Transcription
All right, well, we're gonna get started. I'd like to welcome everybody to the AB sponsored symposium on Hidradenitis Suppurativa: Translating Medicine, Basic Science into Improved Therapies. And I'm Mike Rosenbloom, and my co-chair for this session is Amanda McLeod. And before we begin, we just have some announcements regarding the formality. So, as you know from the other sessions, we ask you always to turn off your cell phones and to not take any pictures during the presentations because we want to really maintain that every speaker can present unpublished data here. We want to make sure that you disclose any conflicts of interest that you have before your talk, both on your slide, and if you don't have it, also in a that you say it out loud. Please stick to your time slot. And for the audience, if you stand up and have questions, please introduce yourself and ask your question. Thank you.
Okay, so, so first up, we have Cathy Smith, who's a principal scientist at AbbVie, and she's going to give a quick introduction. But before Cathy begins, I just want to thank, on behalf of Amanda and myself, AbbVie for sponsoring this research symposium and actually having an interest in this disease. And so they've helped really push things forward in the HS front. And so for that, we'd like to thank them for this for this symposium and the opportunity. And my disclosures are, I work for AbbVie. So the first thing I really wanted to, I wanted to speak briefly because I wanted to first thank Amanda McLeod, who approached me at the IID last year and came up with this idea, and we have pushed this internally. And I also like to thank Priska Honoré, who's head of our dermatology team, who's really supported me and helped me push this through and get the funding for the project. I'd like to thank all of our dedicated scientists who are committed to understanding HS biology. We have some posters. We have a poster here today, and Bob Dunston gave a talk earlier this week. And I also wanted to note that this year, we have 16 abstracts on HS and 7 talks for in this session. Last year, there were 9 posters, and of the posters, there was only one that addressed any aspect of basic science, and that was Angel Bird's poster, and she's going to give a talk today. And we're really excited to have some basic science done in this very important disease. Thank you.
Thank you so much. So let's move to our first speaker, Dr. Michele Loes. And we're really happy that she will give the first talk on this HS R during this HS symposium because she's the godmother of HS research and has really helped in in in putting, I think, this whole area of scientific endeavors forward. And so she's working in the Laboratory of Investigative Dermatology at the Rockefeller University, and she's very active on the Medical Board of the Hidradenitis Suppurativa Foundation. And there's no better person than her than to give this introduction to HS here today. Thank you.
Thank you. First, I also want to echo thanks to the SID for highlighting HS at this symposium. I think, and at this meeting, I think we've heard a lot more about it than before. And to thank the people who asked me to speak, the organizers, and to AbbVie for sponsoring this symposium. So I'm going to start off just sort of framing the problem and where we're at with HS, and then what are some of the steps we're taking to address these gaps that exist. And then give you an outline of where we think pathophysiology is at the moment, so that you can understand the talks that are coming after me. So these are my disclosures. And, and I really want to start out by saying that HS is a neglected disease, and there really is a lot of work that we need to do. And I think we need to do this together. There is something for everyone in this field because we, there is something for basic scientists, outcomes researchers, translational scientists, clinical trials, as well as clinicians and our industry partners, because I think we'll, we know what we need to do, and we'll get there much faster if we work together. So if you like what you hear today, I encourage you to come to our fourth symposium on HS Advances, that will be held in Detroit later this year. And this is a really wonderful community of people dedicated to solving this disease, and the abstract portal is open, and we do have some travel grants for young investigators.
So what is HS? And it is a clinical diagnosis. It is, I like to think of it as typical lesions in typical places. And it's recurrent. So the lesions need to occur for over six months to make the diagnosis. And it occurs in the armpit and groin, and as well as other areas such as submammary, the buttocks, lower abdomen, and, and it's this constellation of symptoms. And it can take a long time for the penny to drop for someone to actually make the diagnosis. So the lesions of HS, well, just well shown in this slide. This is an axillary vein advanced patient with advanced HS. And here you can see the characteristic nodules and abscesses, and then we see dermal tunnels that are characteristic of the disease and indicate progression of the disease. And the tunnels can become epithelialized, and once they're in that state, they're very hard to reverse with medical therapy. So that after needs surgery at that point. So this is what it looks like. And there's a lot of different comorbidities that are associated with HS. We're now understanding that there are a lot of psychological comorbidities, including depression, anxiety, and increased suicidality. So there's a two and a half fold increased risk odds ratio of suicidality in this disease. It's just not really what we used to seeing in a dermatological disease. There are metabolic associations, as well as signs and symptoms in the joints and the endocrine system. And Dr. Amit Garg is really leading work to do epidemiological studies to understand these relationships so that we can better treat these comorbidities.
And the first myth that I wanted to dispel is that HS is rare. It is a common disease. The claims data would suggest that it's about 0.1% of the population, but I think that's a gross underestimate. And that's because there's about a 10-year diagnostic delay to get to be told you have HS. That's a lot of doctors you can see before someone actually tells you what your, what your symptoms are. And Vincent Pagai and John Ingram published a paper recently showing that it's more like 1% if you can really think about the symptomatology in that period of not being diagnosed. So it's quite common. And second is that it, it really does impact on quality of life. And the other name for hidradenitis suppurativa is acne inversa. And even at this meeting, I had someone say, "Isn't it just pimples in the armpit?" And that is not what HS is at all. It really is a debilitating disease that affects people in a substantial way. And this is a word cloud patients wrote their description of their symptoms, and the thing that jumps out at you is pain. And I was actually late to appreciating how pain is one of the dominant symptoms of this disease. And, and that's something that we really need to think carefully about. You know, dermatologists are not really used to treating acute and chronic pain, or acute perhaps, but not chronic. And if pain is, if this aspect of care is something that you want to read some more about, Dr. Serene Patel is a psycho-oncologist who's now at Memorial Sloan Kettering, has written a very good comprehensive review of where pain is. And we really need to develop some guidelines to treat HS-related pain.
