As defined by the United Nations Framework Convention on Climate Change (Farber and Carlarne, 2017), climate change is an unnatural change to our climate that is attributed directly or indirectly to human activity that alters our world’s atmosphere (Farber and Carlarne, 2017) (Farber DA, Carlarne CP. Climate change law. Ohio St Pub Law Work Paper. 2017;419). Plants are altering the pollens and allergens they generate. Air pollutants are becoming more complex and numerous. The ozone layer is thinning. Existing pathogens and microbes are shifting territories (Hauser et al., 2021). New pathogens and microbes are emerging that we’ve never seen before. As exemplified by SarsCoV2, we are all painfully aware of the consequences. Indeed, inflammatory disorders and cancers of the epithelia of the skin, the intestine and the lung are on the rise. This is because, bearing the brunt of these environmental changes are our epithelial tissues – they form the cellular barrier between our body and the outside world.
In studying the stem cells of the skin epithelium over the course of my career, I’ve learned that when epithelial stem cells malfunction, either in making a proper barrier or in communicating with the immune system upon a barrier breach, chronic inflammation occurs. Our first realization came back when I was just embarking on my academic career at The University of Chicago. I had just cloned and characterized the human skin keratins, which form an extensive infrastructure of filaments (cytoskeleton) that protects the epidermal stem cells and their differentiated progeny from mechanical stress. Given that our skin epidermis is at the body surface, this mechanical framework is essential. Indeed, as we showed, patients that lack the stem cell keratin network, composed of keratins 5 and 14, have a blistering skin disorder known as epidermolysis bullosa simplex (Coulombe et al., 1991; Fuchs and Green, 1980; Fuchs et al., 1981; Vassar et al., 1991). The disorder is rooted in the fact that when the stem cells lack this network, they are prone to rupturing upon rubbing the skin – in severe cases, even washing the face, or walking. By contrast, patients with mutations in keratins 1 and 10, which are only expressed in the differentiating progeny generated by the stem cells, have thickened, crusty skin, but are prone to bacterial infections and cancer (squamous cell carcinoma) (Cheng et al., 1992; Fuchs et al., 1992; Fuchs and Green, 1980). Seeking the underlying reasons for this, we learned that the healthy stem cells, recognizing that the skin barrier they made isn’t right, respond in a futile attempt, by proliferating to create excess layers of cells to try to patch the barrier. In turn, since the barrier is defective, pathogens can enter, triggering a hyperproliferative response.
In more recent work, we examined another structural protein of the terminally differentiating cells of the epidermis (Fig. 1). Filaggrin has long been known to be expressed by the late-stage terminally differentiating cells of the epidermis. So-called granular cells because of the presence of electron dense granules in their cytoplasm, these epidermal cells represent the last transcriptionally active progeny of the stem cells. Soon after they form, all nuclei and organelles are lost as the granular cells flatten out to form the dead cells that are sloughed from our body surface, continually replaced by inner layer cells moving outward. In studying the human disorder, atopic dermatitis, affecting up to 3% of the world population, researchers identified mutations in filaggrin (Palmer et al., 2006). At the time, it was thought that this disorder was strictly an immune disorder, based upon mutations in immune cell genes (Kaltoft et al., 1994; Kawashima et al., 1998; Nishio et al., 2001; Osawa et al., 2007; van der Stoep et al., 1993). There has been skepticism as to whether filaggrin mutations even manifest the disorder (Spidale et al., 2020). Indeed, it is a big protein made up of unstructured protein repeat units, and the mutations are scattered throughout the protein, with no apparent consequence to the physiology. Recently, however, we learned that filaggrin undergoes conformational changes that are pH-sensitive and thermal sensitive (Quiroz et al., 2020). In epidermal cells, filaggrin protein begins to be made as epidermal stem cells give rise to differentiating progeny. As the protein accumulates in differentiating cells, it reaches its critical concentration sufficient to induce a conformational change. This change results in the protein transitioning to an oil-like granule which becomes more viscous as the epidermal cells differentiate and the protein accumulates. The granules also interact with K1 and K10 filaments, whose carboxy and amino terminal domains also undergo these liquid phase transitions. The result is a dense viscous network that puts mechanical pressure on the nuclei and organelles, contributing to their loss to form the barrier. The patient mutations create truncated filaggrins that fail to accumulate a sufficient concentration to undergo these liquid phase transitions, and the result is the retention of organelles and the failure to make a proper skin barrier (Quiroz et al., 2020). These data have brought clarity to our understanding of how the skin barrier is formed, and add further evidence that structural defects in the skin barrier can result in chronic inflammatory disorders and increased susceptibility to cancer. Currently AD patients are given immunosuppressive drugs, which have unwanted side effects, and often do not fix the problem. By investigating the basic science of skin stem cells, we hope to be able to uncover biology that will lead to improved therapeutics for disorders like AD.
