Trained immunity: General and emerging concepts

Over the past decade, compelling evidence has unveiled previously overlooked adaptive characteristics of innate immune cells. Beyond their traditional role in providing short, non‐specific protection against pathogens, innate immune cells can acquire antigen‐agnostic memory, exhibiting increased responsiveness to secondary stimulation. This long‐term de‐facto innate immune memory, also termed trained immunity, is mediated through extensive metabolic rewiring and epigenetic modifications. While the upregulation of trained immunity proves advantageous in countering immune paralysis, its overactivation contributes to the pathogenesis of autoinflammatory and autoimmune disorders. In this review, we present the latest advancements in the field of innate immune memory followed by a description of the fundamental mechanisms underpinning trained immunity generation and different cell types that mediate it. Furthermore, we explore its implications for various diseases and examine current limitations and its potential therapeutic targeting in immune‐related disorders.


| INNATE IMMUNE MEMORY FROM AN E VOLUTIONARY PER S PEC TIVE
All living organisms undergo regular encounters with various pathogens from viruses to bacteria, fungi, and worms.In response to these challenges, they have evolved intricate defense systems that include recognition, control, containment, and/or elimination of a majority of encountered pathogens.Central to the host protection against these pathogens is the role played by immune memory, a long-term attribute that allows antigen-specific cells of the immune system to recognize and recall information on defense against previously encountered microbes.This phenomenon ultimately leads to the development of a heightened and more robust immune response upon subsequent infections with the same pathogen, affording enhanced survival to the organism.Until recently, the consensus was that only the T and B cells of the adaptive immune system are able to mount a memory response, due to their ability to recognize specific and diverse antigens.[3] Furthermore, an alternative adaptive immune system evolved in jawless vertebrates, involving variable lymphocyte receptors (VLR) containing leucine-rich-repeat (LRR) segments obtained following somatic gene rearrangement that aid in the recognition of antigens in an immunoglobulin-independent way. 4,5 adaptive immunity is present only in vertebrates, which include at most 3% of living species, it is challenging to assume that the remaining 97% of all organisms do not possess some sort of immunological memory.][8][9][10][11] Interestingly, despite vertebrates developing further down the line from these ancient precursors, innate immune memory mechanisms appear to have been conserved throughout the evolutionary process, independently of the emergence of adaptive immunity. 12Over the past two decades, these observations were further reinforced by an increasing number of studies that described the mechanisms enabling innate immune responses to exhibit adaptive traits that can be modulated by previous encounters with microbial products.This property has been referred to as "innate immune memory" or "trained immunity," a process that contributes to the heightened immune response observed following subsequent infections or sterile triggers of inflammation. 13,14As its induction is triggered by a diverse range of stimuli, there are distinct mechanisms that control trained immunity, making it less specific compared to adaptive immunity.
Nonetheless, this property of innate immune memory still plays a vital and basic role in vertebrates, conferring a faster and more robust response against pathogens and contributing to improved host survival. 5,14deed, experimental studies performed in mice have demonstrated that priming with microbial ligands or sterile inflammation can train the immune system and induce protection against lethal infection.One such ligand is β-glucan, a polysaccharide obtained from the cell wall of Candida albicans, whose administration induces epigenetic and metabolic reprogramming of monocytes that lay the basis for trained immunity-mediated protection against subsequent bacterial infection with Staphylococcus aureus. 15,16Similarly, mice pre-treated with muramyl dipeptide, a constituent of both Gram-positive and Gram-negative bacteria, exhibited resistance to both Streptococcus pneumoniae and Toxoplasma gondii reinfections. 17Further early examples of the protective effects induced by innate immune training include CpG oligodeoxynucleotide priming which protects against experimental sepsis and Escherichia coli meningitis initiation, 18 whereas flagellin exposure was shown to protect against rechallenge with Gram-positive bacterium S. pneumoniae 19 and rotavirus. 20 addition to microbial agents, additional studies have suggested that sterile inflammation induced through the secretion of specific pro-inflammatory cytokines could trigger the onset of trained immunity.In an early study, van der Meer et al. described how priming with a single dose of recombinant IL-1 3 days before infection with Pseudomonas aeruginosa led to increased survival of the mice. 21It is now clear that, unlike the specific targeting observed in adaptive immunity, trained immunity relies on a generalized enhancement of innate immune protection, thus enabling a quicker and more efficient defense against a broad spectrum of pathogens upon reinfection.Following these findings, additional investigations set out to elucidate the specific mechanisms underlying the induction of innate immune memory.The live attenuated vaccine bacillus Calmette-Guerin (BCG) against tuberculosis was first shown to induce non-specific protection against subsequent infections with Listeria monocytogenes and Salmonella typhimurium over half a century ago. 22The administration of the BCG vaccine in SCID mice lacking the ability to generate T and B cells conferred them protection against subsequent infection with a lethal dose of candidiasis. 23,24Similarly, following mild C. albicans infection, Rag2 −−/−− mice without functional T and B lymphocytes and unable to mount an adaptive immune response exhibited resistance to lethal candidiasis reinfection. 25Ultimately, it was established that β-glucan-induced functional reprogramming of monocytes mediated the activation of trained immunity, thus underscoring that cross-protection occurs in a non-specific manner, independent of adaptive immunity. 25Furthermore, a recent study conducted by Zhou et al. described how neonatal mice exposed to BCG in combination with bacterial lipoprotein (BLP) induced trained immunity, contributing to robust protection against cecal slurry peritonitis-induced polymicrobial sepsis. 26Collectively, these studies argue that this non-specific protection is dependent on the increased pro-inflammatory cytokine secretion by innate immune cells, namely macrophages, following the initial stimulation.
In humans, trained immunity was proposed as a mediator for some of the heterologous protective effects against non-related infections elicited by live attenuated vaccines such as BCG, measlesmumps-rubella (MMR), and oral polio vaccine (OPV).8][29] Beneath these broad beneficial effects of live vaccination lie long-lasting functional, metabolic, and epigenetic changes of innate immune cells, stemming from the induction of trained immunity. 30Given that BCG stands out as the most researched vaccine in the context of innate immune training, numerous epidemiological, immunological studies, and clinical trials have explored its ability to protect against various unrelated infections.Examples of these studies involving BCG vaccination include controlled human infection with yellow fever 31 and malaria. 32Understanding the mechanisms that mediate the heterologous protective effect of BCG, and other liver-attenuated vaccines, will allow to decipher the biology of innate immune memory, and design new approaches for the prevention and treatment of diseases.