So this is a very understudied disease. The red line is the number of publications per year in HS. And to put this in context, there are more publications in psoriasis in one year than there have ever been in HS. So we have some work to do. The federal funding has been, there's been $865,000 given for HS grants, and it's so small, but it doesn't even register on the graph compared to psoriasis, that's $1.5 billion. So there have been nine grants to career development awards, one small R03, and some intramural funding. And then recently, Dr. Jocelyn Kirby is the PI of an R13 grant to help support the conference, which is really exciting and has been renewed for this year. So when Ricardo Saavedra is the program official for skin and inflammatory diseases at NIAID, and when he realized this data, he did something about it. So he and Steven Katz were able to put a funding opportunity together for specifically for HS. And I encourage you to think about, you know, your program of research or clinical trials, and, and work with other investigators to bring your knowledge and expertise to study HS, because that's how we're going to move forward. The HS Foundation also has a small grants program, but, you know, we're really excited about being able to provide some funding for pilot studies. And one of these are our awardees so far, and you're going to be hearing from Angel Bird later this morning.
So the clinical studies in HS are also quite limited. There's been 17 completed clinical trials at the end of last year, and that's why the landscape looks like this, with they're only been one FDA-approved systemic biologic treatment for HS, which is adalimumab. So we have, we have some work, some studies to do. I think part of the reason that it's been hard to do these studies is that the clinical course of HS is not really well understood. And Helen Arlook is going to be the principal investigator on a study to characterize patients over time and take biospecimens. That will be a national effort, and I'm really excited about this, this effort called HS-Progress. And there'll be lots of opportunities for participation and collaboration. So we work, that's a really exciting initiative. And the other challenge has been sharing outcomes. So the International Dermatology Outcomes Measurement Group has HS as a subgroup. And this, this is a partnership between patients, physicians, and industry to try and find measures that we, things that we want to measure, and how we're going to do that. And I think that's really going to be able to move this forward as well.
So just in the last part of my talk, I want to sort of set the stage for where we're at in terms of pathophysiology. I like to think about it as, you know, what starts the process, what causes these characteristic lesions, and then how does it develop? Because I think we need to think about this almost separately. There was a really nice review by Eero Prinz and his group, sort of outlining this process. And we think that at the top of the hair follicle, there's some sort of obstruction to that that leads to dilatation of the hair follicle. And his, and ultrasounds are showing that dilatation process. And then there's something that causes this to continue and becomes a cystic space that ruptures laterally into the dermis. And this seems to cause an innate immune response. And this may or may not be different to that of a regular abscess. But then there's something that causes this to progress, and the patients develop these epithelialized tunnels that are very inflammatory. And that process is, is part of very characteristic of the progression of disease. So we don't really understand the role of sweat glands yet. So this happens in areas that are rich for apocrine glands, the armpit and groin, and these apocrine glands drain into the top of the hair follicle. So it could be that they're just innocent bystanders. We really don't know. And we've proposed that there's a number of possibilities for the role of these sort of hollow sebaceous apocrine gland unit that could be causing this, but we don't know yet. And then Amanda MacLeod recently published a very nice paper just last week looking at the eccrine gland genes and proteins, showing that there's dysregulation. So we really need to think about, you know, what are the sweat glands doing in this disease?
And then in terms of the chronic inflammatory process, Joerg Fritz has published two really nice systematic reviews trying to lay the groundwork for where we're at in terms of the cellular composition and the cytokine expression. And there's a diagram in his one of these two papers showing the relationship between these cells. And then I just want to finish by showing you a sort of, we published a paper last year looking at some transcriptomic data and trying to understand how it all fits together. So we think that at the top of the hair follicle, as I mentioned, there's this follicular obstructive process, which we don't know what the primary cause of that is. And there may be a role for the cutaneous microbiome in in this initial initiation phase. And that leads to these a cycle of abscesses that rupture and resolve. And then in the dermis, there are now products that they wouldn't normally be. So there's keratin and cells and dermal bacteria that we think could be very important for driving the next phase of the disease. There seems to be dysbiosis, so alteration in the normal composition of the bacteria that are normally found in the skin. And then they can form biofilms. And this is a picture showing a biofilm aligning a dermal tract. And these biofilms, bacteria that basically covered themselves with material that protects them from being eliminated. And then this might be what drives a persistent immune response that causes damage in the dermis, leading to separation, the tunnels, the fibrosis, and a systemic immune response. And then these are just some of the elements that we've tried to plug in that might explain this process, with there being gram-negative anaerobic bacteria that are found as the disease progresses. And then I've sort of broken up the immune response into that that's shared with other chronic inflammatory skin diseases, and that's which is separate. So we know that there are keratinocyte responses, there are abundant antimicrobial peptides, there's a T helper 17 response, and there are a number of other cytokines that are elevated. So this is, but this doesn't seem to be unique to HS. And we just don't know yet what's the cytokine signature that's unique. But there are elements that seem to be specific to HS. So there's complement dysregulation, there are abundant neutrophils, there is an interferon signature, and there's a humoral response that hasn't really been investigated yet. And when we start to get these sorts of pathways, we can think about what our therapeutic opportunities are. And there are a number that are being directed at these cytokines, which is really exciting. And then there are some that are even being directed at the specific responses. So there are some anti-complement therapies that are being tried right now. But I do think we can't forget about the role of biofilms, and maybe it'll be that we will need to address that in conjunction with these other therapies. But I think that could be important for driving the relapsing nature of HS. And then of course, what is the initiating factor? We need to think about that. And then I want to thank all the people that worked on the paper with me last year, and then Elton Saavedra, who was a fearless leader of the HS Foundation, and who always reminds us to keep the patients front and center in everything that we do. Thank you.
