages specific to the endometriosis, not the macrophages in other areas or in normal endometrium. Again, in cancers, the PD one, PD L one checkpoint inhibitor prevents that immune cells from destroying the tumor. And it's been a mainstay of therapy now where we can start to block that interaction. And it's been tremendous results in many solid tumors. I'll skip right to that many solid tumors that blocking that interaction of PD one with its receptor has shown tremendous success. And there are many commercially available now, PD one antibodies or PD L one antibodies that block this activity and no longer allow tumors to suppress the cytotoxic activity of T cells, activate them and they go on to attack the tumor.
Well, here we looked at what these antibodies might do. In our mouse model, we created endometriosis. We try to anti PD one antibody, and we see it decreases the lesion size. We also look at epithelial sickness thickness. Sometimes the fluid remains just as we know from the endometriomas that that endometrioma fluid may still be there even after successful medical treatment. So we look at also the epithelial sickness to see the effect on the endometriosis itself. And again here, the anti PD one antibody decreased that nicely. We also show that it decreases the number of T cells, the brown standing here, and I don't know how well you can see that from the back of the room, but there are fewer T cells in rather more T cells in the lesions that we treat with this antibody. So again, more activated T cells, more T cells that infiltrate the lesion.
And we think that's responsible for clearing the lesions. Again, we use not only the PD one itself antibody, but the PD L one antibody and the very same thing, decrease in lesions, decrease in lesion epithelial thickness, and more infiltration of activated T cells. So these lesions are being destroyed by overcoming that immune blockade. Here we look at what the PD one are doing, both rather in a macrophage shell line and in human macrophages, primary cells as well. And when we take conditioned media from endometriosis stromal cells, we can see it induces more PD L one expression by the macrophages. Matter of fact, if we tried a lot of things, the one we focused on the most that seemed to have the biggest, most robust effect from that condition media was IL one beta. IL one beta really turned up the expression of PD L one from the macrophages.
And when we use an anti IL one antibody to block the expression or block the activity of IL one beta in the condition media from endometriosis, we're able to block that PD L one expression. When we look, not only does that PD one coming from the macrophages lead to changes in T-cell function, it also changes endometrial stromal cell function. And we show that PD one promotes stromal cell invasiveness here, an invasion assay, you can see that's increased. So we've really got a couple of things going on the endometriosis. A derived stromal cell makes some IL one beta, which activates the endometriosis associated macrophage to make more PD L one. PD L one is increased, it blocks the T-cell activity, so you don't get the cytotoxic T-cell effect on the endometriosis itself. And it also feeds back on the endometrial stromal cells themselves to increase invasiveness.
So it's not only an immune response but a direct effect on the endometrium itself. So I'll conclude, I've got zero minutes. I talked a little bit fast, but I think I timed it just about right. Endometriosis clearly is a disease of immune dysfunction and there are several types of immune cells that include the endometriosis associated macrophages, which really are a different type of macrophage. I've shown you that circulating macrophages, which are more your traditional macrophages are also affected by microRNAs and other means. And T-cells are very clearly affected. And I think many of our future therapies will start to think about treating these immune dysfunctions in endometriosis. And our initial targets include blocking those endometriosis associated macrophages with things like that Tet inhibitor that I showed you, the Tet three molecule called bobcat, which increases T-cell access to the lesions. And I'll end there, acknowledge a lot of the people in my lab, some of whom are here today, and Chen Wong has shown in the insert who is really the Tet three expert who did that work with me. So thank you. Good minute for.
