# How stem cells respond to infection, inflammation and ageing

Enzo Z Poirier

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## To cite this version:

Enzo Z Poirier. How stem cells respond to infection, inflammation and ageing. Nature Reviews Immunology, 2025. halshs-05246201

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# How stem cells respond to infection, inflammation and ageing

Enzo Z. Poirier

Institut Curie, Innate Immunity in Physiology and Cancer lab, PSL Research University,
INSERM U932, 26 rue d'Ulm, 75005 Paris, France

ORCID: 0000-0001-9795-2144

Correspondence to enzo.poirier@curie.fr

## Abstract

Stem cells maintain tissue architecture by replacing differentiated cells at steady state and upon injury. Implementing this cornerstone role requires protection of stem cells from pathogens and from the toxic effects of immune system activation. However, the pro-inflammatory innate immune mechanisms that protect differentiated cells from infection are poorly functional in stem cells. Instead, stem cells employ other specific defence mechanisms, such as antiviral RNA interference. At steady state, the proliferation and differentiation of tissue stem cells is regulated by multiple cell types, including immune cells. Following sterile tissue injury or during infection, the immune response — in addition to controlling pathogens and clearing cell debris — orchestrates tissue repair by fine-tuning stem cell activity, through direct cell-cell contacts and via inflammatory mediators such as cytokines. There is thus a stem—immune crosstalk that is fundamental to the maintenance of tissue homeostasis. Inflammation, which is defined as the age-driven elevation of inflammation and is associated with an altered immune cell composition, profoundly affects this stem—immune crosstalk, impacting the ability to repair tissues and participating in ageing of the whole organism.
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## Introduction

Stem cells populating tissues are responsible for the replacement of differentiated cells, thereby maintaining tissue architecture. As such, stem cells can be considered guardians of tissue integrity. If differentiated cells are replaceable, and therefore expendable, stem cells need to be protected against pathogen threats, as well as from the deleterious effects of immune activation. What guards the guardians? We will explore the stem cell-specific adaptations that shield them from danger. Although the stem cell status requires a certain insulation, the task of repopulating tissue cells calls for fine regulation. At homeostasis, intestinal stem cells divide constantly to maintain epithelial integrity, whereas muscle stem cells are largely quiescent. Upon skin injury, hair follicle stem cell proliferation needs to be activated to repair the damaged epithelium. How is stem cell activity tailored to a tissue's needs? In this Review, we will delve into the pivotal role of immune cells in orchestrating such processes, and explore changes induced by ageing. For the purposes of this article, the term 'stem cell' relates to undifferentiated cells capable of, firstly, self-renewal, and secondly, the generation of one or several types of differentiated cells, and therefore we include progenitors. We will focus on mammalian stem cells, although commonalities among different vertebrate species will be discussed.

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## Cell autonomous immune mechanisms of stem cells

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### Stem cell infection by pathogens

Are stem cells as susceptible to pathogenic infection as differentiated cells? Experimentalists started tackling this question in the 1970s, using homogeneous stem cell cultures¹⁻³. Cells from murine teratocarcinoma stem cell lines appear infectable by Sindbis virus and vaccinia virus, but are resistant to simian virus 40¹,². Depending on the type of stem cell used and the nature of the pathogen, resistance³⁻⁹ or permissiveness¹⁰⁻²² could be documented. In the context of cerebral organoids, which more faithfully recapitulate tissue organisation, neural stem cells are the target of Zika virus¹². Intestinal stem cells within human gut organoids can be infected by human astrovirus 1²². In vivo, mesenchymal stem cells can be infected by murine gamma herpesvirus 68¹⁹. Stem cells are not only challenged by viruses, but also by bacteria and parasites. Haematopoietic

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progenitors display susceptibility to *Bartonella henselae*, but resistance to *Bacillus Calmette-Guérin* (BCG) infection<sup>23-25</sup>, and haematopoietic and mesenchymal stem cells can be infected by the *Leishmania* parasites<sup>26-28</sup>.

Pathogens undeniably have the ability to infect stem cells, with a variability dependent on the experimental system. Yet, the vast majority of these experiments were performed with reductionist approaches that fail to faithfully recapitulate the in vivo situation, including: one, the scarcity of stem cells in a tissue; two, the 3D tissue organisation with extracellular matrix; and three, the inclusion of stem cells within their tissue niche. As such, whether stem cells are a target for pathogens in vivo remains an open question. Of note, many of these experiments were performed with embryonic stem cells rather than adult stem cells, which limits the physiological relevance of the results. An enthralling observation nevertheless arises from this early work: a given stem cell, resistant to viruses, becomes susceptible upon differentiation¹. It suggests that the stem cell status is endowed with a particular ability to counteract pathogens. Could this depend on classical, differentiated cell-like immune pathways?

## Innate immune pathways in stem cells during infection

In mammals, infection with a pathogen leads to the activation of innate immunity<sup>29,30</sup>. Cues of infection, such as double stranded RNA (dsRNA) accumulation in the cytosol, are detected by pattern recognition receptors (PRRs) and result in the production of type I and type III interferons (IFNs), as well as other pro-inflammatory cytokines. Secreted IFNs signal through the interferon-α/β receptor (IFNAR), leading to the transcriptional upregulation of hundreds of interferon-stimulated genes (ISGs), which encode antipathogen effectors<sup>31</sup>. As such, a protective environment is rapidly set up within cells that are competent for innate immune signalling (Figure 1).

Are classical innate immune pathways operative in stem cells? Undifferentiated cells do express PRRs, such as Toll-like receptors (TLRs) and the cytosolic DNA sensor cGAS<sup>32,33</sup>. Yet, it was observed early on that innate immune pathways can be dampened in stem cells<sup>34-40</sup>. Burke and colleagues showed in the 1970s that undifferentiated teratocarcinoma cells lack interferon production and sensitivity, yet both of these characteristics are instated upon differentiation<sup>34</sup>. Embryonic stem cells (ESCs) behave similarly, due to their low expression of mitochondrial antiviral signalling protein (MAVS),
which is an adaptor protein that triggers antiviral innate immune responses, as well as owing to their higher expression of negative regulators of IFN signalling, such as SOCS3³⁵,³⁶. MAVS downregulation depends on the expression of a specific microRNA, miR-673, which prevents MAVS mRNA translation³⁸. Mouse ESCs deficient in miR-673 display MAVS-dependent IFN production in the presence of viral dsRNA. Likewise, induced pluripotent stem (iPS) cells generated from fibroblasts lose the ability to produce IFN upon stimulation with viral RNA, and IFN signalling is attenuated in intestinal stem cells (ISCs) via the expression of interferon regulatory factor 2 (IRF2)⁴¹⁻⁴³.