Thank you so much. So we have time for questions. We do have some time for questions. If there's any questions for Michelle? No, you're good. All right. To bring you back up, Jason Meyer from UCSF. We saw, we had a surgeon who came to visit us to talk about pilonidal sinuses, and what he said is that he thinks it's not really a sinus. And my thought is possibly might have some relationships with HS that he described follicular origin and skin occlusion as being important. Do you have any other thoughts about that or evidence behind that?
Yes, I think he's associated with HS, and I think there is, there may be pathogenic overlaps with HS, but it hasn't really been very well studied.
Ricardo, hey, thank you. So my question is subtly weird. So Ricardo Saavedra, NIH. So, well, we know that there's a bunch of people that are interested in these diseases right now with different angles and projects and perspectives. What are they missing? You, you are the only one who can answer this question. What's the area that you have not seen addressed by Mike Rosenbloom and Amanda McLeod? What would I mean, if any?
I think we, I think we're just at the beginning. So I think there's probably a lot of things that we're not doing. I think we need to, we need to correlate histology, phenotype, genotype, so that sort of basic science understanding of the disease. We need to think carefully about the different phenotypes. It may be that we need to, the treatments will be more personalized depending on the phenotypes. I'm not sure if I'm answering your question, but it's so much we need to do.
You did, you did the etiology. Etiology is something that is very difficult to address, and I don't know if there are people going in that direction. But I think you did an etiology meeting. So the genetics component, we haven't started yet. What explains that you start having these diseases? Something, oh, very, very difficult to answer at this point. Yes, but, but hopefully by working together and everybody that is in the different specialties comes to listen to each other, they'll get ideas for how we can address them.
Michelle, wonderful talk. One of the things that has helped the psoriasis field has been animal models, basically murine models. But are there any murine models in HS that are being used at this point for experimental work?
There are no murine models, and mice don't have apocrine glands, so that's a little bit of a limitation.
I see. Oh, great. Thank you. I would like to introduce our next speaker, Megan Loes. She's from UCSF. She trained with Dr. Rosenbloom, my co-chair, and the title of her talk will be "Elucidating the Immunopathogenesis of Hidradenitis Suppurativa at the Cellular and Molecular Levels." And we're looking very much forward to your talk. Thank you so much for having me here to speak. I'm very fortunate to be presenting this work. Over the past three years, we've been doing this as a collaboration with AbbVie. So that's a relevant disclosure. Sorry, I don't have a slide. I'd like to say personally, I was one of the ranks of people who was unaware of hidradenitis suppurativa before I joined Mike's lab. I had no idea of the impact, and I've gone from that state to being almost horror-struck by seeing these large surgical incisions that we get in from the OR, and the fact that we're still having to use surgery as a treatment for this disease is really striking. So thank you so much to Dr. Loes and Dr. Bird for giving such a great overview of the severity of this disease.
So I'll jump right into the comparison for my talk. We wanted to look at the immunopathogenesis of hidradenitis suppurativa versus another chronic inflammatory disease of the skin, psoriasis. Now, any dermatopathologist will tell you that these two disease states look very different under the microscope. So you can see how psoriasis is mostly limited in its proliferation and inflammation to the epidermis and to the superficial dermis. In contrast, hidradenitis suppurativa goes quite deeply into the skin. Bob Dunston gave a great talk yesterday where he described these deep invasive epithelial tracks that usually originated around hair follicles, but they invade very, very deep, and this inflammation is very striking. So this raises the question, why are we treating these two diseases of skin? Hidradenitis suppurativa and psoriasis actually respond. Both respond to anti-TNF therapy. It's true that anti-TNF is an important treatment modality. It will give 41% of patients a response in terms of the number of their lesions. However, if you look at the two clinical trials on adalimumab with HS and psoriasis, you see a really striking difference in terms of the actual response in terms of disease scores. So patients with HS only see about a 16 to 26% reduction in their modified Sartorius score. In contrast, patients with psoriasis given an 80% reduction in their PASI score, and some of these patients are completely remit in terms of their disease, and you don't really see that when you treat patients with HS with adalimumab. And so this really makes me ask the question, can we assume that treatment approaches designed for psoriasis will be efficacious for HS? And this is really important because right now, there are ongoing trials with anti-IL-17, and that is used really well to treat psoriasis, but we still don't know whether it will work for HS patients. And we might speculate that perhaps these two disease states are very, very different. And so we then ask the question, are there unique immune pathways that can be targeted in HS that we don't see in psoriasis? So perhaps we're missing on some treatments. If a treatment modality doesn't work for psoriasis, we may just throw it out from consideration for HS, and I really don't think that's appropriate.
And so we're really lucky to have a large cohort of patients being seen for HS at UCSF. Dr. Naik is working really hard to recruit these patients, and they've given us six millimeter punch biopsies from their lesional skin and adjacent skin within the same side of the body, but that is not visibly involved. And on a small subset of samples that we looked at with H&E, it doesn't seem to be nearly as inflamed. And in addition to that, we have site-matched healthy controls. When we receive these samples, we put some of the biopsy in RNA later and perform whole tissue RNA sequencing on these samples. And the other set of the part of the biopsy, we subject to overnight digestion with collagenase and DNase, and then we have performed single-cell cytokine analysis with flow cytometry. And fortuitously enough, this was not an original name of the project, but at the same time as we were receiving these samples in the lab, we also received psoriasis samples, and we have performed the same analysis on these samples. And so the first aspect of the talk, I'll cover some of the differences that we see between these two disease states in terms of their cytokine production. So our objective was to characterize the T-cell infiltrate in the lesional skin and in the non-lesional skin by flow cytometry, and we wanted to determine the cytokine production in the CD4 and CD8 T-cell compartment. And concurrent with this, we also analyzed the frequency of regulatory T-cells in the skin. And so to do this, we gated on CD45 as a leukocyte marker, looked at the CD3 T-cell subset, these into CD4 and CD8 T-cells, and the CD4s for their subset into CD4 T effectors and the regulatory T-cells. And so for cytokine production, I'll be talking about production from these two compartments. Again, we were able to obtain data from both lesional skin biopsies as well as non-lesional skin, and we obtained data upon five different cytokines. And this is gated on the CD4 T-cell compartment. So we looked at Type 1 cytokine, interferon gamma, Type 17 cytokines, IL-17 and IL-22, Th2 cytokine, IL-10, and TNF alpha.
