Immunometabolism, Inflammation and Chemistry Intersecting with Greg Timblin

Welcome to the Axial Podcast. Axial is an early-stage investment firm based in San Francisco. We partner with great founders and inventors investing in early-stage life science companies, often when they are no more than an idea. Axial is fanatical about helping the rare inventor who is compelled to build their own enduring business. And now I think we're recording. Um, yep, we're recording. So cool. Greg, great to have you here and have a conversation with you today. I think played a lot of basketball with you and you're so good. You have an epic spin move. Your spin move is like unstoppable. So we were just talking for like first 10 minutes here, but hey, great to have you here and excited to just talk about your story, your research, and what you do now. And maybe to start it off, do a brief intro and we can go from there.

Yeah, no, thanks for having me. And I don't know exactly how epic any of my moves were, but yeah, I do—

Also, Greg is the epic hustler. He will get every rebound, you know, he'll shoot and then he'll— you have to box him out. And it's like, it's a drag to guard Greg. I'm a big fan of guarding him. Like, there's a Friday basketball game at Berkeley. And I'm always a fan to guard Dave Raleigh or something.

For what I lack in, yeah, athleticism, yeah.

Something like that. I'm trying to guard the old professor, not the young freaking sprinter. We will talk about that.

Yeah, no, thanks for having me. And yeah, I could just start out by talking sort of a little bit about my journey. And yeah, so I grew up in a small town in in Nebraska and did all the, you know, sort of prototypical small town things and really into like sports and the outdoors and hunting and fishing and all that stuff. And yeah, I think I was really interested in science early on, and I was really lucky to have a mom who was a— actually taught either kindergarten or first grade. And so that was cool because we had like summers off and it was like library time or museum time or stuff like that. So like she really like fostered like learning and reading and all these things. And my dad was mechanic. He was super like engineering, hands-on. And I kind of never got into that or caught on to that just because I was so busy with sort of sports and science interests. But sort of, you know, because I had that science interest, You know, kind of where I grew up, it's like, oh, well, if you're interested in science, you should become a medical doctor, and you kind of get like pushed that route and told that that's what you should do. And that thinking though kind of changed when I ended up going to— not to the University of Nebraska, the large university with the football team, but I ended up going to a small College, Nebraska Wesleyan University, that's also in Lincoln, Nebraska. And I kind of chose that for a couple of reasons. I kind of— I didn't want to get lost in sort of the big shuffle, like a state school. And my sister had actually went to Nebraska Wesleyan and had a really good experience there. And yeah, and the other thing, it was— it would actually allow me to keep doing sports. So I probably would have rather been a point guard or quarterback in college or something, but I didn't have the skills to do that. But I had kind of picked up on like running late in high school, and the coach at Wesleyan like kind of asked if I wanted to be on the cross-country and track team. So that was kind of another reason, a draw to go, go to Wesleyan. But, you know, I think during my early time there, it became very clear to me that kind of the depth of my knowledge in things like chemistry and biology was, was really thin. And it was like, you know, when we— the first couple of weeks and all these classes went through everything I kind of learned in high school, I was like, oh, there's, there's more. And also it was just incredible to me that people had done the research to figure all these things out. That was just like so interesting and so attractive to me. And it's like, oh, I want to, I want to do that. So like earlier on, I knew that like being a medical doctor, like, wasn't what I wanted to do. I wanted to do research and figure things out. And, and so, yeah, being at this small university where I could pester and harass all the professors in my chemistry and biology courses, I took full advantage of that, and they, they recognized that, and I really owe them a debt of gratitude in terms of, like, helping me get into research. So one professor in particular, Dr. Jodi Ryder, she had, she had done her graduate work at the University of Colorado in the biochemistry department there. So actually she reached out to some colleagues there and was like, I have this student in my chemistry courses that wants to do research. And so I actually spent two summers out there in Rob Kushta's lab in the biochemistry department doing really cool research at the interface of chemistry and biochemistry. So I was synthesizing nucleotide analogs, so like doing actual organic synthesis chemistry and then doing biochemical assays with those to see how polymerases like decided how to incorporate nucleotides. So like I was really interested in this interface of chemistry and biology, and but as I started to think about like what I wanted to work on in grad school, I was I was a little bit lost, but I think the thing that really got me sort of interested in immunology and on the track to do that in my graduate career was— so I was doing this running thing, but I had some injuries and I wanted to come back and run cross-country with my friends for a 5th year of college. So because of that, I had to like find something to do for that summer before the last year of college. And so I actually wanted to do something other than, you know, go back out to Colorado again. So I applied like across the country to all these like summer research programs. And this included this Amgen Scholars Program at UC Berkeley. And the other thing that happened during that time was I took an immunology course. So Dr. Isaacson, at Westland really got me hooked on this. And so when I took this immunology course and started learning about the immune system, that was like, oh, this is, you know, I want to study the molecular and biochemical details of this. And so that was one of the reasons I tried to get into this program at Berkeley, because I knew they had a strong immunology program. And I didn't think I had much of a chance getting into any of these summer programs at a lot of places, but, and I still remember when I asked them Audrey Knowlton, who was like the program coordinator— I think she still is the program coordinator at Berkeley of the Samuel J. Scholars Program— asked her like, you know, like, you know, why did you pick me? Or like, what stood out about my application? She's like, well, we had never heard of your college before, so we thought, we thought you were interesting. So I guess that's, yeah, a lesson to like I'm like, whatever, I'll take it, that works. It's a lesson to just shoot your shot, I guess, like even if you don't think you have a chance to make it somewhere for something. But yeah, that whole summer experience at Berkeley kind of set me up for the path to come out to the Bay Area for grad school, because I just really love the Bay Area and felt at home here and knew it's where I wanted to do my graduate work somewhere out here. Yeah. So awesome.