The relative dampening of innate immune pathways in stem cells could be linked to toxicity issues, as IFN prevents proliferation, impairs differentiation and triggers apoptosis⁴⁴,⁴⁵. Artificially enforcing an innate immune programme in iPS cells translates into aberrant differentiation⁴¹. Of note, a dampened innate immune response is not a universal feature of undifferentiated cells. Neural and haematopoietic stem cells respond to IFN, by the upregulation of ISGs and the exit from quiescence with proliferation and differentiation, respectively⁴⁶⁻⁵⁰. Nevertheless, associating stemness with weakened immune pathways left the field with a conundrum that would remain unsolved for decades. If stem cells are particularly resistant to infection in the absence of canonical immune pathways, which are the molecular mechanisms of defence counteracting pathogens?

## Stem cell-specific mechanisms of defence

Human pluripotent cells express high levels of the ISG interferon-induced transmembrane protein 1 (IFITM1) in the absence of innate immune activation, whereas its paralogue IFITM3 is present in limbal epithelial stem cells⁵¹,⁵². This suggests that stem cells may be endowed with a unique type of immune organisation. Seminal work by the Rice laboratory showed that undifferentiated cells express a core set of ISGs at steady state in the absence of pathogens, while the expression of these core ISGs disappears upon differentiation⁵³. If certain ISGs, such as IFITMs, are present indiscriminately in all stem cells considered, others are specific to a single stem cell type. Mesenchymal stem cells — but not ESCs — express high levels of IFI16. ISG expression is not ablated by antibody-mediated blocking of IFNAR, suggesting that it is independent of tonic IFN signalling. Similarly, neural stem cells (NSCs) devoid of IFNAR and of the receptor for IFNγ
maintain high ISG expression⁴⁶. The molecular mechanism behind ISG expression at steady state, including the putative role of epigenetic regulation, is currently unknown. Functionally, knocking out IFITM1, IFITM2 and IFITM3 in ESCs, as well as knock-down in HSCs, translates into increased infectivity by influenza A virus and several Flaviviridae viruses, indicating that ISGs protect stem cells against viral infection⁵³. Expression of ISGs at steady state is thus a stem cell-specific mechanism of defence (Figure 1). It is however not restricted to bona fide undifferentiated cells, as lung-resident memory T cells express high levels of IFITM3, which is responsible for thwarting influenza A virus⁵⁴. This suggests that ISG expression may be a feature of the stemness transcriptional programme, which is also present in memory T cells⁵⁵. NSCs and ISCs of gut organoids are competent for IFN signalling but nonetheless express a subset of ISGs at steady state, indicating that stem cells can cumulate different mechanisms of defence²²,⁴⁶.

In that line, we and others documented a second, orthogonal mechanism relying on antiviral RNA interference (RNAi)⁵⁶. RNAi is a pivotal immune pathway in plants and invertebrates that shields from RNA virus infection⁵⁷. Mechanistically, a protein of the Dicer family recognises and cleaves viral dsRNA, generating short RNAs termed small interfering RNAs (siRNAs)⁵⁷. Endonucleases of the Argonaute family use siRNAs to degrade viral RNA in a sequence-specific manner⁵⁷. The activity and importance of antiviral RNAi in protecting mammalian differentiated cells against RNA viruses, alongside the canonical IFN response, is still disputed⁵⁷. The pathway is, however, active in stem cells and counteracts infection of murine ESCs by flock house virus and encephalomyocarditis virus⁵⁸,⁵⁹. Stem cells implement antiviral RNAi by expressing a truncated isoform of Dicer1 named aviD (antiviral Dicer), which is generated by alternative splicing (Figure 1)⁵⁶. Compared with Dicer, aviD displays increased capacity to target dsRNA. It is expressed by stem cells within adult tissues such as brain and gut, as well as by ESCs. Expression of aviD is robustly detected in ISCs of the intestinal crypts, but it is undetectable in differentiated enterocytes of the villi. In a brain organoid model, aviD protects NSCs from Zika virus and SARS-CoV-2 infections.

Stem cells of epitheliums, particularly of the skin and gut, are in contact with a plethora of microorganisms that constitute the microbiota⁶⁰,⁶¹. Microbiota plays a beneficial role in ISC biology, via the stimulation of the PRR NOD2, which translates into stem cell resistance to oxidative stress, ultimately favouring cell survival⁶²,⁶³. The
microbiota metabolite indole acetic acid acts on ISCs to prevent proliferation, safeguarding against neoplastic transformation⁶⁴. In the skin, bacteria such as *Staphylococcus aureus* induce the production of IL-1β by keratinocytes, which bolsters repair responses upon wounding⁶⁵. Butyrate, which is another metabolite derived from the microbiota, has been shown to prevent ISC proliferation, but is metabolized at steady state by enterocytes of the villi, thereby safeguarding the ISC pool⁶⁶.

Stem cells are thus shielded from pathogens by unrelated mechanisms, which could be pictured as complementary or redundant layers of protection. Much is left to understand in the field of stem cell immunity: can pathogens target different types of stem cells in vivo? Are quiescent cells more protected than cycling cells? Are there additional mechanisms of defence complementing steady state ISGs and antiviral RNAi? Can stem cell-specific immune defences be counteracted by pathogens? So far, we largely described stem cells as insulated entities shielded by multiple antipathogen strategies. Yet, stem cells are located within a niche, and dialogue with numerous cell types, including immune cells⁶⁷.