So what was really striking to us was being that Mike is very involved in studying regulatory T-cells. So we've observed the frequency of these cells across multiple inflammatory disease states, and we and other groups have published that regulatory T-cells are increased in frequency in psoriasis lesional skin, and we see this in this cohort as well. However, when we look at HS lesional skin, we don't see this increase in regulatory T-cells. So this is again surprising because you expect that inflammation will be followed by an expansion of T-regs in an effort to control that inflammation. However, in these HS lesional skin samples, we really don't see that. And this was completely against our initial hypothesis in starting this project. And so I'll touch back on that point later, but I'd like to also go into some of the differences that we see in terms of the inflammatory cytokine production. So as has been reported by other groups, Th17 cytokines in the CD4 T-cell compartment are increased in HS lesional skin, as they are in psoriasis versus the healthy controls. However, when we look at the CD8 T-cell compartment, we see a striking difference between these two disease states. So psoriasis lesional skin has an expansion of the CD8 T-cells, and these T-cells have also been described by others as a resident cell within the skin that actually remains there even following resolution of inflammation. So in psoriasis patients are administered anti-TNF, and their skin lesion resolves, these CD8 T-cells stay there. However, you know, in lesional skin, psoriasis, again, we see this expansion of this T-cell compartment, but this expansion is absent in HS lesional skin. And again, this has been shown by others in other inflammatory skin conditions such as vitiligo. Also, like this tissue-resident CD8 population, instead, sorry about that, instead, we see a striking difference in terms of interferon gamma production between these two disease states in both the CD4 T-cell compartment and the CD8 T-cell compartment. So, and this really, we didn't see in our lesional psoriasis cohort. What's also interesting is that the non-lesional skin, while not significantly different here, does show a trend towards being increased. So again, this was skin that was not appearing to be inflamed, but in the adjacent site. And though I don't have the data on these slides, we see an increase in other cytokines like TNF that is significant in the CD8 T-cell compartment and the CD4 T-cell compartment in non-lesional skin of HS. And so this suggests to me that there is something going on even when the skin doesn't appear visibly inflamed, and we're certainly interested in probing that further so that we might be able to get at the drivers of skin inflammation versus what's just along for the ride. But to continue, so again, in HS, CD8s are producing interferon gamma instead. And this also is similar to the findings that was done on vitiligo. So I'm not saying that HS is more like vitiligo than psoriasis, but I would like to sort of launch the idea that we don't really treat vitiligo and psoriasis the same way. And so perhaps we shouldn't really treat psoriasis and HS the same way either.
So to review, we see the adaptive immune infiltrate in HS is vastly different from that in psoriasis, and that regulatory T-cells are not expanded in HS skin, that CD8 T-cells are not Tc17 cells, but rather they're Tc1 cells producing a lot more interferon gamma, as are the CD4s as well. So moving back to our pipeline, the other aspect of this talk I would like to cover is our whole tissue RNA sequencing of these skin biopsies. And again, concurrent with this work on HS, we were doing, we have a cohort of psoriasis patients that were sequenced, not with the same, not necessarily at the same time, but were sequenced in our lab. And so I wanted to compare these two disease states at the whole tissue transcriptome level. So for this analysis, we have a cohort of patients who have not been administered biologic therapy, but they are at a severe enough disease state to warrant administration of biologics. And so we took 13 patients from lesional skin, and we have nine site-matched samples from healthy patients. For our psoriasis arm of the study, we have eight patients with conventional psoriasis and nine site-matched samples from healthy patients. But these are different healthy patients because these diseases were primarily occurring on different sites. And so a principal component analysis shows that for each of these analyses, these samples are segregating quite far apart. So you can see the healthy skin in black, these are individuals, patient samples, as segregating quite far from the lesional skin in each disease state. And again, I would like to point out that these analyses were separate. So for each disease state, I am comparing the lesional sample to its internal healthy skin counterparts. So these are not the same healthy controls across these two sequencing analyses. And so there are thousands of genes that are differentially expressed between lesional skin in each condition and healthy controls. And so to compare these two transcriptomic analyses, we wanted to look at the pathways that were shared and were divergent between these two disease states. And so we turn to Ingenuity Pathway Analysis to do this. And so this analysis is showing regulators upstream of differentially expressed genes between HS and healthy controls, and psoriasis versus healthy controls. And so for instance, you see LPS listed there, and that indicates that genes that are influenced by LPS are increased in both HS skin versus healthy controls and psoriasis skin versus healthy controls. So we see a lot of the usual players that we would suspect in terms of shared pathways. There's a large number of immune activating pathways increased in both of these disease states, so a lot of genes downstream of TLR activation, a lot of genes in the interferon family, consistent with Dr. Bird's work, a lot of genes in the IL-17 family, and also some anti-inflammatory regulators, IL-1 receptor antagonist and IL-10 are reduced in these two disease states. I will point out that consistent with our cytokine data, interferon gamma is increased in both, but is more upregulated in the HS skin than it is in the psoriasis skin comparison.