Yeah, I think what makes you truly unique is your balance. You had for like so long balance two things, being a world-class scientist and then training to become a world-class runner and like just the time required and you have to be able to be so efficient. Just like, I mean, I think probably Audrey saw something where it's like you're a hard worker, right? Just a lunch pail hard worker of a scientist. That's probably one thing. Maybe coming from a small school in Nebraska is another way of saying Greg's gonna work hard. And I think that's kind of the common theme in anything you do is like you just, you're all hustle. You're just always, you know, at 100 miles per hour and making things work. And I think that's gonna be a common theme in your research and also just personally. Maybe we can talk more about like how was that initial experience What was that process to choose Berkeley? Was Berkeley just like your number one choice? Was it obvious? And then when you got to Berkeley, how did you think about like, you know, figure out what problems you're excited about? Because you can say I'm excited about immunology, but holy smokes, that's really complex. How did you hone in into like what problems you want to work on? Was it driven by the people? Was it driven by something you always were interested in?

Yeah, I think it was definitely driven by the people and the type of research that we're doing in terms of the folks in the Berkeley and CDA immunology. I always use the term like molecular immunology, like we're going to study the real small nitty-gritty details of the immune system. And so when I kind of started learning about that program and actually worked in the— during that summer, I worked in Nilav Shastri's lab, who unfortunately just recently passed passed away, but I owe him a huge debt of gratitude for being an amazing mentor that summer and then throughout my graduate work. But Nilav basically worked on the very specific details of how peptides get trimmed by peptidases and loaded and presented on MHC class I molecules to be recognized by T cells. And so again, it's kind of back to this, what are the molecular and biochemical details of how the immune system works? When I kind of saw research like that happening at Berkeley, that was kind of a new, that's like a type of environment I wanted to be in. And then, yeah, so once I like started grad school there, and it's kind of funny, even though I kind of discussed or talked about my interest in immunology, I kind of maybe ended up within the immunology department there in the least immunology lab. So I ended up in Mark Schlüssel's lab, who, if you think about it, Mark's lab was more of like a biochemistry slash developmental biology lab because his lab was focused on things like, you know, like the biochemistry of VDJ recombination and how nucleases make DNA breaks, or a lot of the kind of the part of the lab I was working in was more focused on like the development of of B cells from hematopoietic progenitors. So kind of thinking about like stem cell differentiation type problems and then how that goes awry when stem cells transform and become malignant cancers. So I kind of joined the least immunology immunology lab at Berkeley, but yet I was in this environment where you had other labs working on things ranging from innate immunity and host pathogen interactions all the way to anti-cancer immunity and T cells. So it was just like a really great environment to learn about a lot of different aspects of molecular immunology. But also what I became really interested in at Mark's lab, which I kind of didn't know I was interested in before, was just very basic questions around how gene expression is controlled in cells. And that started to become a really fascinating thing to me and how cells make decisions. You know, this kind of the time I was in grad school was around the time when sort of, you know, things like the Yamanaka factors and stem cell reprogramming were all becoming very in vogue. And those type of questions like really excited me and drove my research. And yeah, I think my training in Mark's lab just really gave me a strong foundation in basic molecular biology and And the other thing happened in Mark's lab is that he actually left Berkeley during my third year of grad school. So I was kind of stuck in this decision-making zone where, you know, the people above me, they— the lab was going to be kept open and they could finish their thesis work or their postdoc work. But the people below me had to make decisions to like change labs. And so I was kind of stuck in this place where I had to make a decision about whether I stay and try to push projects forward and take remote mentorship from Mark or whether I change labs. And I decided to stay, and I think that was actually really important in that it made me really like— I think at the time I had this expectation that your mentor sort of would just guide you along and tell you all the things to do, and those things would, would years later lead you to thesis, but I think I took a little bit more ownership and decided that I kind of had to drive projects and make decisions, especially given now that like my mentorship was gonna be remote. So I think it made me sort of a more independent scientist and thinker, and that kind of set me up really well for kind of the rest of my scientific career.