| EPI G ENE TI C S AND CELLUL AR ME TABOLIS M
Upon first encountering various pathogens, innate immune cells can develop immunological memory and functional changes which will ultimately influence their future responses to microbial ligands.As a result, the immune response can be either enhanced, causing the establishment of trained immunity, or repressed, leading to innate immune tolerance.During the establishment of a trained immunity phenotype, a major shift in cellular metabolism and signaling pathways takes place in innate immune cells, influenced by the first stimulus and the signaling pathways that are subsequently activated.The immunological memory conferred by trained immunity has been described to last at least for months 33 and up to one year. 34However, prior epidemiological studies argued that some non-specific effects of neonatal vaccination can last up to 5 years. 35Notably, recent studies have suggested that trained immunity can confer transgenerational effects, akin to the innate immune memory observed in invertebrates.For newborns who received BCG vaccination, the non-specific beneficial effects and survival rates were higher when their mothers had previously undergone BCG vaccination (and scarring), thus highlighting a synergistic transgenerational impact of the BCG vaccine. 27though less specific and shorter in duration than adaptive immunity, the distinct intracellular mechanisms through which trained innate immune memory develops are evolutionarily conserved, conferring a rapid and improved response to pathogens encountered by the host.Several studies to date have determined that epigenetic, transcriptional, and metabolic programs lay at the base of the protective effects of trained immunity. 36Upon stimulation, the activation of gene transcription is accompanied by epigenetic changes associated with several metabolic pathways, the majority of which occur early and precede the trained phenotype. 37These mechanisms are intertwined, and our understanding of the specific pathways involved, and their interactions continues to expand. 38It is important to note that besides the metabolic and epigenetic changes that are common denominators in trained immunity, such as the Akt/PI3K/mTOR pathway and changes in histone methylation and acetylation in promoters and enhancers of pro-inflammatory genes, different stimuli can activate different trained immunity programs.

| Epigenetic reprogramming of innate immune cells
Following the stimulation of innate immune cells, diverse intracellular pathways are activated, which results in upregulated transcription of pro-inflammatory genes and subsequently, increased production of pro-inflammatory cytokines and chemokines. 39terestingly, some of the epigenetic changes that accompany gene transcription during the initial stimulation of cells persist after the elimination of the stimulus, allowing for a quicker and more efficient stimulation of the cells upon restimulation.Subsequently, the induction of trained immunity enables these cells to exhibit a faster and increased transcriptional responsiveness after rechallenge. 40This effect is enabled once the cellular machinery gains access to regulatory gene elements found in specific regions of the genome, which are involved in these processes.In this regard, several stable and durable epigenetic modifications take place, such as DNA methylation or demethylation, the remodeling of chromatin tridimensional structure at the level of the topologically associated domains (TADs), and transcription of long non-coding RNAs. 41o of the processes involved in the regulation of gene expression patterns are DNA methylation and modification of chromatin architecture.DNA methyltransferases (DNMTs) contribute to the repression of gene transcription by recognizing CpG-rich sequences to methylate cytosines.The DNA tails extending from the core histone octamers contain several modifiable amino acids that can be recognized by proteins that bear histone-binding domains.These histone-modifying enzymes can catalyze several different modifications, such as methylation, acetylation, phosphorylation, and ubiquitination.During the induction of a trained immune response, several key epigenetic marks have been described.The first ones are the modulation of histone 3 lysine 27 acetylation (H3K27Ac) and histone 3 lysine 4 methylation (H3K4me1) epigenetic marks at the distal enhancers, with H3K4me1 persisting even at decommissioned enhancers. 25,40,42An additional change is the histone 3 lysine 4 trimethylation (H3K4me3) chromatin mark accumulation on the stimulated immune gene promoters.Indeed, a recent study established that mice lacking Setd7, which encodes the enzyme Set7 lysine methyltransferase that mediates induction of H3K4me1 are unable to develop β-glucan-induced trained immunity. 43Moreover, a different in vitro study has demonstrated how monocytes stimulated with β-glucan exhibited a training-specific H3K4me1 enhancer signature which is pivotal for IL-32 transcription. 44Notably, BCG was found to induce increased H3K27Ac in several trained immunity-specific signaling pathways, such as PI3K/AKT (phosphatidylinositol 3-kinase) pathway, epidermal growth factor receptor (EGFR), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) pathways. 31A different study found that LPSinduced tolerance in macrophages can be partially reversed in vitro following β-glucan treatment, which causes H3K27Ac accumulation at enhancer regions. 42Consequently, this can lead to the reactivation of unresponsive gene transcription.Taken together, these studies demonstrate that several genes play key roles in the induction and regulation of trained immunity following its stimulus-specific induction.Nonetheless, understanding the way these gene regulatory mechanisms induce epigenetic changes that take place at discrete locations in the genome is critical for improving our understanding of epigenetic transcriptional memory.
Immune gene transcription is not continuously active, as it occurs in "bursts," at times when promoters exhibit a permissive phenotype.This causes high gene expression variability between innate immune cell populations. 45Eukaryotes have evolved to reduce stochasticity in gene expression by maintaining the promoters of certain immune genes in an H3K4me3-primed state, where RNA polymerase II is paused, thus allowing rapid transcription upon engagement by signal-dependent transcription factors. 46Previous studies have described long non-coding RNAs (lncRNAs) as emerging key regulatory elements in the long-term epigenetic reprogramming of innate immune cells. 47The chromatin within the eukaryotic nucleus is divided into regions enriched with chromosomal loops, the TADs.Within TADs, these loops bring DNA elements such as lncRNAs and enhancers into proximity to protein-coding genes to regulate their expression. 48creasing evidence suggests that the uniform transcriptional response of immune genes responding robustly to external stimuli across various cell populations may be influenced by chromosomal looping and TAD structure.This is especially relevant for 'H3K4me3-primed' innate immune genes, such as the CXCL chemokines, which include IL-8, CXCL1, CXCL2, and CXCL3.Fanucchi et al. demonstrated how a class of lncRNAs, termed 'immune-gene priming lncRNAs' (IPLs), play a key role in the activation of trained immunity by aiding in the accumulation of H3K4me3 on the promoters of these pro-inflammatory cytokine genes, leading to their epigenetic activation. 49In the same study, the expression of a prototypical IPL named UMLILO (upstream master lncRNA of the inflammatory chemokine locus) was shown to regulate the H3K4me3 accumulation on the CXCL chemokine gene promoters.The H3K4me3 epigenetic priming of CXCL promoters occurs prior to their transcriptional activation, which can be explained by the interaction between UMLILO, the WD repeat-containing protein 5 (WDR5) and mixed-lineage leukemia (MLL) methyltransferases. 49Thus, during a trained immune response, IPL expression is increased, leading to H3K4me3 accumulation at the promoters of genes, which in the end drives the robust trained immunity gene transcription.Interestingly, the IPL-mediated mechanism is shared with other key trained innate immune genes, such as IL-6 and IL-1β.