So the more interesting aspect of this analysis is what's different between these two diseases. So we know a lot of inflammation, inflammatory pathways are shared, but what is different? And so one of the, when I look at the pathways that are up in HS with a Z-score of greater than two and are going in two different directions in psoriasis, I saw a lot of immune regulators that were uniquely active in HS skin. And this is interesting because psoriasis has been described as primarily an activated T-cell driven disease that is sustained by some inflammatory dendritic cells. But we don't really know about what's going on in terms of inflammatory cells, macrophages, or macrophages, dendritic cells. Now, we're learning more about neutrophils in HS. And so we see though some divergent regulators in terms of innate immune activation between these two disease states. So TNF superfamily 11 is Rangel and it promotes dendritic cells. CSF1 and CSF3 are important in macrophage and neutrophil support. And interestingly, IL-3 has been reported to promote IL-3 stimulated dendritic cells, promote CD8 secretion of interferon gamma, which ties back into what we saw in terms of cytokine production. Beyond the immune compartment, we also saw striking differences in terms of fibrotic pathways, where many pro-fibrotic pathways are divergently regulated when comparing psoriasis to HS. And this is really interesting because Dr. MacLeod has just published a paper where she looked at HS pathways and saw pathways up in HS were shared and healing, which is also intimately linked with fibrosis. And so we, we know that psoriatic inflammation really extends deeper than the superficial dermis, but that HS lesions can progress to this hypertrophic scar formation. And so this perhaps is, is supported by our clinical observations, but it's still quite striking that, so psoriasis is really going in the opposite way of HS.
Now, I promised you that I would tie this back to regulatory T-cells. So we have several active projects in the lab, which you heard some of yesterday at Mike's talk, looking at the role of regulatory T-cells in terms of supporting skin homeostasis. And so one of the postdocs in our lab, who has recently gone on to a wonderful new job in a biotech company, Lucky Kalakara, has been working on looking at how T-regs influence fibrosis. And so he's seen that, he's seen that actually they play a very important role. So when you graft skin on a humanized mouse, an NSG mouse, and apply human skin, there is a dramatic increase in alpha-smooth muscle actin staining, which you can see here in brown. When you transfer in regulatory T-cells, this is dramatically reduced. And in our mouse models, we are also able to see the reverse, in which we can deplete T-regs using a Foxp3-Cre-DTR model. So we administer diphtheria toxin, T-regs are reduced, and we see an increase in alpha-smooth muscle actin staining. And so this is striking because in HS, we don't see this increase in regulatory T-cells, but we do see all of these fibrotic pathways. And this leads us to hypothesize that perhaps something going on in the T-reg compartment that is not allowing these cells to control fibrosis and inflammation could result in what we're seeing in our data. Again, this is just a hypothesis. We're certainly looking forward to exploring this further.
So for our future work, we have a lot of interesting things going on. We just finished a first experiment doing single-cell RNA-seq on cells from HS lesions. And so we're looking forward to collecting more samples and really probing what's going on on the single-cell level in terms of the transcriptome. We also have a CyTOF panel up and running where we can immunoprofile HS skin from lesional and non-lesional sites and compare it to healthy skin. And we're seeing, as expected, dramatic changes in the immune compartments. We've also begun work on some models to test compounds, the efficacy of compounds ex vivo and in a humanized mouse model. So for our ex vivo culture model, we are taking skin from HS lesions, digesting it, and then subjecting it to different biologic therapies, and we are able to see changes in CD4 and CD8 T-cell activation in vitro following a short culture period. And finally, we have successfully grafted HS skin upon a humanized mouse, and we're looking forward to proceeding with that so that we can test candidate compounds on a sample that's more physiological.
I'd like to thank all the members of the Rosenbloom lab who've worked really hard, and also Dr. Naik for really driving our clinical arm of this study, not only recruiting patients but clinically phenotyping them and providing really important insight into how we can analyze this data. Our UCSF, our surgeons are also critical in getting us larger excisional samples. Our collaborators at AbbVie have been really important in research discussions apart from the funding. And of course, our patients. We couldn't do this without them. And I'll take questions now. Thank you.
We have time for questions. Ricardo, wonderful, wonderful job, congratulations. So they are very different subtype of diseases, different stages dictated by the number of lesions and how many fistulas you have. Have you seen any correlation with this and the absence of T-reg cells? And what about your T-helper one phenotype? If you can correlate somehow this type of severity with your phenotype?
Um, so we are still, I do not see, well, the T-regs are unchanged. So I didn't see a strong association with the disease severity in a first pass analysis. But I did mention that all of our cytokine data is from patients who are, the data I presented is from patients who are prior to administration of biologics, so they are at a higher severity of disease. And so that's that. I do, however, have more recent data where we have analyzed data from the larger excisional samples from the OR. So these are very severely inflamed sites, and we actually T-reg percentages dropped dramatically in that analysis. So in a biopsy on a patient who's less severe, we see no change in T-regs, but a drop in patients who are more severely inflamed.
So you didn't show the TNF alpha beta? Yeah, yeah. So I can tell you about it. It's basically our average for production of TNF alpha for healthy patients is around 60%, and it's around 90% of CD4 and CD8 in lesional skin of HS patients. So sky-high. But the TNF production in the skin is for healthy patients is much higher than other cytokines. So it's hard to know what is, you know, we suspect that's related to inflammation, but it's hard to know what the appropriate amount of TNF production in the skin really is.
I think that's really fascinating. Thank you for the talk. I just wanted to suggest that it's possible that not all these alterations are pathogenic, and so especially in the case of HS, because I think the distinction between inflammation alone versus actually some features of infection, it's hard to make sometimes. So besides getting data later about interventions and whether they're effective, do you have any other suggestions about how that can be sorted out?
Yeah, how to tell whether something, well, it's not even necessarily pathogenic versus just, it's also what's along for the ride. I think it's hard to sort out. I think that looking at non-lesional skin is really key in these patients versus a healthy patient, because it's that, I think, will tell us what is maybe predisposing sites to inflammation arriving before the whole malady comes in.