## Interplay between stem cells and immune cells

### Stem-immune crosstalk at steady state

At homeostasis, proliferation and differentiation of tissue stem cells is tightly regulated. Hair follicle stem cells (HFSCs) of the epidermis, which are responsible for the maintenance and repopulation of hair shafts, alternate between an active proliferation state (anagen) and a quiescent state (telogen)⁶⁸. Fine-tuning of HFSC activity relies on signals emanating from cells of the niche, unrelated to immunity, that modulate signalling pathways such as Wnt, TGFβ and BMP⁶⁹,⁷⁰ (Figure 2).

Macrophages are instrumental for clearing cell debris via phagocytosis, as well as for detecting pathogen infection and activating innate immunity⁷¹. This eventually sets in motion the adaptive branch of immunity, embodied by antigen-specific B cells and T cells. CD8+ T cells kill target cells after their T cell receptor (TCR) recognizes specific peptide-MHC class I complexes, whereas regulatory T (Treg) cells are tasked with dampening the immune response⁷². In the uninfected epidermis, macrophages are in close proximity to HFSCs and their numbers fluctuate according to HFSC cycles⁷³,⁷⁴. Loss of macrophages
between telogen and anagen is due to apoptosis, which leads to the liberation of Wnt ligands promoting HFSC proliferation<sup>73</sup> (Figure 2). Contrastingly, TREM2<sup>+</sup> macrophages produce oncostatin, an IL-6 family cytokine, that triggers JAK-STAT5 signalling directly in HFSCs and promotes quiescence<sup>74</sup>. Macrophages thus provide signals that regulate HFSC activity.

Treg cell numbers also fluctuate in the epidermis, increasing in telogen and decreasing in anagen<sup>75</sup>. Genetic depletion of Treg cells leads to impaired telogen to anagen transition, as well as impaired HFSC differentiation. Mechanistically, Treg cells favour anagen by expressing the membrane-bound Notch ligand Jagged 1<sup>75</sup> (Figure 2). In addition to modulating the Notch pathway, Treg cells express TGFβ3, which stimulates anagen upon depilation (that is, hair removal)<sup>76</sup>. The HFSC example indicates that essential properties of tissue stem cells — proliferation and differentiation — are partially regulated by immune cells at steady state, through direct cell-cell contacts and via secreted mediators.

This stem-immune crosstalk [G] exists in other tissues, although with modalities that seem largely tissue-specific. Treg cells probe MHC class II molecules expressed at the surface of ISCs<sup>77</sup>. Such interaction plays a functional role, as ablating Treg cells in vivo leads to increased ISC numbers<sup>77</sup>. Subtypes of Treg cells also produce specific cytokines that affect ISC proliferation and differentiation, as demonstrated in the context of gut organoids<sup>77</sup>. Microbiota influences ISC activity, by directly or indirectly tuning the cross-talk between macrophages and ISCs<sup>78,79</sup>. A dialogue between stem and immune cells is also reported in the bone marrow<sup>80,81</sup>, central nervous system<sup>82,83</sup> and mammary gland<sup>84-86</sup>.

If stem cells have historically been considered 'immune privileged' (Box 1), it appears instead than immune cells can be part of the niche and fine-tune stem cell activity. Yet, immune cells carry a destructive power, exemplified by the ability of CD8+ T cells to provoke cell death. Could the stem-immune crosstalk lead to toxicity directed towards stem cells? In a transgenic mouse model expressing high levels of antigen, cycling stem cells such as ISCs can be eliminated by CD8+ T cells<sup>87</sup>. This correlates with robust expression of MHC class I and proteins of the antigen presentation machinery. Oppositely, cells in quiescence such as muscle stem cells (MuSCs) and telogen HFSCs are spared, mostly due to a downregulation of MHC class I<sup>87</sup>. Whilst it is unclear if stem
cells could be CD8+ T cell targets without enforced expression of antigen, these results
suggest that the immune system surveils stem cells. In the haematopoietic
compartment, stem cell presentation of antigens to T cells allows for the immune-
mediated clearance of transformed cells and avoids leukemia development<sup>88</sup>. More
generally, the stem-immune crosstalk plays a role in pathologies such as cancer (Box 2).
Immune cells thus regulate stem cells at homeostasis. Upon infection or sterile injury,
stem cell activity is pivotal to reconstruct tissues. Does the stem-immune crosstalk play
a role in the repair process?

Immune cells instruct stem cell behaviour upon deviations from homeostasis

Sterile injury and infection represent deviations from homeostasis that activate an immune response, with an influx of immune cells in the affected tissue, partly dependent on the increased activity of haematopoietic stem cells (HSCs)⁸⁹. An efficient immune response thus relies on a modification of HSC behaviour, with a so-called emergency myelopoiesis that has been discussed in detail elsewhere⁹⁰. A return to homeostasis implies clearance of pathogens and/or cell debris, as well as restoration of tissue integrity — a process of utmost importance for epithelial barriers. Bolstering stem cell activity is thus needed to provide for a novel pool of differentiated cells. Perhaps not surprisingly, immune cells — the major actor behind sensing and resolution of deviations from homeostasis — instruct stem cells in this matter.

Sterile injury of muscle is repaired by the activation of otherwise quiescent MuSCs (Figure 3). During this process, macrophages are recruited to the site of injury and transiently interact with MuSCs⁹¹,⁹². Ly6C⁺ macrophages secrete the protease Adamts1 that cleaves Notch1 and promotes exit from quiescence of MuSCs⁹¹. In parallel, the cytokine meteorin-like, another product of macrophages, promotes stem cell proliferation and can be harnessed to boost muscle repair⁹³,⁹⁴. Treg cells also home to the site of injury and orchestrate the switch of myeloid cells from a pro- to an anti-inflammatory phenotype, as well as express amphiregulin to bolster muscle repair⁹⁵ (Figure 3). A similar coordination role is played by Treg cells in muscular dystrophy, a class of pathologies characterised by the chronic loss of muscle fibres⁹⁵. In addition to MuSCs, fibro/adipogenic progenitors participate in muscle repair and are mobilised by the cytokines IL-4 and IL-13⁹⁶. In the epidermis, as well as generating hair shafts, HFSCs can
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participate in repair⁹⁷. HFSCs migrate to the wound bed after injury and differentiate into stratified epithelial cells. This process depends on Treg cells, which accumulate during the repair process in close proximity to HFSCs⁹⁸. Treg cell depletion in the skin translates into defective epithelial barrier restoration⁹⁸. In addition, migration of epithelial cells to the wound bed is governed by a metabolic rewiring towards glycolysis, orchestrated by the cytokine IL17A produced by γδ T cells⁹⁹,¹⁰⁰.