Earlier studies observed that most non-coding elements are poorly conserved evolutionarily.This could explain the increased resistance that mice display to various inflammatory stimuli when compared with humans. 50Interestingly, the absence of the UMLILO and IL-8 in the mouse pre-formed chemokine TAD is accompanied by a lack of H3K4me3 accumulation on the promoters of CXCL chemokines, causing these pro-inflammatory genes to be closed to training by β-glucan in murine macrophages. 49Notably, the insertion of the UMLILO genomic sequence using CRISPR-Cas9 homology-directed repair into the Cxcl murine TAD can reverse the closed state of the CXCL chemokine genes uniformly across a population of immune cells.Moreover, UMLILO together with another IPL, termed IPL-IL1, were found to be involved in the long-term increased responsiveness of neutrophils following BCG vaccination. 51Upregulation of IPL-IL1 expression in neutrophils resulted in H3K4me3 deposition on effector function genes, such as cytokine production and killing capacity.
Therefore, these findings support the conclusion that IPLs contribute to increased H3K4me3 deposition on target genes, leading to decreased stochasticity in the transcription of immune genes.The beginning of open chromatin enhancement after BCG stimulation, measured by an assay for transposase-accessible chromatin using sequencing (ATAC-seq) in mice demonstrated that it begins at the level of the hematopoietic stem cells (HSCs) in the bone marrow. 52ring HSC differentiation into various myeloid and lymphoid progenitors, the locations of such marks remain partially preserved. 52us, it is an appealing hypothesis that the H3K4me1 marks which were first developed at the level of HSCs remain present in terminally differentiated myeloid cells.In this way, the trained innate immune phenotype can be maintained by transmitting these marks through DNA replication and the cell cycle in HSCs. 53An overview of the epigenetic processes that mediate the induction of trained immunity at the level of chromatin is presented in Figure 1.
More recently, studies assessing DNA methylation patterns have found differences between the subjects who can induce a robust innate immune response following exposure to trained immune stimuli, and those who lack this ability ("non-responders").Specifically, BCG-treated individuals who reacted to M. tuberculosis infection by inducing enhanced containment of viral replication exhibited a significant loss of DNA methylation on the promoters of immune genes indicative of IL-1β production, compared to the non-responders. 54us, a follow-up study indicated that responsiveness to BCG could be predicted by 43 differentially methylated sites. 55Therefore, these alterations in the DNA methylome could act as predictors of whether an individual can undergo rapid and robust training following exposure to different stimuli.

| Immunometabolic circuits in trained immunity
The intricate mechanisms underlying trained immunity induction, maintenance, and regulation in innate immune cells and their progenitors rely on the integration of metabolic information and transcriptional control via the enzymatic consumption of metabolites in epigenetic reactions. 38,56,57Cellular metabolism and epigenetic reprogramming are closely intertwined and represent the two main pillars mediating the trained innate immune response.The induction of a robust immune response following the first encounter with a microbial or endogenous ligand that induces trained immunity requires large amounts of energy and building blocks for effective chemical processes.Therefore, different metabolic pathways are employed to act as a source of nutrients that fuel epigenetic alterations and remodeling of chromatin architecture in cells, facilitating an enhanced response in the event of a secondary encounter with a pathogen. 30lls in a resting state generally rely to fuel their energy consumption on slow but highly efficient metabolic pathways, such as oxidative phosphorylation (OXPHOS) and fatty acid oxidation.Upon stimulation, different genes involved in aerobic glycolysis, tricarboxylic acid (TCA) cycle, glutaminolysis, and cholesterol metabolism are quickly upregulated.The sudden increase in the energetic and nutritional needs of activated cells leads to the altering of the level of several crucial cellular metabolites. 58As a result, the high energetic and nutritional demands of activated cells, including the induction and regulation of epigenetic and functional changes, are met.It is important to note that the involvement of metabolic rewiring in the induction and maintenance of essential transcriptional and epigenetic processes necessary for the induction of trained immunity is not limited only to enhancing ATP production. 57Importantly, several intermediate metabolites involved in these processes possess immunomodulatory properties, serving as cofactors for chromatinmodifying enzymes. 59We will next explore the various metabolic pathways and their involvement in the molecular mechanisms that lay the basis of trained immunity induction and regulation.
Although characteristic of anaerobic environments, the glycolysis pathway can also be upregulated in the presence of oxygen, termed aerobic glycolysis, following an increase in cellular energy requirements by trained immunity induction.Previous research linked β-glucan-induced training of monocytes with a shift in metabolism towards increased aerobic glycolysis, which in turn leads to enhanced TCA cycle activity. 60The metabolic shift was dependent on the activation of mammalian target of rapamycin (mTOR) through a dectin-1-Akt-HIF-1α (hypoxia-inducible factor-1α) pathway. 60In line with this, blockade of the Akt-mTOR-HIF-1α by chemical inhibitors led to inhibition of trained immunity. 60Consequently, the enhanced responsiveness to secondary challenges is explained by the upregulation of genes that code for pro-inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), and IL-6. 40,60rthermore, a significant amount of pyruvate is transformed into lactate resulting from aerobic glycolysis.A growing number of studies have recently demonstrated that lactate is not just an inactive by-product of glycolysis but can also regulate the immune response exhibited by human monocytes and tumor-associated macrophages. 61,62Zhang et al. recently identified a novel histone modification induced by lactate binding that contributed to the regulation of the epigenetic landscape during macrophage differentiation. 63Stimulation with LPS increases lactate accumulation in murine macrophages, leading to the upregulation of genes coding for cellular homeostasis and downregulation of pro-inflammatory genes. 63However, the mechanism directing the lactate to bind to specific histones at discrete genomic locations remains unknown, F I G U R E 1 Epigenetic processes mediating the induction of trained immunity at the chromatin level.The first stage in this process represents the state of chromatin in unstimulated innate immune cells.Following initial exposure to a stimulus such as Bacillus Calmette-Guérin (BCG) or β-glucan, the cells become activated due to the deposition of chromatin marks and downregulation of DNA methylation.During this process, the chromatin begins to unfold, causing the secretion of pro-inflammatory signals.In the absence of a stimulus, the resting "trained" cells still possess some of these epigenetic changes.Secondary stimulation with an unrelated stimulus will result in a heightened and more robust immune response due to increased gene expression and transcription factor binding.H3K27ac, histone 3 lysine 27 acetylation; H3K4me1, histone 3 lysine 4 methylation; H3K4me3, histone 3 lysine 4 trimethylation.Image created with BioRe nder.com, with permission.and its role during the induction of trained immunity remains to be demonstrated.These studies altogether outline the dual role that glycolysis plays regarding immune response activation: first, it is involved in energy necessary for generation, and second, the increased lactate production through aerobic glycolysis is likely to modulate the inflammatory response in innate immune cells.