I have a question too. It was a great talk, was a great overview over the kind of like immunophenotype of HS and psoriasis. But we talked or learned earlier that there's a big prevalence of African-American populations that actually suffer from HS. So my question is, in your analysis, was African-American skin included? Was it controlled for? Was it sex and age and gender controlled for on all levels?
Right. We do have a, so we do have a large cohort. So I've looked at the representation in our HS cohort, and it's the characteristic of the representation of minority populations and there's a, and the disease state. So the healthy skin, we don't, unfortunately, the way we get our healthy skin is discarded from OR. So we have eliminated demographics information. We do match in terms of site and gender, but the immunophenotype would there was no difference or were there differences?
No difference between, oh, we I don't think we've done that analysis. We are definitely looking at gender differences, and there are some gender differences and site differences.
Right. No, I mean, it seems like there's so many, I mean, alluding to the question that Ricardo asked earlier about, you know, what can be studied, I mean, even from yours and Dr. Bird's talk and Dr. Loes' talk, there's so many, you know, immune cells, pathways that that I think we don't understand. So I think that's really great. But let's get one more, one more question, maybe.
Maggie, great talk. I was just wondering about, and Victor, maybe again, so it's like, it's really good that you did this analysis, but I'm wondering if there's any plan to compare HS potentially with atopic dermatitis? And here, I mean, it sounds crazy, but like, if you look from like, 30,000 feet up, there's some similarities there, yet the diseases are completely different. You know, for example, non-lesional skin in both diseases really not normal. They're multiple immune cells, multiple immune pathways involved. There is a parent role not just for the immune system, but also for keratinocytes in both diseases, etc. So just wondering if there is any idea to maybe compare these and try to see what, what's different between these two?
Certainly. Um, so I think we're lucky because UCSF, the research support, they're so strong that we do get a lot of patients from these different disease states, and that's ongoing work in the lab. I mean, certainly from the cytokine analysis, it's some of our standard procedures in the lab, we can definitely do that. But I think extending that even further to our CyTOF panel is going to be really important, and that's definitely in the works.
One brief and last question. We really, we are behind schedule, but we love the discussion, so that's why if you don't want to stop it. But one brief question. Thank you. It's usually from a higher father speedo. Very nice talk. Quick question to Yoder Sir. No, the HS, you know, African Americans have a high frequency in. Did you assess white people or you know, African relativity and the child comparison? The phenotype, they are different, you know, phenotype.
So I haven't done that formally, but it's in the plans to do so. And certainly, so we can't really do that comparison versus our healthy, but we can do it internally and segregate into different groups.
The your data says so why do people are ever kind, many people hold both, both always together for this analysis? Yes.
Thank you. Okay, thank you so much. That was great. So, so next up, we have Heidi Kong, who's going to talk about a very important aspect of the disease, the microbiome.
Afternoon. Thank you to the organizers, the funding support for this symposium, and inviting me to participate because it really highlights the increasing interest and the increasing research going on in this disease, which, as we've heard, has many unmet needs. So I'll be talking about the skin microbiome in hidradenitis suppurativa. I have no conflicts to disclose.
So I have divided my talk into two parts: What is known about the human skin microbiome, and why am I doing that? First, because it's important to understand what is normal so that we can then decide what do we need to do to study a disease. And then I'll finish off by talking about the skin microbiota that are found in hidradenitis suppurativa. So first, definition of microbiome. What is it? So it's the collection of bacteria, fungi, and viruses, and their collective genomes. So not only what is there, but what might they be doing there? And for those who are not familiar, because they're going to be talking about different studies, what is different between microbiome studies versus culture-based studies, which have been going on for over a century? So what we did back in 2007, when the Human Microbiome Project was initially being discussed and how that might be applied to different body sites and people, took two swabs from one single site on a healthy volunteer, on our healthy volunteers, sent one to the lab at the culture lab and the NIH microbiology lab in the clinical center, and they plated it on multiple different types of agar, aerobic, anaerobic conditions. And then the other one, we sent for direct sequencing. And in comparing the direct sequencing data versus the genomic data from all the isolates that were cultured out, we can see that there are differences. So you're going to see throughout this talk bar charts, bar and pie charts, but the essential message here is the zero to 100% relative abundance. Each of these colors represents a different microbial taxa. And so here we're talking about bacteria. And on the left-hand side is the direct sequence data, on the right-hand side is the culture data. And what is noticeable between these differences, these two bars, is from the culture data, there is much more orange, which is Staphylococci. So in regular culturing, we can see that there's some bacteria that will grow quite readily and out-compete the slower growers. And especially when we start talking about, and I'll talk about a little bit more, that the anaerobes people have mentioned in HS, this is important because it may be difficult to grow some of those. So what we're getting is unique information.
So to give you a sense of what is known over the body, we originally looked at 20 different skin sites. These were selected because they were sites of predilection for certain skin diseases. So these were not randomly selected. They were importantly, the important sites for certain skin diseases. And so we initially looked at bacteria, and we showed that there are site-specific differences, meaning that you could have two sites that were several centimeters apart, and you would see different bacterial communities. So this is where we think about, okay, if we're going to study a disease, it's very important to be very consistent in the site that you're sampling from one patient to another, or if you follow a patient over time, from one time point to another. Because fungi are also important in generally in dermatology, we also developed a pipeline to look at this in skin. And here we showed using the internal transcribed spacer region one, or ITS1, we studied fungi on skin at these same body sites. And we use ITS1 because it was helping us to differentiate and speciate Malassezia. That's what we see here in purple. And so most of the body is Malassezia. The feet are much more diverse fungal-wise. But even with Malassezia, if we get down to the species level, we can see that there are certain body sites that are much more Malassezia globosa predominant, and that some that are Malassezia restricta predominance. So we still see the site specificity when we're looking at bacteria and fungi.