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These examples indicate that the very immune response activated by injury orchestrates tissue repair, by influencing stem cell proliferation and differentiation. This holds true in infection. In the lung, the initial infection-driven inflammatory response is swiftly followed by a wave of repair, entrusted to stem cells called alveolar type 2 (AT2) cells, responsible for repopulating AT1 cells of the epithelium¹⁰¹. Injury results in an influx of interstitial macrophages that localise in the vicinity of AT2 cells and drive the transition towards damage-associated transition progenitors, via the production of the cytokines IL-1β and TNF¹⁰¹,¹⁰². Ly6G⁺ macrophages also play a part, presumably through cytokine secretion acting directly on stem cells¹⁰³. After influenza A virus infection of the lung, Treg cells produce amphiregulin that signals to mesenchymal cells, which in turn regulate the proliferation of AT2 cells¹⁰⁴.

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In summary, immune cells not only counteract pathogens, but deal with the aftermath of infection by coordinating tissue repair. Only when inflammation is resolved can repair begin, which indicates the need for a tight temporal control of stem cell activity. As Treg cells' primary function is to dampen the immune response, it is interesting to note that these cells seem to take a central role in the stem-immune crosstalk. Placing the onus of stem cell control on Treg cells presumably fits with the role of these cells in orchestrating the return to tissue homeostasis after infection or injury. In the gut, ISCs respond to multiple cytokines produced by T cells, such as IFNγ, IL-10 and IL-17, which will either favour maintenance of the stem cell pool or differentiation⁷⁷. These cytokines originate from different types of T helper cells, whose numbers and activity vary depending on the nature of infection or injury. In the steps of Biton and colleagues, it is tempting to speculate that a finer granularity exists in the stem-immune crosstalk, in which stem cells would be regulated differently depending on the type of pathogen, via the activation of different subpopulation of T cells (and other immune cell types)⁷⁷. This may allow to better tailor stem cell activity to repair the pathogen-specific damages

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caused to the tissue. Immune cells would thus act as a ruler of tissue damage and instruct stem cells to perform the appropriate repair tasks. This flux of information is bi-directional, as stem cells also influence the activity of immune cells, as discussed below.

Stem cells instruct immune cells

In the epidermis, HFSCs that migrate to the wound bed show increased expression of CD80, a co-stimulatory molecule expressed by antigen-presenting cells, such as dendritic cells and macrophages, which participate in T cell activation¹⁰⁵. Genetic ablation of CD80 in HFSCs translates into decreased Treg cell accumulation at the site of injury and impaired wound healing. CD80 favours the expansion of lymph node-primed, newly arrived Treg cells from the circulation, resulting in a Treg cell-based anti-inflammatory programme. HFSCs additionally increase production of the chemokine CXCL5, which recruits neutrophils into the wound, instating an antipathogen barrier across the breached epithelium¹⁰⁵. Ultimately, Treg cell populations expanded via CD80 dampen the pro-inflammatory activity of neutrophils. A choreography of immune cells is thus orchestrated by HFSCs to support wound healing.

Epidermal stem cells (EpSCs) upregulate the membrane-bound Skint molecules upon wounding, which participates in maintaining a network of γδ T cells pivotal for repair¹⁰⁶. This depends on the activation of HIF1α in stem cells by wound-induced hypoxia¹⁰⁷. EpSCs produce IL-24, which acts in an autocrine and paracrine manner to promote repair. The authors propose that, akin to myeloid cells detecting pathogen cues and activating the immune response, EpSCs detect wounding cues such as hypoxia and produce cytokines that orchestrate repair. Stem cells thus could, in certain contexts, substitute or contribute to typical immune functions. In that line, HFSCs also perform phagocytosis, in a non-redundant manner with respect to professional phagocytes such as macrophages¹⁰⁸.

In the gut, ISC expression of MHC class II has been documented, although the ability of ISCs to influence CD4+ T cells in vivo is unknown⁷⁷. Stem cells of the olfactory mucosa that maintain a population of primary sensory neurons (responsible for the sense of smell) respond to acute inflammation via the production of a variety of cytokines and chemokines that attract immune cells¹⁰⁹. NSCs secrete vascular endothelial growth factor (VEGF) that modulates the activity of microglia (the brain macrophages) with
unexplored effects on the immune response¹¹⁰. The overall ability of stem cells to
influence immune cells remains largely unchartered.

*Inflammatory memory in stem cells*

Infection and sterile injury trigger an inflammatory response that can epigenetically reprogramme cells at certain genomic positions linked to immunity, essentially resulting in an inflammatory memory¹¹¹. This phenomenon identified in immune cells has been coined trained immunity¹¹². The Fuchs laboratory explored this concept in the context of EpSCs, responsible for production of skin epithelial cells¹¹³. Topical application of the TLR7 agonist imiquimod leads to a first, transient event of inflammation on mouse skin. A second mechanical injury, introduced after resolution of imiquimod-driven inflammation, heal faster in mice that received treatment compared to control mice. Previous inflammation thus accelerates wound healing. At the genomic level, imiquimod treatment reprogrammes EpSCs, with the establishment of stably open chromatin on loci coding for immune proteins, including for the inflammasome absent in melanoma 2 (AIM2). Secondary wound healing is increased by AIM2-dependent secretion of IL-1β. Reprogramming of EpSCs relies on STAT3 and the transcription factor pair FOS and JUN¹¹⁴.