Systems biology analysis of innate immune cells also identified several amino acids whose metabolism is enhanced following βglucan-induced trained immunity in monocytes.The metabolism of glutamine into glutamate, α-ketoglutarate, and succinate semialdehyde provides substrates such as fumarate and succinate that fuel the TCA, being, therefore, crucial for training induction. 37These intermediates can also influence the activity of innate immune cells by themselves.Interestingly, fumarate by itself can induce innate immune training by inhibiting the lysine demethylase enzyme KDM5, thus increasing the trimethylation of histones at H3K4me3 at the promoters of the pro-inflammatory genes IL6 and TNF. 37This increases chromatin accessibility for the binding of transcription factors that aid in the generation of enhanced responsiveness following secondary LPS stimulation, proving that immunometabolism and epigenetic reprogramming are intertwined in the generation of the trained immune response. 37Furthermore, α-ketoglutarate generated from glutaminolysis has been described to act as a metabolic regulator, instructing anti-inflammatory macrophages to activate and polarize through H3K27 demethylase JMJD3-mediated epigenetic reprogramming. 64Additionally, α-ketoglutarate production was shown to contribute to the endotoxin tolerance acquired on proinflammatory genes during the LPS priming phase in macrophages. 64 the contrary, Tannahill et al. outlined how LPS-induced succinate accumulation can act as a signal to increase IL-1β secretion in bone marrow-derived macrophages through the HIF-1α proinflammatory signaling pathway. 65Citrate, another TCA cycle intermediate, can regulate the expression of nitric oxide, reactive oxygen species (ROS), and prostaglandin E2 (PGE2) in the cytosol of M1 macrophages. 66Upregulation of the metabolite itaconate following LPS priming of macrophages causes succinate dehydrogenase inhibition, and this was shown to reduce mitochondrial respiration, reactive oxygen species production, pro-inflammatory cytokine release, and inflammasome activation. 66,67These itaconate-induced metabolic and functional changes take place after the level of anti-inflammatory transcription factor NRF2, which targets genes involved in antiinflammatory and anti-oxidant response, is stabilized. 68Thus, NRF2 suppresses the expression of genes encoding IL-1β and IL-6 and inflammation is reduced.It is important to note that the tolerant phenotype induced by itaconate can be reversed through the induction of trained immunity with β-glucan.This is possible due to β-glucan's ability to inhibit immune-responsive gene 1 (IRG1) protein expression, the enzyme responsible for the production of itaconate, leading to the upregulation of succinate dehydrogenase expression. 69 addition to aerobic glycolysis upregulation and modulation of the TCA cycle, the enhanced expression of genes coding for proteins of the cholesterol synthesis pathway is another process described to take place following β-glucan-induced trained immunity. 40 support this, a study combining transcriptomic and epigenomic analysis proved that inhibition of the cholesterol synthesis pathway by fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, prevented training in monocytes. 70It was revealed that the accumulation of mevalonate, an intermediate in the cholesterol synthesis pathway, aided in the generation of the trained innate immune response through the insulin-like growth factor 1 receptor and upregulation of the Akt-mTOR pathway. 70In a subsequent investigation, individuals with hyperimmunoglobulin D syndrome (HIDS), an autoinflammatory syndrome characterized by mevalonate accumulation within monocytes, exhibit a persistent trained immunity phenotype.This is marked by heightened cytokine production, along with a notable upregulation of glycolysis and mTOR pathways. 70The synthesis of fatty acids promotes an inflammatory phenotype of macrophages by activating the NLRP3 inflammasome. 71Furthermore, trained immunity induced by aldosterone activates the mineralocorticoid receptor, triggering fatty acid synthesis.This process culminates in elevated trimethylation of H3K4me3 on genes associated with the fatty acid synthesis pathway, accompanied by enhanced IL-6 and TNF production. 72Further cementing this finding, a follow-up study found that aldosteroneprimed cells pre-incubated with a fatty acid synthesis inhibitor prior to rechallenge led to impaired pro-inflammatory cytokine secretion. 72The metabolic process involved in the induction of trained immunity can be observed in Figure 2.

| CELL P OPUL ATI ON S THAT MED IATE TR AINED IMMUNIT Y
4][75][76] In contrast to T and B lymphocytes that bear antigen-specific receptors obtained through gene rearrangement, a characteristic of adaptive immunity, innate immune cells lack this specificity.However, they do express pattern recognition receptors (PRRs), which can recognize their specific pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and trigger a subsequent immune response. 77,78Following the engagement of a PRR with their respective stimulus, cells undergo long-term epigenetic reprogramming and metabolic rewiring, which ultimately leads to an enhanced immune response upon non-specific restimulation attained through increased pro-inflammatory cytokine production as a main mechanism. 53Among the main PRRs involved in the induction of trained immunity, dectin-1 plays a crucial role in recognizing β-glucan.Additionally, NOD2 serves as the receptor for muramyl peptide, a component found on the cell envelope of the bacteria Mycobacterium Bovis in the BCG vaccine. 33Engagement of other pattern recognition receptors on the surface of innate immune cells is expected to induce different trained immunity programs, with a systematic assessment needed in the future.
In addition to immune cells, a growing number of studies indicate that non-immune cells such as epithelial cells, fibroblasts, and stromal and epidermal stem cells possess memory-like characteristics specific to trained immunity.In this section, we will sequentially explore each of these cell types and their capacity to induce innate immune training.

