International Medical Conference Endometriosis 2025:
Endometriosis 2025: Your Mother Should Know, Your Doctor Should Know Better!
The Role of Neutrophils in the Onset of Endometriosis - Katherine Burns, PhD
Alright, so now I have the pleasure of introducing Dr. Catherine Burns, who's an assistant professor at the University of Cincinnati College of Medicine. She's specializing in environmental health and reproductive biology. Her research focuses on the impact of environmental factors on endometriosis and women's health, aiming to uncover how exposures influence disease progression and treatment outcomes. Dr. Burns.
Hello everybody in, as everybody has said, thank you for having me. Thank you for me being here. And one of the things that I was actually sitting here thinking about is years ago I met Dr. Taylor at a meeting and I just thought he was a celebrity and I still do. But the idea behind some of this is how I'm actually standing in a meeting now talking to so many of you. So hopefully we can learn something today and share what we're doing. So we're going to talk about a different immune cell, the neutrophils. And one of the things that my lab has done and worked on for a number of years is to use a mouse model of endometriosis. And I want to point out that not all mouse models of endometriosis are created equal. So one of the things that we did was very carefully use the endometrium to figure out how to develop lesions in the mouse model.
So what we did is we looked at how these tissues attach form angiogenesis. They eventually then pattern and have changes in immune modulation that occur throughout this. But then there's proliferation and pericrine signaling. One of the things that really stood out to me, because I had a number of people tell me when I first started this work, endometriosis is an estrogen dependent disease. Don't study estrogen receptor knockout animals. That's exactly what we did. And what we found is that it doesn't matter if you have estrogen at the very beginning of lesion development, there's no differences. So that's what we're going to talk about today and how we're looking at some of the cells that are very early on, the macrophages in the neutrophils, your neutrophils are going to be the cells that are your first responders. So we really hypothesized that pro angiogenic neutrophils are necessary for endometriosis attachment.
So we use menstrual fluid as an investigative tool. In our study design was to look at healthy controls in women with endometriosis. We included women aged 18 to 45 having regular menses. And for endometriosis they had to have laparoscopically confirmed disease. And you can see here are list of exclusions illusions. So one of the things that we've done differently than some studies have done is to collect menstrual fluid on day one and day two to see what is happening between these two different days. We collected for about eight to 12 hours on both days, we had patients transfer their menstrual fluid to a 50 mil conical. They kept it cold in their refrigerator and brought it to the lab within 12 to 24 hours.
And this is an example of what we've done. So what we did was a lot of trial and error to figure out how to be gentle enough with the cells not to kill the neutrophils because neutrophils are very fragile, I guess is the best way to say it. So we filter them carefully through different meshes, different strainers. We then run two different sets of per call gradients, and we then find that there is the immune cell layer that we remove. We then take them for spectral flow cytometry in which we use about 25 to 28 color markers to look at different innate and adaptive immune cells in the menstrual fluid. But one of the things that I'm most excited to show you is looking at cytosines or differentials where we've used and stained with modified gim sustaining.
So this is what menstrual fluid looks like. If you take out just all the other cell types, but the white blood cells and what we find are neutrophils and they're very happy, healthy neutrophils. In women with endometriosis, they are two to three lobed. And occasionally you'll see some, if I can point it correctly, that are maybe for lobed. But you also then see macrophages that are present and they have some level of activation, but not too much. Here's our endometriosis patient. This floored us because we didn't know what this actually meant. And this is what I'm so excited to show everybody about today because it took me years to get permission to be able to show this set of data. So what we have is the nuclei are multi lobe with three to five lobes. They have various sets of ES that you can find both within the polymorphonuclear nucleus and in the cytoplasm.
The nuclei themselves are much more condensed. They have, as I mentioned, these VAEs and then the macrophages. If you see the macrophages here, they're larger, they're more activated, they're foamer, and oftentimes they are more fragile again than as seen over here in the healthy patient. So we thought maybe we found something wrong. We kept going and kept going, and we keep finding the same phenotype is present in women with endometriosis. So our next step was to say what happens in the peritoneal cavity of women with endometriosis? And we find that same neutrophil phenotype is occurring in the peritoneal cavity.
So from here then we decided to use spectral flow cytometry. And this is just an example of one of our gating strategies, but we're looking for aged and pro angiogenic neutrophils. And our initial gating strategy is for CD 66 B and CD 1 93 negative to exclude eosinophils. And what we find is there's really no difference between endometriosis and control. However, if we then look at the specific, excuse me, cell types, specifically the age neutrophils, which are now CD 16 negative and CXCR two positive, we see about a twofold increase in the cell type looking then at the pro angiogenic age neutrophils with now looking at VEGF R one receptor, we see about a two and a half fold increase in the level of these neutrophils suggesting that there's something different with the neutrophils and endometriosis and that this may play a role in disease. Oops.