Yeah, and when Mark left, was it— did Did you join TJ's lab as well, or was that concurrently?

Yeah, so Mark kept the lab open for 2 years at Berkeley, but then towards the end of that time, I needed like an actual physical space to keep working in. And so I ended up joining TJ's lab for the last year of my grad work, in part because I also had a fellow student in my class who was kind of working on very similar questions, like how a transcription factor controlled cell fate decisions during B cell development. So it was like a logical place to kind of end up and it was actually kind of reinvigorating in ways to kind of go from this lab that was winding down and it was like not the most maybe fun place to be doing your thesis work, but then jumping into this sort of—

Yeah, for context, Robert Teegen, I think he biologically characterized the first transcription factor, like AF1 or something.

I forgot the details.

And then on this theme of, you know, as a scientist growing, right, he's this mentor, but then he has to become more independent over time. Tijen is definitely the deep end of that water where at least that post, he was present HHMI at the time, I think. And so, yeah, he was very hands-off lab.

Yeah, but no, very thankful for Tijen letting me join his group for that year. And it was kind of, yeah, reinvigorated me a little bit to be around that group. And it was a lot of fun.

One question I have about Tijen's lab, was Claudia still there?

Claudia was there. Yeah.

Claudia. There's these legends of these labs. Always remember I do a podcast with Claudia. There's always these kind of like people who actually run, who actually make a lab successful. And like people like, I forgot her last name, but Claudia and a few other folks who kind of drive success. But cool. And then maybe we can talk more. Then you have this juncture in your career where you're running and you're having success in running. I've seen you run. You're really fast, you have great, you know, your arm motion, you've got like Usain Bolt arm motion. I don't know, I don't know, what do you call this when the arm, like the, what do you call this, uh, when the arm goes back? Uh, there's probably a term for it, but you know, swinging arm carriage, arm swing, you know what I mean? I played football and they were always yelling at me for arm swing. Um, that's the most important part of running actually, it's like them, like you have to train yourself. Um, and after grad school, maybe you talk about decisions you have to make about what you wanted to do. And do you want to run? Do you want to advance your scientific career? And how do you manage those? And you figure out a way to manage both those things. Some people end up having to choose one or the other. You end up having both. So how did you end up doing that?

Yeah, so kind of what Josh was alluding to is I kind of kept running throughout grad school, and there was like, you know, various clubs around Berkeley. It was actually a really great environment because you had a lot of guys like me who had like ran, uh, in, you know, small colleges and wanted to keep training. And so like I trained with a group of guys as part of this like Strawberry Canyon Track Club that was all like, you know, you had 8, 9, 10 guys that, um, were all either doing their grad work or postdocs but were like still pretty committed to like training together. And there's like a pretty intense like local circuit around the Bay Area of like road races and cross-country races, and we kind of committed to like doing that for a few years, or at least as long as, you know, our partners and girlfriends wouldn't be mad that we're spending too much time running or something. Um, but yeah, so I kept doing this and like, uh, it was going pretty well. I was actually staying healthy and, um, I kind of ran a— so, so there's something called the Olympic Trials Marathon. It's basically this, uh, race where if you run a certain time in either the marathon or half marathon, you are allowed into this race, and then all those people are put onto the starting line to run, and then the top 3 guys go to the Olympics. But if there's like a very wide range of abilities that make it into this race, so you can like barely qualify and then you'll go get beat by 10 minutes in this trials race, or you can be like a pro runner, but it's kind of like a badge of honor for amateur runners to like qualify and make this race. So I think, I think at least 2 runners, for instance, from my, from my college, Nebraska Wesleyan, have qualified in my time, if not even more. So it's, yeah, it's just something to like put on your resume and say you did. Oh, I met this qualifying time and made it to the Olympic trials. And so I kind of ran, uh, I'd never done much sort of longer distance running, but towards the end of my grad school, I ran a kind of a half marathon time that made me think, oh, if I, you know, committed to this and trained for a couple years, I could maybe come close to meeting one of these qualifying standards to get into this race. So it's like, so if I wanted to actually do this, like how would I make this work? And so what I ended up doing is I knew that the immunology department at Berkeley had actually hired to replace Mark Karu Saijo, who was this incoming new professor. She had done her postdoc in Chris Glass's lab at UC San Diego studying microglia and neurodegenerative disease. So she was coming in to start a lab in the immunology department. And so I just reached out to Kairu and I said, hey, would you, you know, want to hire me as your lab manager, lab technician, or whatever, and I can help you get this, the lab set up? I want to kind of focus on, um, and, and, uh, um, uh, oh yeah, when, when this Olympic trials, which, uh, was I think in 2016, is comes around and either make it or don't make this qualifying time, then I'll go interview and move on and find a postdoc somewhere. She's like, sure. So I basically ended up joining Karu's lab as her lab manager and helped her kind of get the lab set up. So kind of keeping my mind in science, but kind of focusing on running. And it was actually kind of a really, it was really a beneficial time because to be honest, I actually didn't really know what I wanted to work on in my postdoc. I was kind of feeling a little bit lost again. So it kind of gave me a chance to think about that. And it also gave me a chance to like learn some new skills actually. So like Karu kind of asked me to, so the Glass Lab is kind of famous for doing a lot of like these next generation sequencing things like GroSeq and ChIP-seq and whatnot, RNA-seq. And so she kind of asked me to learn all these things so that they would be kind of up and running in her lab. And so it was actually a beneficial kind of, time for me when I could take the time to learn those types of things and employ them in my own toolkit and use them going forward. Yeah. So that's awesome.