| Immune cells involved in trained immunity induction
Following an infectious or metabolic challenge, the cellular basis of innate immune memory has been mainly studied at the level of the functional reprogramming of myeloid cells, such as monocytes, macrophages, dendritic cells, and mast cells.Early indications of the adaptive characteristics of monocytes and macrophages emerged from the investigation of LPS-induced tolerance. 79These studies revealed that specific changes in chromatin structure through histone modification are linked to the silencing of genes coding for inflammatory molecules. 79In contrast, the genes encoding antimicrobial products were upregulated.It was later revealed that monocytes and macrophages stimulated with C. albicans, or its fungal cell wall component β-glucan, show increased production of pro-inflammatory cytokines upon a second challenge with an unrelated pathogen, essentially exhibiting a trained immunity phenotype. 33This phenomenon was thereafter shown to be accompanied by changes in the epigenetic landscape of the cells as mirrored by histone marks such as H3K4me1, H3K4me3, or H3K27Ac, 25,40 as described above.Moreover, a trained immune response was also recently observed in monocytes and macrophages exposed to parasites. 80re recent work demonstrates adaptive characteristics in dendritic cells as well, which displayed increased protection against Entamoeba histolytica. 81Moreover, DCs obtained from miceinduced training following initial exposure to the fungal pathogen Cryptococcus neoformans showed increased pro-inflammatory cytokine secretion upon secondary challenge. 74To prove that epigenetic reprogramming was driving these effects, mice were treated with a histone methyltransferase inhibitor, which led to the inhibition of their capacity to mount a trained immune response. 74long-lasting protection through trained immunity cannot be explained through effects on monocytes alone, as they have a short half-life in circulation, between 1.5 and 7 days. 82Previous studies looking at the effects of trained immunity following BCG vaccination 52 and β-glucan administration 83,84 have uncovered how newly generated cells following myelopoiesis from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow present an innate immune memory phenotype.Indeed, BCG-vaccinated individuals displayed changed HSPCs transcriptional programs 3 months after vaccination compared to non-vaccinated subjects, which was F I G U R E 2 Metabolic processes that underlie the induction of trained immunity.Primary exposure to a stimulus (e.g., β-glucan) initiates a series of intracellular cascades that trigger the upregulation of various metabolic pathways.These pathways include glycolysis, tricarboxylic acid (TCA) cycle, OXPHOS, glutaminolysis, as well as fatty acid metabolism.The metabolites that are obtained from these pathways, such as fumarate and acetyl coenzyme A (acetyl-CoA), play a crucial role in the modulation of enzymes involved in epigenetic rewiring, such as histone demethylases and histone acetyltransferases.PAMPs, pathogen-associated molecular patterns; PRRs, pathogen recognition receptors; mTOR, mechanistic target of rapamycin; α-KG, α-Ketoglutarate.Image created with BioRe nder.com, with permission.associated with a bias towards myelopoiesis and granulopoiesis. 85milarly, HSCs pre-exposed to LPS presented increased myelopoiesis, leading to long-term conservation of epigenetic marks and increased responsiveness of associated immune genes upon secondary stimulation. 86nate lymphoid cell populations, such as NK cells and innate lymphoid cells (ILCs) have also been shown to possess memory characteristics following activation with pro-inflammatory cytokine combinations.Although NK cells do not possess specific antigen receptor genes for the recognition of pathogens, they rely on a limited repertoire of activating and inhibitory NK receptors encoded in the germ line, which they use to recognize MHC class I and class I-like molecules. 87Thus, the small number of NK-specific receptors causes these cells to make use of other activation signals, including DC-derived cytokines. 88,891][92][93][94] In mice, the MHC-like glycoprotein m157 encoded by MCMV has been shown to engage the activating NK cell receptor Ly49H. 95,968][99] Upon reactivation, these NK cells swiftly degranulate and start secreting cytokines, leading to enhanced immune protection upon secondary viral challenge when transferred into an animal host. 90,100A similar memory-like protective function of NK cells is found in humans and is generated after the UL40 peptide encoded by HCMV is recognized by the activating NKG2C receptor of NK cells after being presented on the nonclassical MHC molecule HLA-E. 101,102Adding to this, it was found that Ad26 vaccination induced antigen-specific NK cell memory in primates, which was maintained 5 years after vaccination. 103en though they lack the gene segment rearrangements that generate antigen-specific recognition receptors in lymphocytes, the properties of memory NK cell immune responses after MCMV/ CMV infection are functionally closer aligned with T-cell dependent immune memory than macrophage-specific trained immunity.
However, both NK cells and ILCs can also exhibit trained innate immunity characteristics.Previous studies demonstrated how mouse and human NK cells primed with IL-12, IL-15, and IL-18 displayed increased interferon-gamma (IFNγ) production lasting several weeks, accompanied by a more robust response after restimulation. 89,104Importantly, these cytokine-preactivated memory-like NK cells were demonstrated to possess robust responses against leukemia. 105Although more resembling T-cells, NK cells specific to CMV infections still require pro-inflammatory cytokine signaling to differentiate into memory NK cells which possess enhanced protection against secondary infection, a trait specific to trained immunity. 106,107In addition to CMV and pro-inflammatory cytokines, BCG vaccination was found to induce long-lasting enhanced cytokine production by human NK cells following rechallenge, suggesting that they partially play a role in the non-specific protection by BCG against C. albicans. 73 addition to NK cells, a recent study illustrated how liverresident group 1 ILCs (ILC1s) expand locally and exhibit long-term persistence following MCMV infection, leading to the acquisition of stable epigenetic, transcriptional, and phenotypic changes that are preserved a month after the resolution of infection. 75In this context, the innate immune-like memory behavior of ILC1s was dependent on both antigen specificity due to the recognition of MCMV-encoded glycoprotein m12 by the NK1.1 receptor, and on the production of pro-inflammatory cytokines. 75Moreover, ILC2s were shown to display memory characteristics by successfully mediating severe allergic inflammation following initial stimulation by inhaled allergens, 108 which resembles previous studies showing NK cell memory obtained after hapten sensitization. 109During repeated pregnancy, uterine NK cells were found to possess a unique transcriptomic and epigenetic signature conferred by open chromatin around the enhancers of IFNG and VEGFA, leading to improved placentation. 110ken together, these observations suggest that NK cells may lay at the evolutionary crossover between innate and adaptive immune memory responses.

| Trained immunity induction in non-immune cells
Interestingly, studies performed in recent years hint also at the ability of non-immune cells, such as fibroblasts, stromal and epithelial stem cells to display memory features, a concept termed "extended trained immunity" 111 or "inflammatory memory". 112The inflammation-induced immune memory that has been recently observed in epithelial stem cells is especially relevant since tissueresident stem cells are known for their ability to regenerate and maintain their population during homeostasis, thus contributing to tissue repair. 113,114During their response to tissue damage, epithelial stem cells exhibit versatile behaviors, which are attributed to their cross-talk with the local niche microenvironment.When homeostasis is disturbed by threats such as infection and inflammation, epidermal stem cells are prompted to remodel their chromatin dynamics and change their gene expression program to survive the stressful environmental change. 114As a result of these stressful encounters, stem cells gain an epigenetic memory similar to trained immunity in innate immune cells, allowing them to better cope with subsequent tissue barrier breaches. 1157][118] Additionally, these receptors sense when perturbations in the niche disrupt skin barrier function, which allows adjacent stem cells to remodel their transcriptome to recruit other immune cells and contain the breach. 119It was determined that cells in the damaged niche communicate with the nearby stem cells, which in turn upregulate chemokine genes that recruit distinct immune cell repertoires, allowing them to repurpose their functions.In the end, this creates an effective feedback mechanism, where the newly recruited immune cells coordinate with stem cells, instructing them to engage in wound healing. 119sides stem cells, other non-immune cells that display innate immune memory-like behavior are the fibroblasts.Kamada et al.   recently demonstrated that priming with IFNβ leads to the development of epigenetic memory in fibroblasts, leading to enhanced transcription of interferonβ-stimulated genes (ISGs) upon restimulation. 120This specific transcriptional memory response by ISGs was acquired through the hastened recruitment of RNA polymerase II to their gene loci. 120Although there are already numerous studies that have explored the capacity of various cells to exhibit trained innate immune phenotypes, the full functional implications of these processes on diverse cell types and tissues are yet to be entirely understood.