Our studies of the skin and its stem cells have led us deeper into the biology of chronic inflammatory disorders, which as a cohort, include not only psoriasis, atopic dermatology and chronic wound healing disorders, but also inflammatory bowel disease and asthma. These are all disorders of barrier epithelia at the interface between the body and the external environment (Niec et al., 2021). A common feature of these disorders is that the epithelial hyperproliferation often occurs at flexural regions (elbows, knees for skin disorder, for example), and it typically comes and goes. Upon the next assault, it often occurs in the same spots and with increasing severity. And curiously, the secondary trigger need not be the same as the initial irritant. The first stimulus might be poison ivy, while the next one might be a pathogen or other irritant.
In 2017, we decided to probe deeper into the biology that underlies these curious phenotypes. In watching how wounds heal, we noticed that if the skin of animals had been exposed briefly to an irritant that triggers an immune response known as a “Th17” inflammatory response, and then wounded a month after the skin pathology had returned to normal, the skin always healed its wound faster if it had been pre-exposed to inflammation (Fig. 2). Even 6 months after the initial inflammatory stimulus, the skin still responded more quickly to heal its wounds faster. We then exposed naïve skin to a yeast infection, a “Th2” inflammatory response, and a wound as primary and secondary stimulus. Each time, inflammation conditioned the skin to heal wounds better (Naik et al., 2017).
We thought at first that immune cells would be involved. Indeed, B and T cells can permanently rearrange their receptors so that they can recognize a pathogen the next time they encounter it. Indeed, this is how vaccines work. However, when we repeated the experiment on mice that lacked all B and T lymphocytes, we again found that after we exposed the skin to an inflammatory stimulus, let the pathology return to normal and then wounded the skin, it always healed the wound faster. We also looked at whether innate immune cells, including macrophages, might be involved. However, after eliminating these immune cells, we began to wonder whether the epidermal stem cells themselves might be the ones to harbor this memory. Indeed, this turned out to be the case (Naik et al., 2017).
First, we looked at the transcriptional profile of the epidermal stem cells before treatment, at the height of inflammation and after the inflammation had resolved. Very few genes were changed at the transcriptional level. However, when we turned to chromatin, we found a different story. While >10,000 chromatin sites became open soon after the inflammatory stimulus was administered, >1000 of these sites – mostly in gene regulatory regions known as enhancers – remained open long after the stimulus was withdrawn. In testing these open sites for activity, we learned that they harbored inflammation-sensing activity, and in mice, had the capacity to activate a reporter gene following an inflammatory stimulus (Naik et al., 2017) (Fig. 3). Moreover, the genes associated with these “inflammation sensors” were rapidly activated upon a secondary stimulus, e.g., wounding.
In the past several years, we’ve now addressed three major questions: How is this memory established? How is memory retained? And how is memory recalled? In the first set of experiments, we simply scanned the transcription factor motif frequency of these inflammatory sensors and compared it to the motif frequency of the many genes that closed back following inflammation and the many genes whose expression was insensitive to inflammation (Larsen et al., 2021). Stat3 and Fos:Jun (AP1) sites were markedly enriched in these memory domains. Looking at inflammation, we learned that Stat3 is phosphorylated and rapidly activated following Th17 inflammation and FOS is rapidly induced as well. FOS’s obligatory heterodimerizing partner, Jun, was already present in homeostatic epidermal stem cells. Using “CUT and RUN” technology to map whether these transcription factors bind to the DNA, we discovered that the inflammation-sensing chromatin is silent in homeostatic skin, but rapidly opened upon the Th17 response. pSTAT3 and FOS:JUN bind to these sites. By ablating these transcription factors in the skin epidermis, we learned that pSTAT3 is required to act as a “pioneer factor” in opening the chromatin at these sites, while FOS:JUN is necessary to remodel the chromatin to recruit RNA polymerase and transcribe the genes (Larsen et al., 2021).