Turning to the haematopoietic compartment, mice infected intravenously with BCG are better protected against subsequent challenge with *Mycobacterium tuberculosis*, largely through the production of macrophages superior in targeting bacteria²⁵. In HSCs, the initial BCG infection leads to establishment of memory at inflammatory loci that is maintained in newly produced and differentiated macrophages. The PRR agonist β-glucan similarly reprogrammes HSCs, resulting in a bias towards the production of myeloid cells, which increases resistance to chemotherapy-induced toxicity¹¹⁵. Mice injected with the TLR agonists poly(I:C) or lipopolysaccharide reprogramme HSCs via the chromatin factor c/EBPβ, resulting in increased survival following *Pseudomonas aeruginosa* challenge¹¹⁶. Hematopoietic progenitors can similarly be trained by *Mycobacterium avium* infection¹¹⁷. During pregnancy, *Yersinia pseudotuberculosis* infection results in stable modifications of chromatin accessibility in fetal epithelial stem cells, which is dependent on the production of IL-6 by the mother, as well as on an influx of Th17 cells in the gut lamina propria¹¹⁸. Infection-driven reprogramming of fetal stem cells bolsters immune responses in the offspring, with
increased autoimmunity as a tradeoff. In humans, recent data suggest that chronic rhinosinusitis leads to a memory of inflammation in epithelial progenitor cells¹¹⁹. There is thus a long-term reprogramming of stem cells after antenatal or postnatal inflammation.

How much can these cells remember? Repair of skin wounds is tackled by EpSCs, assisted by HFSCs that migrate from the hair bulge¹²⁰. HFSCs that migrated adopt characteristics of EpSCs, but maintain memory of the bulge, their niche of origin. Functionally, EpSCs from HFSC origin display increased capacity to respond to signals that are typical of the HFSC niche. Stem cells are thus reprogrammed by a variety of cues in addition to inflammation and store diverse, cumulative memories¹²⁰. It is tempting to speculate that the accumulation of a patchwork of memories progressively leads to the generation of subsets of stem cells not reprogrammed with the same information, and therefore functionally distinct. AT2 cells of the lung, for example, exist in subsets that respond differentially to injury¹⁰¹. Note that although we focus here on mammalian stem cells, the stem-immune crosstalk is conserved in other animals (Box 3).

## Dysregulation of the stem-immune crosstalk during ageing

Stem cell exhaustion represents one of the hallmarks of ageing¹²¹. Aged stem cells show reduced repair capacity, partly due to cell-intrinsic accumulation of mutations and dysregulations in epigenetics and proteostasis¹²¹⁻¹²³. Cell-cell interactions within the niche also play a role. The decrease in MuSC regenerative capacity originates from a niche-driven deficiency in Notch signalling, coupled with excess TGFβ production¹²⁴,¹²⁵. The immune system plays a part in ageing of the organism. Deleting the DNA repair endonuclease ERCC1 in the haematopoietic compartment leads to accumulation of DNA damage in immune cells and premature senescence¹²⁶. Even if this accelerated form of ageing is restricted to the bone marrow and immune progeny, ageing propagates to non-haematopoietic, ERCC1-competent tissues. An aged immune system thus promotes ageing of the entire organism¹²⁶. What are the characteristics of an aged immune compartment? Older individuals display chronic inflammation, also termed inflammageing, which corresponds to elevated levels of secreted inflammatory molecules in the blood and tissues (for example, cytokines and IFNs, such as TNF, IL-6,
IFNγ), as well as changes in immune cell profiles<sup>121,127</sup>. Inflammation [G] affects the stem cells at steady state, as well as during the loss of homeostasis, such as in injury.

### Inflammation affects stem cells at steady state

There is an age-dependent decline in the ability of HFSCs to regenerate hair, which is related to increased inflammation in the skin<sup>128,129</sup>. IL-1α, produced in part by epithelial cells, accumulates in the epidermis of old mice. Linked to this inflammatory milieu, HFSCs display sustained JAK-STAT signalling, and JAK inhibitors rescue the proliferative defect of HFSCs<sup>128</sup> (**Figure 2**). HFSCs isolated from young and old mice demonstrate an equal propensity to maintain colonies in culture<sup>129</sup>. When transplanted on young individuals, HFSCs from young and old mice regenerate hair in similar amounts, whilst young HFSCs grafted on old individuals fail to do so<sup>129</sup>. These results indicate that, in the skin, the inflamed environment constitutes a cell-extrinsic source of stem cell decline.

Chronic inflammation in the *Nfkb1*-deficient mouse model results in lower survival, cachexia and weight loss<sup>130</sup>. Tissue repair is affected in the liver and the gut, which has a malabsorptive phenotype that is linked to reduced ISC proliferation. Both these defects are rescued by the non-steroidal anti-inflammatory drug ibuprofen<sup>130</sup>. Inflammation also increases in the HSC niche with age, with endosteal mesenchymal cells producing higher levels of IL-1β and TNF<sup>131-133</sup>. Sustained delivery of IL-1β in young mice renders the HSC niche ‘old’, whilst IL-1β blockade prevents ageing, demonstrating that the inflammatory milieu affects HSC regenerative capacity<sup>133</sup>. Microbiota also plays a role, as its composition changes with age, and can regulate HSC function by modulating inflammation<sup>134,135</sup>.

Inflammation is not always toxic to stem cells. In the brain, ageing translates into increased inflammation of the NSC niche, with a drop in total stem cell numbers, together with an increased proportion of quiescent NSCs<sup>136</sup>. Aged NSCs show an increased IFN signature<sup>46</sup>. Antibody-mediated blocking of the pro-inflammatory chemokine CXCL10 results in a decrease in quiescent NSC numbers, establishing that brain inflammation may preserve the stem cell pool by favouring quiescence. sFRP5, an antagonist of Wnt, also plays a role in this process, showing that regulation of the stem cell pool does not only rely on inflammation<sup>136</sup>. Age-related inflammation can thus have intricate effects on stem cells, with an inhibition of proliferation — and therefore of tissue repair — coupled
with a preservation of the stem cell pool, through quiescence. In that line, inhibiting JAK-STAT signalling in aged mice rescues HFSC proliferation, but comes at the cost of reducing the total number of these proliferative stem cells¹²⁸.