So what we did is use our mouse model of disease, and this is just a quick overview. We have a donor animal that is GFP positive. We take that animal's uterus, use the endometrium and put that into the peritoneal cavity of the host's mouse. So importantly, CXCR two is important for recruitment of neutrophils out of the bone marrow. And what we wanted to do is see how the recruitment of neutrophils are involved in the attachment of lesions. So we have four different groups. We pretreated the donor mice with IgG and then the host mouse also. Then HI IgG. We have IgG or anti CXCR two in the host animal. The donor animal then was treated with anti CXCR two or IgG, and then both the host and the donor were treated with anti CXCR two.
Our first set of experiments are looking at how the depletion changes the recruitment and attachment of these cells at 24, these developing lesions at 24 hours. So if you look at the images, we're looking at bright field GFP and then merge. We use a GFP labeled mouse model so that we can follow the tissue and be able to see the smallest of lesions. The attaching lesions are in red because they often appear more colorful if you will. They're pink, they have blood spots, whereas the lesions that are unattached are often very white and will just peel right off and come out of the peritoneal cavity. So quantitating that and looking at the attaching tissue versus the unattached tissue is directly an inverse correlation. And what we find is that when the host is treated with anti CXCR two, we see a significant decrease in the development or attachment of these lesions.
What was interesting is that even though the donor tissue, the uterus was treated with anti CX C two, it didn't have much an effect. So suggesting that the tissue in the developing lesion itself is not as important, it's what is being recruited through the peritoneal cavity. And to do that, what we looked at then is again, flow cytometry mice are much easier than humans. We're able to do phenotyping of the uterine with, I'm sorry, with the neutrophils with LY six G. We do sham animals to see what baseline is happening with a surgery of inducing endometriosis. And with anti CXCR two, you can see a decrease in the neutrophil infiltration looking then at what happens with the total neutrophils in the peritoneal fluid, as we would expect in a similar to women with endometriosis, we see a significant increase in the level of neutrophils in the peritoneal cavity.
We block this with anti CXCR two and suggesting that these recruitment is involved in the process. We then also looked similar to the human data analysis is to look at CXCR four, which is important in looking at pro angiogenic neutrophils. And very similarly we're seeing with the anti CXCR two treatment that we're blocking the recruitment of these neutrophils. So what does this mean? So the question then says, what is happening and how are these lesions forming and what is happening in these mice? So these are what the lesions look like when we histologically evaluate them at 24 hours. This staining here that is the dark purple color is staining for S 100 a eight or staining for neutrophils. And as you can see, as we go across the different treatments, there's a significant decrease then in the recruitment of neutrophils into these lesions suggesting that the recruitment is really important in the development and attachment of disease.
We then went on to look at neutrophil extracellular traps because these are what neutrophils, part of what neutrophils do are to do phagocytosis, degranulation and also NETosis. And part of NETosis is releasing the DNA as well as around these histones to create a level of neutrophil elastase and mye peroxidase. So what we did is we went and looked at the lesions specifically and also at the peritoneal fluid of these mice. And what we find is, again, correlative to who has been treated with anti cxcr two differences in the ELA or neutrophil elastase and my peroxidase. And what was interesting to us is that there's a shift between the responsiveness in the peritoneal fluid as well as the lesion compared to ELA and MPO suggesting that there's a different mechanism of action that's happening within the lesion itself versus the peritoneal cavity.
Our next step then was to look and see what we could find and understand as part of lesion attachment. And some of what we did is years ago we had created a cell line from some of our endometrioid lesions in the mouse and we used that cell line to then do an adhesion assay and with the adhesion assay then we treated the cells with peritoneal fluid of these mice with each of these different groups and looking across at some of these standard adhesion molecules and matrices. What we were able to find is that there's some binding to fibrin, but specifically we're seeing very specific neutrophil mediated response to fibrinogen. And I have other pieces and parts that we're not necessarily going into, but this is our overall proposed mechanism of potentially how the neutrophils are playing a role in disease development. So we have neutrophils that age or become pro angiogenic neutrophils, the development of the nets, what they secrete and how then they provide this provisional matrix through fibrinogen, which then we think is probably cleared out through the next different phases of disease development to form probably more of a collagen type fibrotic matrix.
But we have this provisional matrix suggesting that that is where we have some of the ability to see that first adhesion and attachment that's happening. And one of the other things that we were disappointed, but I guess shouldn't be a surprise because hypotheses are often wrong, but what we find is that there are specific signals that are coming from the lesion tissue itself and communicating with the peritoneal cavity. And I think I've touched on everything there. And with that, then I would just want to thank the people that are in the lab and the funding that has made this happen. So thank you.