I think, I mean, sometimes, you know, like, I think maybe running helps you, you know, give you space. And I'm assuming you're running— I run, but I run like 3 miles. That's all I can run. Beyond that, it gets very boring. And I'm assuming Someone like you, I have friends who run long distance as well. It's a time to reflect. What do you, when you're running, what do you think about? Are you just thinking about, sometimes when I'm running, I'm like, man, I need my breath. But someone like of your, you know, of your talent, um, when you're actually running on the 7th mile, are you like in the zone or are you thinking, are you like reflecting on things? Yeah. That's, that's, uh, yeah.

I mean, it depends on the day and like what you're doing and how hard you're running. But if I'm just out for like a, casual run. Like, no, I mean, like, I'm more than likely I'm thinking about science, and it's my time to think and reflect on that and kind of talk to myself and, and, um, you know, talk through hypotheses and why or why not that things may be this way or not this way.

Assuming running helps you. I mean, I have friends—

oh, for sure. Yeah, all my—

I have so many, you know, like whether you spoke of this, I have so many friends who are world-class chemists And they almost all run like really high-level running. But maybe we can transition towards your key work. You had a Nature Metabolism paper that got published in 2020, and I remember kind of seeing it kind of near the end of it. And it was really— you had this preprint and you got to Nature. And so you really made some awesome discoveries around the role of mitochondrial kinesis. In macrophage metabolism and fate and function. And just for some quick background, mitohormesis is just kind of quite simply, you know, mitochondria create kind of reactive oxygen species, things that could be damaging. And a little bit of damage is actually valuable. Too much then ends up becoming harmful. That's kind of the hormetic response. So you kind of establish this kind of phenomenon, macrophages. And I think it's a great story. Maybe you can talk about how that project got started because you're in Sanjo's lab, but Sanjo's focused on microglia. She has no business in this field. And so, you know, how did you get from this starting point that's kind of related but kind of not connected to this whole different field? I think that's kind of the great part of the story.