What then happens after inflammation, when STAT3 and FOS are no longer there? Without FOS, transcription of the genes associated with memory domains shuts off. However, once the chromatin was open, not only JUN but also several other stem cell transcription factors gain access to the chromatin and bind. In addition, one histone modification in particular, H3K4me1, also gained access to the chromatin, and in contrast to H3K27ac, this mark persisted in the memory state. Hence the memory is retained because there are stem cell factors and histone modifiers that bind and remain bound to the memory domain once inflammatory transcription factors had opened it.
How is memory recalled? In this case, since the chromatin is open, STAT3 is no longer required, and if we ablate Stat3 after memory has been established, it does not prevent transcriptional activation following stress. However, in order to activate transcription at these sites, FOS must be induced. FOS, however is induced in response to a wide variety of different stresses. Within 4 hours after a general stressor, FOS is induced, and binds to the memory domains, chromatin is remodeled and the associated genes are transcribed (Larsen et al., 2021). Moreover, when we re-analyzed all the published data on inflammatory memory to look for parallels to this mechanism we unearthed, we found a remarkable conservation – in all cases, AP1 factors, FOS/JUN appear to be integral to memory establishment and recall, while the initial transcription factor involved in opening chromatin appears to differ – Stat1, Stat4 or even NFkB. These factors appear to be integral in choosing what genes will be associated with a particular memory, whereas FOS/JUN appear to be general stress-induced factors crucial to remodel the chromatin in memory establishment and memory recall.
These findings begin to answer many long-standing puzzles. Since the 1930s it has been known that plants that survive one pathogen are often resistant to other pathogens that they’ve never seen before. Infants have long been vaccinated against Bacille Calmette-Guérin (BCG) acquiring resistance against tuberculosis. Epigenetic memory applies the lessons learned from one experience towards a new experience. Memory has an evolutionary advantage in enhancing protection against harmful microbes and also wound repair, as all stem cells must be mobilized to repair tissue injury. (However, it can also be maladaptive as in the case of chronic inflammation).
Since our original publication reporting the existence of epigenetic memory of inflammation in a stem cell, it has now been shown that hematopoietic stem cells have memory (de Laval et al., 2020) and multipotent hematopoietic progenitors possess inflammatory memory (Christ et al., 2018; Mitroulis et al., 2018). It has also been shown that BCG educates cells against tuberculosis (Kaufmann et al., 2018). Importantly, however, we now know that memory extends to other types of epithelial barrier cells: airway epithelia bear epigenetic memory of asthma (Ordovas-Montanes et al., 2018) and intestinal epithelia bear epigenetic memory of gut pathogens (Lim et al., 2021). In addition, epigenetic memory can be inherited. If a mom mouse eats a bacterial pathogen, the inflammation can be transmitted to the fetus through the circulation, and the offspring then carry the epigenetic memory into adulthood (Lim et al., 2021). In the case of pancreatic epithelium, exposure to inflammation predisposes the tissue to acinar ductal metaplasia, increasing the risk of pancreatic cancer (Del Poggetto et al., 2021; Quiroz et al., 2020).
These findings raise many questions: are there different kinds of memories? Are memories cumulative? In the last year, we addressed these questions by generating a wound model in mice in which we specifically mobilized hair follicle stem cells to exit their niche, migrate upward, confront the wound and associated inflammation, undergo a fate change to epidermal stem cells and repair the missing epidermis. Thereafter the hair follicle-derived stem cells behave as epidermal stem cells. Using chromatin landscaping at high throughput level, we showed that at each stem along this journey, the hair follicle stem cells retain epigenetic memories of their experiences: Memories that they used to be hair follicle stem cells and are now epidermal stem cells; memories that they migrated; memories that they encountered inflammation and signs of epigenetic adaptation indicative that the stem cells now have a different set of tasks than they had before (Gonzales et al., 2021). These memories have profound consequences. I’ve already discussed the consequences of inflammatory memory. But the stem cells also harbor memories that they used to be hair follicle stem cells. This memory confers increased plasticity to the stem cells: when challenged, they can make both hair and epidermis, while naïve epidermal stem cells only make epidermis. They also bear memories of migration. When challenged, the wound-memory stem cells can migrate much faster than their naïve counterparts.