| CENTR AL VS . PERIPHER AL TR AINED IMMUNIT Y
The first studies to outline the mechanisms underlying long-term induction of innate immune memory focused on the memory response that occurs in bone marrow immune progenitor cells, where the trained phenotype is passed on to circulating innate immune cells, now designated as centrally trained immunity. 52,83,85More recent work has illustrated how tissue-resident macrophages can also develop innate immune memory, now described as peripheral trained immunity. 121Peripheral trained immunity occurs after bone marrow progenitors are first exposed to a trained immunity-specific stimulus, leading to their reprogramming and differentiation into mature peripheral innate immune cells, which display an enhanced inflammatory response upon restimulation.An example of this is the increased secretion of IL-1β by peripheral mononuclear immune cells that occurs 3 months post-BCG vaccination, after a secondary challenge with C. albicans. 85The upregulated pro-inflammatory response upon rechallenge was explained by an increase in DNA accessibility in the proximity of inflammation-associated genes such as C-X-C motif chemokine ligand 6 (CXCL6) and retinol-binding protein 4 (RBP4). 85e first studies that functional reprogramming induced by BCG vaccination occurs at the level of myeloid progenitor cells in the bone marrow were done in mice, in which BCG vaccination skews HSCs in the bone marrow towards myelopoiesis using IFNγ signaling, leading to the generation of trained differentiated cells. 52A genetic polymorphism at the promoter region of the IL32 gene has been suggested to impact these BCG-mediated effects. 44milarly, administration of β-glucan induced the selective expansion of myeloid-biased cells such as CD41 + HSCs, thus promoting myelopoiesis and leading to an enhanced immune response to secondary LPS stimulation. 83At the base of this trained innate immune induction in mice by β-glucan lay mechanisms such as the elevation of IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, together with the reshaping of glucose metabolism and cholesterol biosynthesis pathways. 83The role of HSCs for the induction of trained immunity was in parallel demonstrated after metabolic challenge in mice using a Western-type diet. 122Taken together, these studies suggest that the ability of short-lived innate immune cells to acquire memory, leading to long-term trained immunity, should be attributed to the reprogramming of progenitor cells in the bone marrow.In addition to HSPCs, additional work showed that respiratory epithelial progenitor cells adopt a more stem-like state during human allergic inflammatory disease, and the alterations in their accessible chromatin exhibit varying abilities to revert to their initial state upon the withdrawal of the stimulus. 123cent studies have furthermore examined the induction of trained immunity in tissue-resident immune cells, and how the regular function of tissues is maintained during the functional reprogramming of these cells.Several tissues that act as a barrier between the inner and outer environments, such as the skin, lungs, and intestine are constantly exposed to stimuli that initiate training.
Previous studies proposed that acute respiratory viral exposure to adenovirus, as well as latent gammaherpesvirus, can induce longterm training of lung immunity. 121,124Adenovirus infection was shown to confer robust protection against bacterial infection, following the generation of long-term memory in alveolar macrophages independently of circulating monocytes and bone marrow progenitors. 121Alveolar macrophage memory was associated with increased MHC II expression, as well as increased secretion of chemokines and neutrophilia upon restimulation.Notably, further analysis demonstrated that the tissue-specific training phenotype required priming with IFNγ produced by CD8 + T cells, and the maintenance of memory alveolar macrophages was dependent on the polarization of CD8 + T cells. 121The involvement of CD8 + T cells and their products in the training of lung-specific innate immune cells had, therefore, established a link between the innate and adaptive immune systems, providing evidence for a new concept, later known as "noncanonical tissue-trained immunity". 125Furthermore, mice chronically infected with gammaherpesvirus displayed reduced severity of house dust mite-induced asthma. 124Differently from adenovirus exposure, the training phenotype following gammaherpesvirus priming relied on the continuous generation and maintenance of regulatory alveolar macrophages derived from monocytes, which granted protection against the onset of an allergic response in the lung.Taken together, these two recent studies underline the significance of tissue-specific induction of trained innate immunity, which is constantly adapting to outside stimuli.It is, therefore, essential that further research is aimed at better understanding the mechanisms behind the training of tissue-specific immune cells and their role in the protection against infections, as well as inflammatory diseases and cancer.
Additionally, innate immune memory was recently reported in microglia, which are resident macrophages of the central nervous system and are vital for normal brain function.Microglia have been shown to display innate immune memory characteristics after a challenge with LPS. 126Similarly, pre-treatment with β-glucan was shown to activate microglia without inducing significant cytokine expression and was ultimately proven to induce immune training after LPS rechallenging. 76Therefore, these cells exhibit a classical inflammatory response, which is different from alveolar macrophage-induced trained immunity described above.Another study associated the activation status of microglia with the deposition of histone 3 lysine 4 dimethylation (H3K4me2) in the promoter and enhancer regions of pro-inflammatory genes. 127This finding was able to explain the ability of microglia to return to a steady state once the priming stimulus is removed, at the same time responding better to subsequent stimulation due to acquired epigenetic memory. 127The interactions between central and peripheral immune factors are also important in the elucidation of the mechanisms behind the long-lasting effects of innate immune memory.Evidence of this can be found in the brain, where LPSinduced peripheral inflammatory cytokines cause trained immunity induction, leading to differential epigenetic remodeling of brain-resident macrophages lasting for at least 6 months. 126nsequently, communication between central and peripheral immune players is crucial for the induction and maintenance of a prolonged trained immunity phenotype.

| IMP ORTAN CE OF TR AINED IMMUNIT Y IN D IS E A S E
Until recently, infections used to be the most common cause of death throughout the world, as recurring epidemics of influenza, cholera, smallpox, and the bubonic plague led to the death of large parts of the adult population.In line with this, infections are one of the most important evolutionary pressures in both humans and other species, with immunological memory being one of the most important mechanisms counteracting the effects of infections.
While adaptive immune memory has been shown for more than half a century to have important protective effects, recent studies have argued that innate immune memory also contributes to the protection of the host against reinfection.This is most evident in species that lack adaptive immunity or in newborns that do not yet have functional adaptive immunity.In contrast, while trained immunity is beneficial for resolving infections (and probably for immunosurveillance in cancer), its aberrant activation can lead to a maladaptive effect, potentially contributing to chronic inflammatory diseases such as atherosclerosis, rheumatic diseases, and neurodegenerative disorders.Therefore, induction of trained immunity can play a role both in maintaining health if activated at the right time and in the right place or promoting disease if inappropriately induced.