Yeah. Yeah, so, so 2016 came and went and I was interviewed for some postdocs in other places, but kind of nothing really interested me. And so when 2017 rolled around, I had to decide what I wanted to do. I actually ended up just staying in Kairou's lab and transitioning over to being a postdoc. And part of the reason for that was because we had stumbled upon this sort of interesting and mysterious observation very recently. And that was— so, so Kairu had been studying sex hormones, which normally work through sex hormone receptors. She had primarily been studying estrogens and how they work through the estrogen receptors in microglia to modulate their sort of inflammatory tone and and just sort of demonstrated that could, supplementing specific estrogens could beneficially modulate like neuroinflammatory disease. And we had sort of had reasoning to ask a similar question for not for microglia, the macrophages of the brain, but just for macrophages in general, 'cause they actually express some different nuclear hormone receptors than microglia. I'm talking about the macrophages peripherally in the body. So basically just did a screen of, endogenous estrogen metabolites and were asking how they influence macrophage inflammation, such as like inflammation driven by like toll-like receptors. That was kind of the screen. And this was like an important question to ask in general because this field was actually kind of conflicting in terms of whether people thought estrogens were pro or anti-inflammatory. And then also this was also during a time when And people still are making this push that we need to actually be more equitable in how we study biological male and female inflammatory responses, because we know that the immune responses in biological males and females are very different, and that sex hormones probably play a big role in this. So we should probably know more about how estrogen influences inflammation. And so we did a screen where we put all these endogenous estrogens on to macrophages, treated them with LPS, this toll-like receptor ligand that induces like a pro-inflammatory macrophage phenotype, and we found that this particular class of estrogen metabolites, these hydroxyestrogens, were really anti-inflammatory. But the surprising thing came when I did the experiments where either— or I did the experiment in macrophages that lack the estrogen receptor, and we found the the anti-inflammatory activity of these hydroxyestrogens was actually still there, meaning that it was an estrogen receptor or hormone receptor independent effect that we were observing. So we're kind of left with this puzzle and it's the puzzle that I kind of committed to trying to figure out when I decided to stay in Kairu's lab and do a postdoc there. Yeah, kind of how I started to relate to, to mitohormesis and these hormetic responses is that when I started looking at literature about what was known about these hydroxyestrogens, it became very clear that they had this reputation for being evil because they, they could themselves like directly oxidize proteins, or they could actually do this, do this thing called redox cycling where they can snatch electrons and give them the oxygen and make their own reactive oxygen species. So I kind of initially was like, Oh crap, this is like some toxic effect where these things are just causing oxidative damage to the macrophages and maybe killing them, and that's why we're seeing this anti-inflammatory effect. But that wasn't the case. So actually what I ended up talking about in the paper is how the hydroxyestrone-treated macrophages actually, because of this oxidative stress that it generated, actually become more resistant to to subsequent oxidative stress. So it wasn't the case where this was a toxic effect. It was this hormetic effect that Josh kind of alluded to. But, you know, the way we related this to macrophage metabolism was that, you know, this was— I kind of had a hunch that sort of macrophage metabolism was involved in the anti-inflammatory effect, but kind of at the time, looking through the literature, it was it was kind of unclear about how exactly that worked. And that was, I think, in a large part because immunologists, for whatever reason, love to do like overnight time points. So they like to like stimulate macrophages with things and then the next day like look at cytokine production or gene expression. But if you looked at the people who were studying things like the acute macrophage pro-inflammatory gene transcription downstream of things like toll receptor ligands they would study these things at minutes or hours time points, because that's when changes in— initial changes in metabolism would happen and things like opening of chromatin and initial transcription of pro-inflammatory genes would happen. So there was like this discordance between sort of how the macrophage phenotype was being studied, but yet all this stuff was happening a lot faster than people really appreciated. So I kind of had to work backwards and look at how these hydroxyestrone were affecting this really initial metabolic reprogramming in these macrophages. And we basically found that it's sort of this oxidative stress that they were causing by either directly oxidizing proteins or generating their own reactive oxygen species seemed to be disrupting some specific metabolic fluxes that were important for supporting the inflammatory response. Namely, it was inhibiting the utilization of glucose by mitochondria in the macrophages because the mitochondria early on during the inflammatory response are sort of this biosynthetic hub where they take glucose and make things that support an inflammatory response, including this metabolite acetyl-CoA, which is important for the first step of the inflammatory response, which is acetylate histones so that the pro-inflammatory genes could be turned on. So yeah, we sort of characterized how these hydroxyestrogens were causing this oxidative stress, both sort of acutely suppress that flux, and then they would also cause this hormetic response that would sort of totally rearrange macrophage metabolism and shut down that mitochondrial glucose utilization, which is something that actually naturally happens as macrophages, when they are exposed to like a toll-like receptor ligand, they sort of undergo this inflammatory response, but then they shift over to this tolerized phenotype. And so we sort of set up this model where this oxidative stress might be important for promoting this sort of metabolic shift in the macrophages that enforces this tolerance. And, and, um, yeah, it's a conceptually, um, you know, people always ask me, they're like, well, you know, ox— ROS and oxidants are always thought to be pro-inflammatory in macrophages. Like, how can it be that you're saying they're anti-inflammatory? And I guess I'm not saying that those things have to be mutually exclusive because, you know, a lot of biological pathways, for instance, like NF-kappa-B signaling— like, NF-kappa-B signaling happens, but it also has negative feedback built into it. So like, for instance, some of the targets of NF-kappa-B are actually like negative regulators of NF-kappa-B signaling. So I figured If biological pathways like NF-kappaB signaling can be both activating and simultaneously repressing, then why can't signaling by reactive oxygen species be the same way? So I definitely think certain aspects of ROS production during early inflammatory responses by macrophages do promote things like inflammatory signaling. There's some great work by a friend, paper that actually talks about how oxidants support aspects of pro-inflammatory signaling. But I figured, why can't it be both? Why can't those oxidants also be involved in feedback that shuts off the inflammatory response?