What's also important in in other diseases are viruses. And so what we did was shotgun metagenomics, which allows you to take a sample, shear all the DNA, sequence all that DNA, so you can study together instead of individually bacteria, fungi, now we can look at bacteria, fungi, and viruses, specifically DNA viruses and bacteriophages. And in these pie charts, the orange is bacteria, tiny part fungi are red, and you see much more variability in the green, and that is what we observe is that there are differences with regards to even amongst these the communities of different types of microbes. That bacteria and fungi have much more site specificity as opposed to viruses, where we see much more differences across the body and across people. So what is the take-home point for these studies is that it's site-specific, and that some features and
Which may be important in something like HS is that we see some common features. So in the oily of survey show sites, we see that there are much more *Cutibacterium*, or also known as *Propionibacterium*, formerly known as *Propionibacterium*. The moist sites or creases, we have much more *Corynebacterium*. And then for the dry sites, we see much more variability over the person and between people.
What is important is that the purity, the time of puberty, is also a time that we see shifts. So while this disease in general, we look at adults, so it's important to be looking at the right controls. But if you're looking at a pediatric population for another disease, then it is important that you have the right controls because we see differences.
So another point that may be important in HS is how stable is it? Because what was not known, really had not been studied systematically, was are the skin microbes on the skin constantly changing? Because if it's constantly changing, then it would suggest that these are transient. They're there, they're gone the next moment. And then so are they really relevant? Because if it's there, it's possible that the human body is interacting with the microbes, or are the microbes potentially doing something to the host?
So this study we performed was to bring back our healthy volunteers at least a year after they come to see us initially, and then ask them to return a month after that, so we could get at least one to three years of a timeframe of what changes might be happening. And then a month after that. And here's one person to give you an example of what we saw. Each box has about three bars. Those three bars of three different time points. Each box represents one skin site that was consistently sampled on that individual all three time points. And within those three bars, you can see that there is some stability from one time point to another, some remarkable stability, which suggests is that our skin microbiome in a healthy population is fairly stable. But you do see some site-to-site variation, suggesting that it depends on where you are sampling on the skin sites, on the anatomical skin site.
Comparing that to two other individuals here, we see that if you go vertically on one of those skin sites, you see variations. So that when you're looking at bacteria, fungi, and viruses, that it is somewhat individualistic. And that would be important to think about when you look at a disease. So in general, the skin microbiome in healthy individuals is relatively stable and somewhat individual.
So now let's move to hidradenitis suppurativa with that background in place. And so what has been shown, we've talked about is that it's an inflammatory skin disease with this recurring abscess, these tunnels and scarring, with this purulent drainage in skin folds. And so important to think about, okay, what are the characteristic sites that are affected in this disease? Early culture studies have shown that there is *Staphylococcus aureus* that can be cultured, but also more importantly, anaerobes in many of these studies. And I've just only highlighted a few here. There are several other studies that have been published. And so interestingly, you see that antimicrobial approaches, antibiotics, are are part of the therapeutic regimens. I believe, you know, that's despite that, and it's still frustrating for patients, and especially we hear pain and other issues are a major component in this disease. So these, this disease is not being effectively managed. But if we believe that microbes are important, by understanding this, we can potentially provide deeper understanding of the pathophysiology, which we've heard about today, is very important. But that there potentially are different ways to manage these patients, which is important.
So there have been some studies that have compared culture versus 16S sequencing on this. With this group from France, they looked at national and non-lesion skin in different skin folds and hidradenitis suppurativa. Their cultural studies showed that 83% of the lesion-all swabs were and had anaerobes, whereas 53% of the non-lesion skin grew anaerobes. Comparing that to 16S sequencing, this group showed that there were anaerobic gram-negative anaerobic rods and that were predominated. And the general that were predominant were *Cratella* and *Porphyromonas*. We'll see some consistency with this. And that this other group, Ring et al., they published quite widely in HS, and they did the study of 16S. And in this one, they were studying specifically the tunnels. So unroofing the tunnels and taking the contents of that and doing 16S sequencing. And so what they show here in blue, you'll see in blue bars, again, haven't and *Porphyromonas* predominate. What's interesting is that they also in a prior study compared healthy volunteer skin versus HS skin, and they showed that there was less *Cutibacterium*. So what one might consider a general commensal, potentially on on healthy individuals, it was less than hidradenitis suppurativa. And in fact, if you look at the bar chart here on the on the right-hand side, you don't see it listed.
So in looking at the the studies, there's a continuing interest. And there are two abstracts that are being presented at this meeting today, Haley Nayak's abstract, as well as Andrew Schneider and Amanda Nelson's group. And so encourage you to talk to those prism abstract presenters as well. And so I'm just going to talk a little bit more about Haley Nayak's study. So this is just a snapshot of some of the data from her cohort. And looking at on the left-hand side are the health and volunteers that she recruited and have studied, as well as the hidradenitis suppurativa patients on the right-hand side. Each of these bars represents one of these individuals. On the top part, we see in the inguinal crease, and on the bottom part are axilla samples. And there and additional samples that she studied. And the important thing about this population is that she was very careful when she designed this study to ensure that these patients did not have potential confounding topicals, systemic agents, and so truly trying to understand what is the natural history of this disease. And these patients were also carefully phenotyped and staged with the Hurley stage. And the the analysis of the analysis was done by my postdoc, Jay. And so what we can begin to see, just to highlight the the major taxa here, in light blue is *Corynebacterium*, and dark blue is formerly *Propionibacterium*, now *Cutibacterium*. And so the other thing I want to point your eye out lies too is the dark orange and the light orange, again, *Cratella*, *Porphyromonas*. Some commonalities here across these different cohorts, whether they're from different parts of Europe or from the US. And so but what the other point is, is that you see that from the different skin sites, you're going to see different populations. And so that's also important to consider that where you're sampling is is important.