| Trained immunity in infections
The most relevant benefit of trained immunity induction is the broad protection against infectious agents, which is conferred through long-term memory adaptions.Probably the best-studied vaccine that outlines this property of the innate immune system is the BCG vaccine.Although initially developed as a vaccine against tuberculosis, numerous studies have proven that its priming ability induces immunological memory, shielding the host against secondary infections other than tuberculosis, such as parasitic, viral, and bacterial infections. 34,128One study outlined how BCG-vaccinated volunteers presented accelerated phenotypic NK cell and monocyte activation following malaria challenge infection which contributed to reduced parasitemia, indicating a potential for malaria vaccine development strategies. 32Besides malaria, the priming of human monocytes to BCG led to improved protection against different Leishmania species in vitro via upregulated secretion of ROS and IL-32. 129Adding to this, studies using Leishmania infection mice models have demonstrated that BCG vaccination decreases lesion size and parasite load in infections caused by L. braziliensis, while parasite load is decreased in the spleen, liver, and bone marrow in mice infected with L. infantum. 129other randomized trial assessed the effects of BCG on the Vi polysaccharide typhoid fever vaccine, which is known for inducing immune tolerance. 130It was determined that prior BCG administration partially reversed innate immune response inhibition by the typhoid vaccine. 131 relation to viral infections, initial exposure of monocytes to BCG induces IL-1β-mediated functional reprogramming and protection against subsequent infection with an attenuated yellow fever virus vaccine, leading to decreased viremia. 31In addition, BCG vaccination in the ACTIVATE clinical randomized trial has shown to inhibit respiratory tract infections by 80%, which are mainly of viral etiology. 132Based on these studies, the hypothesis that trained immunity could be employed to reduce susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proposed. 133An initial observational study suggested that BCG-vaccinated individuals display a lower incidence of sickness and extreme fatigue during SARS-CoV-2 infection, 134 while a BCG revaccination study in Greece (ACTIVATE-II) showed a lower incidence of COVID-19 in the elderly. 135[144] While no effect of BCG vaccination on the total number of infections was observed, these studies were too small to be able to conclude whether BCG was able to protect against disease severity, although a potential beneficial effect on overall mortality has been suggested. 145It is important to note that the study of Moorlag et al observed that vaccination with BCG improved IL-6 and humoral responses against SARS-CoV-2 after COVID-19 diagnosis. 142Previous work using an in vivo mice model demonstrated that BCG administration failed to reduce morbidity and mortality against SARS-CoV-2, while protection against Influenza was successful.Additional research also demonstrated high production of IL-1β and TNF following PBMC exposure to BCG, additionally to IL-6.The lack of protection against SARS-CoV-2 infection was attributed to the ability of the virus to produce pulmonary vasculature damage, facilitating its dissemination to other organs such as the bone marrow, a crucial setting in BCG-mediated trained immunity. 146Ideally, the molecular mechanisms behind BCG-mediated training against SARS-CoV-2 should be better understood in order to successfully boost the innate immune response and reduce the severity of infection. 133 addition to BCG, influenza vaccination was also shown to induce trained immunity through transcriptional and cytokine production regulation of circulating immune cells, while at the same time reducing systemic inflammation. 147This led to an increased innate immune response against various viral stimuli, including decreased COVID-19 incidence among employees of a large Dutch academic hospital. 147Furthermore, a study looking into the potential of vaccination with AS03-adjuvanted H5N1 pandemic influenza vaccine to induce innate immune training found that vaccinated individuals exhibit increased protection against infection with the heterologous Zika and Dengue viruses. 148The increased resistance to infection was associated with increased chromatin accessibility at interferon response factor loci in monocytes and DCs, leading to enhanced expression of genes. 148veral animal and human studies have outlined the ability of various other stimuli to induce trained immunity and protect against reinfections in the host.Previous studies using mice models uncovered that macrophages trained with β-glucan increased protection against Leishmania braziliensis infection via increased production of IL-1 and IL-32. 44Similarly, IL-1β signaling and reprogramming of hematopoietic progenitor cells in the bone marrow were shown to guide the protective effects of β-glucan priming against M. tuberculosis. 149Nonetheless, research into the ability of other pathogenassociated molecular patterns to induce trained immunity has been quite limited.In the lungs, infection with live attenuated Bordetella pertussis led to protection against the influenza A virus through immune tolerance caused by attenuation of cytokine-mediated inflammation. 150Innate immune memory was also achieved through initial stimulation of human PBMCs with Plasmodium falciparum-infected red blood cells or the malaria crystal hemozoin, which caused them to hyperrespond upon secondary TLR ligand challenge. 151rthermore, the reversal of epigenetic changes induced by immune tolerance through trained immunity can prevent immunoparalysis in sepsis patients, leading to increased protection against secondary infections. 42,152,153In this context, it was found that S100 alarmininduced immune programming leads to training, thus protecting newborns from neonatal sepsis and immune tolerance. 154Lastly, a recent study uncovered the ability of cell wall peptidorhamnomannan (PRM) of the pathogen Sporothrix brasiliensis to induce proinflammatory cytokine production, such as IL-6, TNF and IL-1β in PBMCs. 155The induction of cytokines required CR3 and dectin-1 receptor activation, therefore, exhibiting close similarities to the classical mechanism of β-glucan-induced trained immunity.
Collectively, this evidence proves that trained immunity can be induced by a variety of stimuli, therefore, making it an attractive therapeutic target that can be employed in the prevention of secondary infections.