So I think that's kind of this non-intuitive discovery, and it's just like, it's consequence of this really profound observation of like, okay, these hydroxyestrogens are promoting an anti-inflammatory phenotype independent of the estrogen receptor. And so like, you have to spend years, all this experience. I can imagine all the like excitement, but the grind to have to figure out, okay, how's this actually working? And that's kind of the exciting part of science. And then, you know, you have, you have to kind of rebuild almost, um, what, you know, like sometimes people start from a target and they can figure it all out. Based on existing discoveries. You kind of have to rebuild it. You have to rebuild the universe yourself based on this one observation that was non-intuitive, and you do all these experiments to validate and double-check. And that's kind of the hard part. And I think maybe one thing we could dive a little deeper into is more around, like, macrophages exist in two main states. M1 and M2. I always forget which one's pro-inflammatory. I think it's M1.

Or yeah, so yeah, people— yeah, this is, uh, you're saying dirty words to some people now because people are trying to get rid of that nomenclature. But actually, I'm like, whatever, like, it's— I'm fine with it because I think, um, I think the two main states—

I think macrophages exist in various other states, but the two main states I was taught is M1 and M2. Maybe it's changed now.

Yeah, so it's thought that these inflammatory macrophages, which I was studying, they can— they're definitely good. They can help you initiate the inflammation that helps repair a wound or helps you get rid of a pathogen. But it's thought that those M1 macrophages are the ones that go off track and promote disease-driving inflammation and things like cardiovascular disease or arthritis or neurodegeneration or like early on in a septic shock response.

Yeah.

But then you have this on the other hand, you have these, these pro-repair macrophages that would be normally important for things like healing a wound or healing damaged tissue after an infection. But that since because those macrophages are inherently have a different job, not to, to do inflammation but to do wound repair, those macrophages can maybe be bad in certain contexts. So for instance, like in Context like cancer, those macrophages might be not supportive to anti-tumor immunity. And in processes like cancer, you might actually want to— that's been pretty— I think it's pretty well appreciated. You might want to actually promote a pro-inflammatory macrophage phenotype in cancer. So yeah, just, it was, as you said, sort of a situation where I had sort of a pharmacological phenotypic effect, but I had to kind of like work backwards in maybe a non-intuitive way to kind of figure out what's going on. And, and, um, it's so hard.

That is so hard. I mean, that's like some, like, that's like science that was done in the '60s, you know, like there's some old school, like, non— like just not knowing, and just like just seeing a phenotype and have to figure it all out. Like, that is like brutally hard. Most, most, most of the time now you have a target, you kind of know what you were looking at. And you can kind of build from there. But maybe we can talk more around like how your work kind of like tips the balance and kind of, not kind of, increases our understanding of these different macrophage states and how they're relevant in disease. And so, you know, your work kind of shows that a little bit of stress can shift the, shift the, scales of macrophage or shift the state of macrophage towards an anti-inflammatory state. And what does that mean for disease and maybe our understanding of immunity? That like a little bit of stress can actually shift at least a macrophage to a different state.

Yeah, so yeah, a couple of thoughts on that. The first thing I was gonna say in kind of relation to all of this is that, you know, I owe like a huge debt of gratitude to like the folks that had worked out all these mitohormetic mechanisms, which had primarily been worked out in like yeast and C. elegans. So I started reading— it was funny, I started reading a whole bunch of literature by this guy named Michael Ristow, who had kind of coined the term mitohormesis in some of his C. elegans work. And then it was funny, he ended up writing like a preview of the paper and sort of asking bigger picture questions that you're kind of alluding to. But Yeah, I think it raises this— it was sort of going back to what I said about thinking about reactive oxygen species in general and whether they are pro or anti-inflammatory. And like I'm saying, I don't think those things are mutually exclusive, but, you know, it's very clear in clinical trials across multiple inflammatory diseases that what we think are antioxidants have not been successful in any of those. So that says that You know, my work kind of raises the question that not only might those antioxidants not work, but they might actually be detrimental to the process of how ROS might promote suppression of inflammation naturally. So that's kind of, I think, a larger kind of bigger picture concept that my work kind of raises some questions about. And then, yeah, the other Just from like a therapeutic standpoint, it kind of, as you said, raises the, this counterintuitive notion, as you say, that like a little, you know, we don't really, just as when I initially read about these hydroxyestrogens and started seeing that they were evil because they cause oxidative stress and that I was like, oh my gosh, like what am I getting myself into? But maybe we should actually not be afraid of pharmacologies that do things like that. So it's kind of an atypical way of thinking in terms of like, if you could design maybe molecules that cause stress in the right place and right time.