How do stem-immune cells interactions change during ageing? CD8+ T cells expressing IFNγ accumulate in the brain of old individuals¹³⁷. A subpopulation of NSCs that expresses the IFNγ receptor displays increased STAT1 signalling and proliferates less. This suggests that T cells accumulating with age may prevent NSC proliferation¹³⁷. In the gut, ageing correlates with impaired ISC proliferation and an increased percentage of differentiated cells in the tissue, skewed towards the secretory lineage¹³⁸. Purified ISCs from young and old mice display an inflammatory transcriptional signature, including higher expression of MHC class II. Cytotoxic CD4+ T cell and group 2 innate lymphoid cells that express IFNγ accumulate in the lamina propria of old mice, and the aged transcriptional signature of ISCs is reverted upon IFNγ blockade. These results suggest that ISCs drive the accumulation of CD4+ T cells through MHC class II interactions — as documented elsewhere⁷⁷ — which in turn increases inflammation and perturbs the proliferation/differentiation balance¹³⁸. Of note, chronic inflammation unrelated to ageing similarly affects HSCs¹³⁹ and NSCs of the olfactory mucosa¹⁰⁹.

Ageing of the immune system thus affects stem cells at steady state in diverse ways, impairing proliferation and differentiation. A characteristic of older individuals is the poor ability to repair tissues after injury. Is there a role of the stem-immune crosstalk in this process?

## Inflammaging affects stem cells during repair

The number of MuSCs and ability to regenerate muscle fibres decreases with age¹⁴⁰⁻¹⁴². It correlates with the establishment of an inflammatory environment, characterised by the accumulation of monocytes, macrophages and T cells, as well as by an increase in inflammatory cytokines such as IL-6¹⁴⁰⁻¹⁴⁵ (Figure 3). Young mice transplanted with old (but not with young) bone marrow display reduced MuSC numbers, whilst old mice transplanted with young bone marrow show increased muscle fibre cross-section¹⁴²,¹⁴⁶. This points towards a role of inflammation in altering MuSC activity, in part due to macrophage-driven production of the cytokine osteopontin¹⁴⁷. In a model of muscular dystrophy, transplantation experiments show that old MuSCs have a reduced capacity to
generate myofibres, which is related to increased expression of members of the JAK2-STAT3 pathway<sup>140</sup>. Inhibiting JAK-STAT translates into symmetric division and increased engraftment in transplantation experiments, with a boost in muscle repair following injury<sup>140</sup>. Alterations of MuSC repair properties during ageing are thus explained by a conjunction of cell-intrinsic modifications and cell-extrinsic inflammationing (Figure 3).

In the context of tissue repair, complex tripartite relationships between inflammation, stem cells and ageing have been shown to exist in the lung<sup>148</sup>, pituitary gland<sup>149</sup>, central nervous system<sup>150</sup> and skin<sup>106</sup>. Aged skin is characterized by slower re-epithelialization, due to decreased proliferation and impaired migration of keratinocytes to the wound bed<sup>106</sup>. Old keratinocytes fail to upregulate Skint1, a membrane-bound protein that drives the accumulation of γδ T cells involved in wound repair. This is due to decreased IL-6 accumulation in the wounds of old mice. Consequently, complementation with IL-6 bolsters wound healing in young and old mice alike<sup>106</sup>. Inflammation thus broadly affects stem cells.

Stem cells can be a source of inflammation

In the gut, aged ISCs show increased expression of MHC class II, with accumulation of CD4+ T cells and concomitant reduction of CD8+ T cell<sup>151</sup>. Contacts between ISCs and CD4+ T cells rise in number, presumably through direct MHC class II-TCR interactions<sup>77,151</sup>. Organoids generated from isolated ISCs maintain an ‘aged’ transcriptional signature for weeks, in the absence of immune cells, and this is associated with a primed chromatin state for the binding sites of the inflammatory transcription factors STAT1 and RELA. During ageing, ISCs are reprogrammed in a cell-intrinsic fashion and endowed with a superior ability to respond to inflammation<sup>151</sup>. This may result in ISCs driving inflammation, by attracting higher numbers of CD4+ T cells, which in turn produce IFNγ, establishing a feed-forward loop of inflammation. These results need to be put in perspective with the ability of stem cells to remember inflammation by epigenetic reprogramming. Hypothetically, adaptation of tissue stem cells to be reprogrammed by inflammation could, in the long run, considerably affect chromatin organisation as to transform stem cells into a source of inflammation in older individuals. Such considerations remain highly speculative, and the participation of stem cells in ageing-associated inflammation remains to be thoroughly assessed.
Conclusions and perspectives

In relation to their pivotal role in maintaining tissue architecture, stem cells are protected by specific mechanisms of defence: the expression of ISGs at steady state and antiviral RNAi. Given the importance of these cells, it is likely that additional, unknown mechanisms of immune protection exist. These pathways may not be present ubiquitously, but rather restricted to certain types of stem cells. One may expect stem cells of epithelia, such as ISCs, to be especially geared up against pathogens, given their exposure to exogenous microorganisms. Identifying these protective mechanisms will be an exciting development of the field. Another open question is the ability of pathogens to infect stem cells in vivo. In a tissue loaded with viral particles, are stem cells infectable or completely protected? Do pathogens drive stem cell demise, resulting in impaired tissue repair? Is there such a thing as stem cell-specific inborn errors of immunity, in which genetic mutations alleviate stem cell protection against infection?

Stem cells pair the use of specific cell-intrinsic defence mechanisms with a sustained crosstalk with immune cells, among which macrophages and Treg cells seem to take the centre stage. One could argue that the macrophage–stem crosstalk makes sense, due to the role of the former as sentinels of infection and wounding. In that framework, cells that sound the alarm upon rupture of homeostasis also give the initial push for repair. Then, efficient repair occurs only after resolution of inflammation, which largely depends on Treg cells. This stem–Treg cell crosstalk would allow for a properly timed repair process and avoid a too early start. Yet, these a posteriori justifications appear rather unsatisfactory, given that the role of numerous immune cell populations in the stem–immune crosstalk are largely unknown. For example, the interplay between stem cells and B cells remains to be deciphered. Attempts to construct a unified model of stem–immune crosstalk should also be put into perspective with the heterogeneity observed between stem cells of different tissues in terms of firstly, their activity (cycling, quiescent, or alternating between the two), secondly, the composition of immune cells which varies between tissues, and thirdly, the presence and composition of the microbiota. Model tissues such as skin and gut are pivotal to unravel the specifics of the stem-immune crosstalk. Yet, the field lacks a more exhaustive picture of the stem-
immune crosstalk. Working towards this goal would allow us to elucidate which stem-
immune interactions exist across tissues, and determine which interactions are tissue-
specific. This effort would allow us to construct a grammar of the stem-immune crosstalk
and allow to determine if certain aspects are governed by universal mechanisms.