In closing, since our studies in 2017, the field has exploded, as each day, new memories are uncovered in new cell populations. How long do these memories last? We’ve followed memory for up to 6 months in the skin stem cells of mice (equivalent of 5-6 years in humans). With the ability to accumulate memories, and the longevity of stem cells, these findings raise concern for our future. Neurons are long-lived and non-dividing – the prime situation for harboring memories. Increasingly it is becoming clear that neurodegenerative disorders such as Alzheimer’s Disease are associated with inflammation in the brain, raising the question as to whether inflammatory memory in neurons may be at the roots of these disorders. Epigenetic memory of inflammation also raises potential consequences in aging. Will we show increased susceptibility to inflammatory stimuli that we’ve never encountered before? Could epigenetic memory explain why the COVID19 response has often be more severe in aging individuals? And what happens in a world where allergens, pollens, pollutants and other irritants are ever on the rise? Our next challenge will be to come up with therapeutic strategies to erase the bad memories and keep the good ones.
Literature Cited
Cheng, J., Syder, A.J., Yu, Q.C., Letai, A., Paller, A.S., and Fuchs, E. (1992). The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-specific epidermal keratin genes. Cell 70, 811-819.
Christ, A., Gunther, P., Lauterbach, M.A.R., Duewell, P., Biswas, D., Pelka, K., Scholz, C.J., Oosting, M., Haendler, K., Bassler, K., et al. (2018). Western Diet Triggers NLRP3-Dependent Innate Immune Reprogramming. Cell 172, 162-175 e114.
Coulombe, P.A., Hutton, M.E., Letai, A., Hebert, A., Paller, A.S., and Fuchs, E. (1991). Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses. Cell 66, 1301-1311.
de Laval, B., Maurizio, J., Kandalla, P.K., Brisou, G., Simonnet, L., Huber, C., Gimenez, G., Matcovitch-Natan, O., Reinhardt, S., David, E., et al. (2020). C/EBPbeta-Dependent Epigenetic Memory Induces Trained Immunity in Hematopoietic Stem Cells. Cell Stem Cell 26, 657-674 e658.
Del Poggetto, E., Ho, I.L., Balestrieri, C., Yen, E.Y., Zhang, S., Citron, F., Shah, R., Corti, D., Diaferia, G.R., Li, C.Y., et al. (2021). Epithelial memory of inflammation limits tissue damage while promoting pancreatic tumorigenesis. Science 373, eabj0486.
Farber, D., and Carlarne, C.P. (2017). Climate Change Law (Concepts and Insights). Foundation Press.
Fuchs, E., Esteves, R.A., and Coulombe, P.A. (1992). Transgenic mice expressing a mutant keratin 10 gene reveal the likely genetic basis for epidermolytic hyperkeratosis. Proc Natl Acad Sci U S A 89, 6906-6910.
Fuchs, E., and Green, H. (1980). Changes in keratin gene expression during terminal differentiation of the keratinocyte. Cell 19, 1033-1042.
Fuchs, E.V., Coppock, S.M., Green, H., and Cleveland, D.W. (1981). Two distinct classes of keratin genes and their evolutionary significance. Cell 27, 75-84.
Gonzales, K.A.U., Polak, L., Matos, I., Tierney, M.T., Gola, A., Wong, E., Infarinato, N.R., Nikolova, M., Luo, S., Liu, S., et al. (2021). Stem cells expand potency and alter tissue fitness by accumulating diverse epigenetic memories. Science 374, eabh2444.
Hauser, N., Conlon, K.C., Desai, A., and Kobziar, L.N. (2021). Climate Change and Infections on the Move in North America. Infect Drug Resist 14, 5711-5723.
Kaltoft, K., Pedersen, C.B., Hansen, B.H., Lemonidis, A.S., Frydenberg, J., and Thestrup-Pedersen, K. (1994). In vitro genetically aberrant T-cell clones with continuous growth are associated with atopic dermatitis. Arch Dermatol Res 287, 42-47.