So yeah, I think what makes your work truly unique in the history of like science immunology in particular is I see worm, worms, right? There's always been all these experiments for a few decades around ROS, reactive oxygen species, and playing around with lifespan of worms, right? A little bit of ROS can actually increase the lifespan of worms by X amount. There's always cool data points out there. And then your Nature Metabolism paper, at least in my perspective, and knowing the field decently well, is like, you kind of were the first to connect that observation that was in worms and yeast to like a human cell, a macrophage, you know, it's kind of this like big leap and it's non-intuitive leap. And I think kind of opens up a whole can of worms of like how much damage is valuable, you know what I mean? And how much stress is useful to change an immune cell's fate and function. And maybe you talk about more, how do you Think about translating that work, right? This idea of hormetic drugs seems really like an evil term, like a no-no term for some drug developers. But how do you think about this idea of how do you translate these discoveries into maybe something that could potentially become a cancer drug down the line? And what are the challenges to try to drug a hormetic pathway or take advantage of it?

Yeah, so that's kind of one thing I've been interested in, and I think it gets challenging into thinking about whether this hormetic response that I described is caused by generalized stress or whether there is actually more specific targeting of specific protein or proteins and that that's kicking off this hormetic response. And because I think when you talk about a generalized stress response, there's, yeah, like you said, the drug developers or the people in pharma, they always want a specific target and biomarkers. And yet I'm describing kind of a bigger picture holistic response, but maybe it's not actually that way. And maybe there are actually specific targets that are being hit, say, by these hydroxyestrogens that are actually, it's actually the driver of this response. And that's actually something I've been investigating a little bit outside of my academic work. And yeah, basically trying to know if there is actually a specific target of these hydroxylations. If we knew this target and information about it could actually help us design therapeutics to either inhibit or degrade that target and sort of kick off the same response that that I'm describing in the, in the Mycormesis paper. Yeah. Cool.

I think there's just a lot of work left to be done here and it's very non-intuitive. That's the thing. Immunology's already hard and now you're adding this whole other thing on top of it. It just makes it so much harder to grok and figure out. And so maybe we can transition more towards like what you're doing now. You published a paper earlier this year on macrophage metabolism and what you do, and you kind of moved to UCSF to do a postdoc. Then we can talk about research there and what you're working on.

Yeah, so, you know, if I had to sort of like boil down what excited me most about, um, kind of following up on the mitohormesis work— so the, the mitohormetic work kind of talks about how these hydroxyestrogens sort of disrupt or short-circuit this mitochondrial glucose utilization, this sort of anabolic metabolism that the macrophages use to mount an inflammatory response. But I actually became more interested in this observation we made that this supplementation of macrophages with this metabolic cofactor, coenzyme A, which actually would overcome the anti-inflammatory effects of the hydroxyestrogen by basically reinvigorating mitochondrial glucose oxidation. So basically helping macrophages utilize glucose to do the anabolic things they need to do to build an inflammatory response. We basically found that this, not only could this CoA supplementation like reverse this pharmacological effect that I discovered, but I became really excited when I found that this CoA supplementation could actually sort of reinvigorate these tolerized or M2, to me they're kind of the same flavor of macrophage, these sort of like immunosuppressed macrophages that are more worried about suppressing inflammation and rebuilding a tissue. It could actually, CoA supplementation could actually sort of revert them back to the inflammatory state where they could utilize glucose. In the mitochondria to mount an inflammatory response. And that to me was very exciting from a— because in some ways I almost think that's a harder problem to crack, and that's— and maybe a more important problem to crack because, you know, the mortality in terms of cancer or things like late-stage sepsis and the immunosuppression that happens there, it's a really large burden. So if you can figure out a way to sort of reinvigorate innate immunity with this CoA supplementation, that was something I really wanted to follow up on. And so that's kind of what I've been doing. That was one of the reasons I moved to Val Weaver's lab at UCSF, because she'd had— she's taken a big interest in how these anti-inflammatory, pro-repair, pro-resolving macrophages support and drive tumorigenesis. And so I kind of came to Val and said, you know, I think I have a way to sort of, via this CoA supplementation, a way to reinvigorate the pro-inflammatory response of these macrophages and sort of reorient them back to a more inflammatory state. And I want to know if that could work as an anti-cancer therapeutic. And also I've kind of kept in touch with plenty of friends in Berkeley and so I've also been working with them. So it's thought that in a lot of like chronic infections, especially infections where the pathogen likes to counterintuitively use live and replicate inside of macrophages, that this similar sort of immunosuppression and tolerance happens in those macrophages and allows those macrophages to create a replicative niche. And so I also have been working with Russell Vance here at UC Berkeley to ask the question about whether this CoA supplementation, which we've shown in Val's lab can make a macrophage more inflammatory anti-tumor, whether it could also make a more inflammatory antimicrobial macrophage. And so we've been working on this CoA supplementation and how it helps macrophages restrict and kill Legionella pneumophila, which is this intracellular bacteria that the Vance lab uses to study host-pathogen interactions. So yeah, I've basically been doing the other side of the coin of now I'm not trying to suppress inflammation, but I'm trying to now promote an inflammatory macrophage phenotype. But I think bigger picture, going back to like, you know, what is the larger term implications of all this? Like, I think now that I've convinced myself that I can sort of suppress or promote these phenotypes via manipulating macrophage metabolism, I hope that this makes, you know, I think as you said, this is like a, Immunology is already complicated enough, and then you throw metabolism on top of it. I hope this makes kind of people, you know, maybe appreciate the power of modulating metabolism in terms of controlling cellular phenotypes, and that it's not a passive player in things, but it actively controls the cell fate decisions of these cells. Well, yeah, I think I didn't know—