Acknowledgements

My apologies to colleagues whose work was omitted due to space constraints. I am
grateful to members of my team, to scientists of the Immunity and Cancer Unit of Institut
Curie and to the Reis e Sousa lab of the Crick Institute for fruitful discussions and critical
reading of the manuscript. This work is supported by an ERC Starting Grant (STEMGUARD
101075865), by a grant "Impact Santé" supported by Agence Nationale de la Recherche
under France 2030 (ANR-24-RRII-0005) on funds administered by Inserm, by funding from
the program France 2030 launched by the French Government (ANR 11 LBX 0043, part of
the IDEX PSL ANR-10-IDEX-0001-02), by the Major Research Program of PSL Research
University "DEVINE" launched by PSL Research University and implemented by ANR with
the reference ANR-10-IDEX-0001, by a Jeune Équipe grant from the Fondation pour la
Recherche Médicale (ConvAJE202110014409) and by funding from Institut Curie.

Competing interests

The author declares no competing interests.

**Peer review information**

*Nature Reviews Immunology* thanks A. Ditton, A. Martin-Villalba, Y. Miao, T. Ohteki and the other, anonymous, reviewer for their contribution to the peer review of this work.
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## Figure legends

**Figure 1. Stem cell-specific immune mechanisms**

Stem cell-specific mechanisms of defence comprise antiviral RNA interference (RNAi), driven by the expression of antiviral Dicer (aviD) and Argonaute 2 (Ago2), protecting against RNA viruses. aviD expression is undetectable in differentiated cells. Stem cells also express interferon-stimulated genes (ISGs) such as IFITMs, at steady-state. In differentiated cells, ISG expression depends on IFN production downstream of pathogen detection by pattern recognition receptors (PRRs). Whereas stem cells are protected by additional mechanisms of defence is unknown. IFITM, interferon-induced transmembrane protein; ISG15, interferon-stimulated gene 15; MX1, myxovirus resistance protein 1; RSAD2, radical S-adenosyl methionine domain containing protein 2.

**Figure 2. Crosstalk between hair follicle stem cells and immune cells at steady state.**

HFSCs alternate between a quiescent phase (telogen) and a proliferative phase (anagen). Macrophage-derived oncostatin promotes quiescence whilst WNT secreted during macrophage apoptosis favors anagen. Secretion of TGFβ3 and activation of the Notch pathway by membrane-bound Jagged 1 allow regulatory T cells (Treg cells) to tune anagen. Inflammation translates into accumulation of IL-1β in the epidermis, which inhibits HFSC proliferation via JAK/STAT. JAK inhibitors (JAKi) rescue the proliferation defect. BMP, bone morphogenic protein; JAK, janus kinase; STAT, signal transducer and activator of transcription; TGF, transforming growth factor; TREM2, triggering receptor expressed on myeloid cells 2; WNT, wingless-related integration site.

**Figure 3. Crosstalk between muscle stem cells and immune cells during repair.**

Muscle stem cells (MuSC) are responsible for repair of tissue injuries, through exit of quiescence and activation of proliferation. Macrophages secrete the protease Adamts1 that cleaves Notch to abolish quiescence, as well as the cytokine meteorin-like involved in boosting proliferation. Treg cells home at the site of injury and participate in MuSC-driven repair. Old individuals show decline in repair capacity, partly due to inflammation, including via osteopontin secretion by macrophages and via the accumulation of pro-
inflammatory cytokines in the tissue. MuSCs also display age-driven, cell-intrinsic
increase in expression of inflammatory mediators.

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**Box 1: The immune privilege of stem cells**

Transplantion of genetically non-identical material from the same species (that is, allografts) face the possibility of rejection, largely through the activation of T cell-mediated immunity¹⁵². Back in the 1950s, it was observed that allogeneic skin grafts are rejected when placed on the body wall, but not on the cornea, indicating that certain locations of the body are more prone to tolerate transplants ; such locations were named ‘immune privileged’¹⁵³. The concept of immune privilege was then applied to stem cells upon the realisation that ESCs and mesenchymal stem cells would be retained for weeks upon grafting¹⁵⁴. This was attributed to their low expression of MHC molecules, which was shown later to depend on the cycling state of the stem cells⁸⁷. More recent work describes the molecular basis behind the crosstalk between stem cells and immune cells. HSCs persist in allografted mice for a month, relying on Treg cells that mediate a local, immunosuppressive environment¹⁵⁵. These Treg cells, part of the HSC niche, preserve cells from oxidative damage and favor quiescence by generating extracellular adenosine⁸¹. In the skin, Treg cells protect HFSCs from excessive inflammation driven by IL-2¹⁵⁶. There is thus an immunomodulatory function of the niche which protects stem cells from the toxic effects of the immune system in certain contexts.

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**Box 2: Subversion of the stem-immune crosstalk in pathology**

Cancer stem cells represent a subpopulation of cells that have the capacity to initiate
tumour formation, typically when transplanted in a naive host<sup>157</sup>. Such cells can occur in
multiple malignancies, including leukemia and solid cancers<sup>158</sup>. Being endowed with
stem cell characteristics and a stemness transcriptional programme correlates with a
decrease in the antitumoral immune response<sup>159</sup>. This suggests that the stem-immune
crosstalk may be active in cancer stem cells and participate in thwarting the immune
response. In triple-negative breast cancer, tumour cells that escape T cell targeting are
quiescent, mirroring the behaviour of regular tissue stem cells, although without MHC
class II downregulation<sup>87,160</sup>. Other examples include downregulation of TLR4 in

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glioblastoma stem cells, as well as activation of STAT3 in breast cancer stem cells through close interactions with macrophages¹⁶¹,¹⁶². STAT3 also plays a role in non-small cell lung cancer and in pancreatic cancer, in which settings it is activated by Th17 cell-derived IL-17 or IL-22, respectively¹⁶³,¹⁶⁴. In the brain, infiltrated plasma cells favour glioblastoma stem cell growth through IgG secretion¹⁶⁵. Hijacking the stemness programme also allows a subset of cancer cells to dampen the effect of immunotherapies¹⁶⁶. Moving away from cancer, transplantation efficiency can be hindered by unwanted stem-immune interactions, as allogenic bone marrow transplant can translate into targeting of intestinal stem cells by T cells, partly through IFNγ and STAT1 activation¹⁶⁷,¹⁶⁸.