Kaufmann, E., Sanz, J., Dunn, J.L., Khan, N., Mendonca, L.E., Pacis, A., Tzelepis, F., Pernet, E., Dumaine, A., Grenier, J.C., et al. (2018). BCG Educates Hematopoietic Stem Cells to Generate Protective Innate Immunity against Tuberculosis. Cell 172, 176-190 e119.
Kawashima, T., Noguchi, E., Arinami, T., Yamakawa-Kobayashi, K., Nakagawa, H., Otsuka, F., and Hamaguchi, H. (1998). Linkage and association of an interleukin 4 gene polymorphism with atopic dermatitis in Japanese families. J Med Genet 35, 502-504.
Larsen, S.B., Cowley, C.J., Sajjath, S.M., Barrows, D., Yang, Y., Carroll, T.S., and Fuchs, E. (2021). Establishment, maintenance, and recall of inflammatory memory. Cell Stem Cell 28, 1758-1774 e1758.
Lim, A.I., McFadden, T., Link, V.M., Han, S.J., Karlsson, R.M., Stacy, A., Farley, T.K., Lima-Junior, D.S., Harrison, O.J., Desai, J.V., et al. (2021). Prenatal maternal infection promotes tissue-specific immunity and inflammation in offspring. Science 373, eabf3002.
Mitroulis, I., Ruppova, K., Wang, B., Chen, L.S., Grzybek, M., Grinenko, T., Eugster, A., Troullinaki, M., Palladini, A., Kourtzelis, I., et al. (2018). Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity. Cell 172, 147-161 e112.
Naik, S., Larsen, S.B., Gomez, N.C., Alaverdyan, K., Sendoel, A., Yuan, S., Polak, L., Kulukian, A., Chai, S., and Fuchs, E. (2017). Inflammatory memory sensitizes skin epithelial stem cells to tissue damage. Nature 550, 475-480.
Niec, R.E., Rudensky, A.Y., and Fuchs, E. (2021). Inflammatory adaptation in barrier tissues. Cell 184, 3361-3375.
Nishio, Y., Noguchi, E., Ito, S., Ichikawa, E., Umebayashi, Y., Otsuka, F., and Arinami, T. (2001). Mutation and association analysis of the interferon regulatory factor 2 gene (IRF2) with atopic dermatitis. J Hum Genet 46, 664-667.
Ordovas-Montanes, J., Dwyer, D.F., Nyquist, S.K., Buchheit, K.M., Vukovic, M., Deb, C., Wadsworth, M.H., 2nd, Hughes, T.K., Kazer, S.W., Yoshimoto, E., et al. (2018). Allergic inflammatory memory in human respiratory epithelial progenitor cells. Nature 560, 649-654.
Osawa, K., Etoh, T., Ariyoshi, N., Ishii, I., Ohtani, M., Kariya, S., Uchino, K., and Kitada, M. (2007). Relationship between Kaposi’s varicelliform eruption in Japanese patients with atopic dermatitis treated with tacrolimus ointment and genetic polymorphisms in the IL-18 gene promoter region. J Dermatol 34, 531-536.
Palmer, C.N., Irvine, A.D., Terron-Kwiatkowski, A., Zhao, Y., Liao, H., Lee, S.P., Goudie, D.R., Sandilands, A., Campbell, L.E., Smith, F.J., et al. (2006). Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nat Genet 38, 441-446.
Quiroz, F.G., Fiore, V.F., Levorse, J., Polak, L., Wong, E., Pasolli, H.A., and Fuchs, E. (2020). Liquid-liquid phase separation drives skin barrier formation. Science 367, eaax9554.
Spidale, N.A., Malhotra, N., Frascoli, M., Sylvia, K., Miu, B., Freeman, C., Stadinski, B.D., Huseby, E., and Kang, J. (2020). Neonatal-derived IL-17 producing dermal gammadelta T cells are required to prevent spontaneous atopic dermatitis. Elife 9, Feb 17;19:e51188.
van der Stoep, N., van der Linden, J., and Logtenberg, T. (1993). Molecular evolution of the human immunoglobulin E response: high incidence of shared mutations and clonal relatedness among epsilon VH5 transcripts from three unrelated patients with atopic dermatitis. J Exp Med 177, 99-107.
Vassar, R., Coulombe, P.A., Degenstein, L., Albers, K., and Fuchs, E. (1991). Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease. Cell 64, 365-380.