So looking at this on a more detailed level, looking at more the numbers of the relative abundances on the left-hand side, that's the *Cutibacterium* species. And we can see in the consistent with prior studies in the HS patients, in in Hayley's patients, that the *Cutibacterium* has a really low relative abundance in the HS patients compared to controls. And then in the middle, we see the gram-negative anaerobes are higher in HS. And we also see that gram-positive positive anaerobic cocci are higher in HS, particularly in the inguinal creases, as compared to the the axilla. So we can see that there are differences here. And these patients, what is interesting in Hayley's cohort, we can see that in blue are healthy volunteers, in red are the hidradenitis suppurativa patients. But having the rich metadata and being able to score them, she has she scored them based on her least age one, two, and three. And the triangles are early stage one, and they segregate a cluster with the healthy volunteer samples. And as the severity increases, we see that they begin to shift away from what is considered a healthy microbiome. So that we do see that this is important and to incorporate these these type of metadata and to really characterize these patients very carefully.
So in conclusion, that clinical culture and management suggest there's a role for microbiota in hidradenitis suppurativa pathophysiology. That there's expanding interest, as evidenced by this entire symposium here today, in the use of genomics to examine hidradenitis suppurativa microbiota. And that antibiotic bacteria predominate, particularly in the more severe patients. And that it differs based on disease severe. But there are challenges here in studying this disease in trying to looking at all the studies that have been published and they're currently ongoing. How do you define your collection method? What are the lesion type? Where are you going to sample them? What types of lesions are you doing? Abscesses, are you going to doing a tax scan? Are you going to do tunnels? And it's important again, have the clinical phenotyping be a major part of these types of studies. And there was already an allusion to other body sites because as in other studies, other diseases I study where we actually are looking at oral gut microbiome, because it's these are often systemic problems. And so trying not to only focus on skin, but to begin to look at other body sites will be important. And it'll be important in trying to drive some of these mechanistic diseases. But to acknowledge the the study that Haley has diligently been working on for several years. And to thank those patients who've given given of themselves are healthy volunteers for our studies and hers. And then also just a few of the people that have worked on the skin microbiome projects over the last decade plus, my close collaborator, Julissa Gray, National Human Genome Research Institute. And this is a photo from our last Cherry Blossom picnic in DC. But thank you very much for your attention, and happy to take any questions. Thank you.
Hey, we have time for a couple of questions. Just to know that we have to be out of this room, not out of this room, but in about seven minutes. So I think we have some time. That's great. Oh, thank you so much. It's very helpful. So I asked this question to Haley the other day, the increases on our board and anaerobes. So it my question is really the we're it's the skin on lesion more oily, less right? Is it the company, the change of the skin that might explain that anaerobes or something coming from inside from the fistula that might explain what you are observing? So that's the question is whether or not what is driving these anaerobes is it that there's something uniquely different about the skin in these patients, or is it potentially something about the lesions themselves, the type of lesions that are based being sampled? And that's a really good question. And I think it needs to be done more broadly to actually do some of the very, very, very, very early patients. But it would suggest that there is a role for the types of lesions that are involved because as you see the severity, those are the patients that are going to have the tracks, the sinuses. And so that may be why there is that increase in the anaerobes. But it does need to be more carefully defined. And maybe having a larger cohort of individuals who are early, early stage to determine because at this point, you're right, is this the chicken or the egg? Is this driving it, or is this a consequence of the disease and the scarring and the types of lesions that you see later enacted?
Okay, good. Amazing data. One thing that I've always been interested in knowing is when it comes to studying the microbiome, do you think that there would be a difference in looking at more a patient with HS and say, the South Mississippi versus a patient that's up this way in DC and Baltimore? Do you think that you will see a difference in that in the role of an environment, so to speak? So in the other early studies, when we looked at just healthy volunteers, we had our cohort naturally or in the DC region, and we had other other investigators in the Human Microbiome Project, they were in Colorado and other parts of the US. Yet that we saw commonalities. And so in general, in healthy individuals, we do see that their shared similarities. However, when you start to look at different populations, are those populations of patients that you're looking at, have they been managed differently? Are they using different things? And so that is one of the, I think, a critical component of Dr. Nayak's approach is trying to really avoid some of those possible confounders that could alter the microbiome.
You can, in interest of time, we can take one short question because we we have another session that comes after here that's the Herman Berman lecture. So we don't want to take any. So a brief question. One thing, more of a comment, one thing real quick. The question came out, what are we missing about HS? We simply can't. We recently completed, it's probably the most detailed histologic assessment of the disease. And everything we're leading, realized one, this is not a primary inflammatory disease. It's a primary epithelial invasive disease. I think that explains an awful lot. There is no evidence really that this disease starts with an inflammatory process, rather extends from an infiltration of the epithelium deep into the tissues. And therefore, when we treat anti-inflammatories, but really doing, we're treating a secondary component of primary disease process. And this really explains why there's so little effect by these anti-inflammatories. You treat the inflammation, but not the epithelial component of the disease. And we have a poster in this. I'd love to debate this because I know it's a very different concept of HS. Or in 868, I'd be happy to debate this with anybody because I really believe this is why we're missing an awful lot about this process. Thank you.
Okay, let's thank all our speakers, Kathy Smith from Abby, Michelle Lowe's, Angel Bird, Mega Lowe's, and Heidi Kong. This was a great session. This was the first like this of this kind at at an SAT meeting, particularly AB for sponsoring this session. And Mykros Employment and myself like for having the honor to to to organize and tear the session. It was a lot of fun, and I think there should and hopefully will be more in the future. Thank you everybody.