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**Box 3: Conservation of the stem-immune crosstalk across vertebrates**

Immune regulation of stem cell activity is not restricted to mammals, but conserved in vertebrates such as zebrafish, suggesting that it may be a general feature of this clade. In *Danio rerio*, haematopoietic stem cell (HSC) activity is regulated by pro-inflammatory mediators such as IFN and TNF, whilst direct HSC-macrophage interactions tune the clonality and quality of stem cells¹⁶⁹⁻¹⁷³. Inflammatory signals similarly regulate neural stem cell activity¹⁷⁴. Age-driven changes in immune homeostasis seem also conserved in vertebrates, as a transcriptomics signature of innate immune activation could be detected in old humans, mice, rats and killifish¹⁷⁵. Whether inflammation affects the activity of stem cells globally across vertebrate species remains to be explored. A fascinating fact emerges when correlating immunity and regeneration: vertebrates able to fully regenerate body parts, such as salamanders, display a more ‘inefficient’ immune response compared to higher vertebrates¹⁷⁶. This suggests that a trade-off exists between evolving a more potent immune system and being equipped with stem cells that can regrow segments. What is the role of the stem-immune crosstalk in regenerative species, and how did it evolved in vertebrates? Regulatory T cells have been shown to play a role in organ regeneration in zebrafish, but the immune mechanisms at play in other vertebrates are largely unchartered¹⁷⁷.

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## Adult stem cells

These cells, located in specific niches within adult tissues, are undifferentiated cells that generate tissue-specific differentiated cells upon division. Contrary to ESCs, adult stem cells can give rise to a limited number of differentiated cell types. Depending on the tissue, adult stem cells are constantly cycling (intestinal stem cells), alternating between periods of cycling and quiescence (hair follicle stem cells of the skin) or quiescent without the activation of tissue repair (muscle stem cells).

## Embryonic stem cells (ESCs)

Pluripotent cells isolated from the inner cell mass of blastocysts (early-stage embryos), which can be differentiated in cells of the three primary germ layers. These cells have been widely used as a stem cell model, including in infection studies, due to their relative simplicity of isolation and culture. One disadvantage of using ESCs for pathogen infection relates to a poor physiological relevance, as such cells are rarely in contact with pathogens in natural settings. Nevertheless, ESCs have been an invaluable tool for the stem cell immunity research field, and allowed to discover biological mechanisms which existence was later confirmed in adult stem cells.

## Inflammation

This umbrella term refers to increased inflammation gradually occurring with advanced age, in the absence of infection. There is increased levels of pro-inflammatory cytokines (interferons, interleukins) in the blood and tissues of aged individuals, as well as changes in the composition of immune cells, with a bias towards the myeloid lineage to the detriment of the lymphoid lineage.

## Stem-immune crosstalk

This refers to the interactions between stem cells and immune cells that modulate their respective activity. It can be mediated by direct cell-cell contact, or via the secretion of soluble mediators such as cytokines. If research largely focused on immune cells
regulating stem cells, more recent data document that stem cells can also influence immune cells.
## Highlighted references

¹Swartzendruber & Lehman

An early work that documents how differentiation renders cells permissive to viral infection.

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³⁸Witteveldt et al.

The authors provide the molecular basis of IFN attenuation in ESCs, which downregulate MAVS, a pivotal actor of the immune signalling cascade.

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⁴¹Eggenberger et al.

Why would stem cells be incompetent for IFN signalling? This paper shows how artificially inducing an innate immune signature is detrimental to iPS cells.

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⁵³Wu et al.

Identification of an important stem cell-specific mechanism of defence relying on the expression of ISGs at steady-state, independently of canonical IFN signalling.

⁵⁶Poirier et al.

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A stem cell-specific mechanism of defence relying on the expression of aviD, an isoform of Dicer that implements antiviral RNA interference and protects against RNA viruses.

⁷⁵Ali et al.

The authors document how Treg cells of adaptive immunity fine-tune the activity of HFSCs through direct modulation of the Notch pathway.

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⁷⁷Biton et al.

ISC proliferation and renewal is modulated by different cytokines produced by subpopulations of T cells.

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⁸⁷Agudo *et al.*

Cycling -but not quiescent- tissue stem cells can be targeted by CD8⁺ T cells through MHC class I-TCR interaction, suggesting that stem cells can be surveilled by the immune system.

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⁹⁵Burzyn *et al.*

This work documents how Treg cells home to wounded muscles and secrete amphiregulin to promote repair.

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¹⁰⁵Luan, Truong, Vuchkovska *et al.*

Upon wounding of the skin, HFSCs regulate the repair process by communicating with Treg cells and neutrophils, through CD80 and Cxcl5 expression, respectively.

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¹²⁹Ge *et al.*

The authors utilise transplantation assays of HFSCs, comparing young and old animals, to demonstrate that the regenerative defects of aged HFSCs originate primarily from cell-extrinsic inflammation.

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¹³⁶Kalamakis *et al.*

During brain ageing, inflammation safeguards the NSC pool by favoring quiescence.

¹⁵¹Funk *et al.*

During ageing, the crosstalk between ISCs and CD4⁺ T cells takes a wrong turn and participates in propagating inflammation in the gut.

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¹⁷⁴Kyritsis *et al.*

In zebrafish, inflammation is pivotal to regenerate the brain after injury. The stem-immune crosstalk is thus conserved beyond mammals.

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