congrats on collaborating with Russell Vance. He's like the legend in sea gas and innate immunity. And I think in general, studying the metabolism of immune cells is very ripe and open to field to then maybe make drugs to modulate that metabolism of those immune cells and ultimately which will affect not only cancer, but any inflammatory disease. So the implications and the value is very broad. I think the hard part is trying to— the immune system is very context-dependent. And so you get to study this macrophage in this environment, the state. And so it's very hard labor. But if you can figure out the right one, I think there's a lot of potential discoveries and drugs to be made. But the next few years, maybe we'll wrap it up. Next few years, you've been building up, right, from Nebraska, you know, getting exposure to chemistry, then immunology, you know, becoming an expert in macrophage metabolism. What do you see the next few years? Do you want to go a particular direction? Do you want to, like, say, you want to, like, make a drug candidate? Do you want to try to explore another field of macrophages, or Do you want to go to different immune cells? What do the next few years look like?

Yeah, that's an interesting question that I'm kind of trying to figure out right now. So I've kind of been on the job market since last year and I've been applying for jobs and have had or have some interviews lined up for like academic positions. Um, uh, yeah, so that's kind of one route I'm pursuing. Um, but yeah, at the same time, I think I've been very fortunate, and from the same point that kind of all my research has involved sort of these like pharmacological effects that have, you know, beneficial biological effects in the systems I've been studying. And so that's kind of got me really interested in the world of biotech and pharma and drug development and like Okay, I have these interesting effects, how would I actually turn them into therapeutics? And so I've also been, I'm kind of alluded to with the hydroxy estrogen stories, I think on the side kind of pursuing avenues and ways to make that a reality in my own company. And so yeah, what the future holds is very uncertain right now, but yeah, just say I have some options and I'm excited about all the options and whether they work. Separately or together, I don't know how it's gonna go.

I saw it work. I think a common theme for you is just like the mental work, just the hard work. You know what I mean? Always, always give it your all in anything you do, and I think that's gonna— you've made some really awesome discoveries. I think that's kind of just trying to unravel that more and more, and I'm sure it's gonna unveil some really great opportunities, maybe in academia, maybe in an industry setting, in startup. I think, you know, I think, I think as an entrepreneur, you have so much potential. Well, maybe both. And so I think, I think I'm, I'll be really excited. We'll touch base in like 2 years and then we'll see what's going on.

Right.

And I think it's kind of the idea, right? We'll do some updates every now and then. But Greg, thanks for doing this. It's just kind of really hear your story. I think you have an incredible unique story out of all the scientists I meet and kind of be able to balance so much in your life. And mainly being a, being a scientist is already hard enough. Doing world-class science is like more than a full-time job. And then be able to like train for run. Oh, one last question. I actually never know the answer to this either. How did you place? Did you get to the qualifiers? How did you place?

No, I ended up, uh, I ended up getting injured and having to have a hip surgery. So yeah, it was—

oh, okay.

I have an issue with overdoing it sometimes.

It's my other problem. Overdoing it. Uh, we got to get you to qualifiers then.

Yeah, that might be— those days might be—

those days are over behind me. Yeah, I'm assuming they have— I had a friend of mine, there's like the over-50 Olympics or something. Yeah, maybe, maybe.

Yeah, no, I'm, I'm just trying to keep my body together, and then I can maybe make a— there's a thing that they call like masters running. Yeah, I think Greg can make a comeback there.

Yeah, make a comeback in the masters running. I think, I think that's kind of the— that's the game plan now, is the masters goals. Yeah, exactly. I think you could do it. Okay, I'll see you at Kezar probably one of these days. Um, but great to do this, and we'll do the— and, and, uh, we'll, we'll do this a few years from now and get the update.

Awesome. Thanks, Josh.

Thanks, Greg.