| Trained immunity and autoinflammatory diseases and allergy
The heightened immune response induced by trained immunity can, in some cases, cause a detrimental hyperinflammatory effect, resulting in the onset of several allergic and autoinflammatory diseases. 156One such disorder is atherosclerosis, which develops because of chronic, low-grade vascular inflammation induced by the immune system.Endothelial cells, smooth muscle cells, and circulating immune cells are all involved in creating a pro-inflammatory microenvironment in the vascular wall that contributes to the formation of atherosclerotic plaque. 157To support this, human observational studies have shown that monocytes from patients with symptomatic atherosclerosis exhibit increased pro-inflammatory cytokine secretion followed by metabolic and epigenetic changes when compared to healthy controls. 158Follow-up studies confirmed that monocytes derived from patients with coronary atherosclerosis exhibit a pro-inflammatory profile and heightened glycolytic activity, persisting even after in vitro differentiation into macrophages. 159tably, monocytes obtained from patients suffering from familial hypercholesterolemia who have not been treated manifested a trained immunity phenotype.The innate immune memory, marked by increased pro-inflammatory characteristics, lasted for up to 3 months following initiation of lipid-lowering therapy with statins, implicating bone marrow involvement. 160A similar hyperinflammatory monocyte phenotype was observed in patients with elevated levels of Lp(a), coinciding with heightened vascular wall inflammation detected through FDG-PET/CT (fluorodeoxyglucose positron emission tomography/computed tomography). 161Importantly, this pro-inflammatory state did not revert to baseline even after 90 days of Lp(a) reduction following PCSK9 treatment. 162Ultimately, Lp(a) has been demonstrated to be capable of inducing hematopoietic reprogramming. 163reover, patients with mevalonate kinase deficiency, an autoinflammatory disorder characterized by the accumulation of mevalonate, experience periodic attacks of sterile inflammation. 70,164is is at least partly caused by the induction of an inappropriate state of trained innate immunity, as monocytes isolated from these patients displayed increased expression of pro-inflammatory cytokines, together with metabolic remodeling and the upregulation of histone methylation.It was determined that mevalonate-mediated training requires the activation of IGF1 receptor and mTOR, which contribute to epigenetic rewiring of inflammatory pathways, providing certain reasoning behind the inflammatory attacks exhibited by these patients. 70milar to the classical induction of trained immunity in HSPCs by vaccinations, HSPCs within the bone marrow obtained of patients suffering from systemic lupus erythematosus (SLE) possess transcriptomic remodeling and myeloid skewing abilities. 165This resulted in a surge in circulating neutrophil numbers, persistent inflammatory phenotype, and ultimately enhanced cardiovascular mortality. 165Also, in the context of systemic inflammation, it was found that epigenetic and metabolic rewiring induced by uric acid priming causes PBMCs from gout patients to produce higher amounts of pro-inflammatory cytokines following restimulation with LPS alone or in combination with monosodium urate (MSU) crystals. 166,167Moreover, Jeljeli et al. used a mouse model of systemic sclerosis, which is a chronic autoimmune inflammatory disease, to investigate the effects of different types of trained immunity on disease progression. 168They found that LPS-induced immune tolerance led to the amelioration of fibrosis, while BCG exposure led to hyperinflammation and worsened prognosis. 168ese findings contribute to the understanding the way innate immune tolerance can be utilized therapeutically to activate or inhibit the immune response to effectively react against autoinflammatory diseases.
Several investigations have recently highlighted the potential role of innate immune training in allergy, where it was again described to have both adaptive and maladaptive outcomes.
Interestingly, allergic children were found to exhibit increased pro-inflammatory cytokine secretion in the first years of life, compared to nonallergic children, who displayed a progressive age-dependent expression. 169,170Nonetheless, the epigenetic and metabolic mechanisms behind this trained immunity-like state in allergic children are still unclear. 171Some adaptive effects of innate immune training against allergy have also been described.
Whole-cell pertussis vaccination in infancy was shown to confer protection against food allergies, and large studies of randomized controlled trials assessed the effectiveness of neonatal BCG vaccination against the development of allergic diseases. 172Trained immunity is conferred through long-term epigenetic changes, however, the precise role of these changes in the development of allergic diseases is still unclear, and further research is needed to better differentiate between the protective and the detrimental effects of training in these immune disorders.In this respect, it is very important to point out that it is very likely that different trained immunity programs induce either protection or exacerbation of allergic disorders.

| Trained immunity in neurodegenerative disease
The induction of trained immunity has been linked to detrimental effects on the progression of neurodegenerative diseases.
The likelihood of developing these disorders of the neurons rises significantly with age, and increasing evidence points to chronic inflammation as being one of the main underlying causes. 173croglia, the specialized immune cells of the brain, have already been described earlier in this review for their ability to mount trained immunity via long-term metabolic and epigenetic remodeling.Various challenges, ranging from bacterial infections 173 to stress 174 have been demonstrated to induce long-term immune memory of microglia. 175The onset of long-lasting immune memory in these brain-resident macrophages has previously been shown to exaggerate cerebral inflammation and β-amyloidosis production in a mouse model of Alzheimer's disease. 126Following this concept, epigenetic reprogramming in microglia led to mTOR activation through increased β-amyloidosis, which was also accompanied by downstream HIF-1α signaling.These characteristics are consistent with the well-known in vivo model of innate immune training of myeloid cells. 176,177Supporting these findings, another study uncovered how infections of neonatal mice led to immunological training, which contributed to synapse damage and cognitive impairment due to β-amyloidosis produced by primed microglia. 177Subsequently, this gave rise to long-lasting susceptibility to neurodegenerative diseases that arise later in life. 177Considering this evidence, a systemic neuroinflammatory response due to the induction of trained immunity can cause longterm epigenetic changes in microglia, which may explain the development of neurodegenerative disorders later in life.However, the maladaptive effects of microglia immune memory were shown to be reversible.Through the establishment of histone acetyltransferase HDAC1/2-dependent tolerance in microglia, Datta et al.   showed that memory impairment is avoided, thus neurodegeneration was slowed down. 178ditionally, vascular brain pathologies associated with systemic inflammation, such as cerebral small vessel disease, have been shown to contribute to increased cognitive decline and onset of dementia. 179By studying the cytokine secretion patterns in PBMCs and monocytes from patients with cerebral small vessel disease after ex vivo stimulation, one study found enhanced IL-6 and IL-8 expression. 180This was consistent with the trained immunity-specific proinflammatory phenotype and ultimately linked to the severity and progression of the disease.Nonetheless, the precise mechanisms tying innate immune memory to the development of cerebral small vessel disease are still largely unknown.There is increasing evidence regarding the involvement of trained immunity in "inflammaging," a condition characterized by chronic, sterile inflammation associated with advanced age. 181An example of this is the age-dependent remodeling of innate immune cells to overexpress certain trained immune-specific molecules such as IL-8 182 and CCL1, 183 leading to excessive inflammation.Therefore, a deeper understanding of the maladaptive programs of trained immunity caused by aging may contribute to the development of novel therapeutic interventions for slowing down or even halting neurodegeneration in elderly patients.

| Cancer
Cancer remains a major cause of death around the globe, and as the aging population continues to rise in numbers, its prevalence is only expected to increase.Therefore, the efficient activation of both the innate and adaptive immune responses is crucial for the elimination of tumor cells from the organism.Traditionally recognized for its role in enhancing the host response to recurrent infections, trained immunity has now demonstrated its potential to contribute significantly to the fight against cancer.[186][187] The establishment of innate immune training following vaccination has been identified to lay the basis of BCG's anti-tumor effects, with autophagy playing a significant role in conferring its protective effects. 1880][191][192][193] More recently, the induction of trained immunity following pre-treatment of mice with β-glucan was found to correlate with a notable reduction in tumor growth. 194This effect was linked to transcriptomic and epigenetic alterations of granulopoiesis progenitors, leading to a distinctive reprogramming of neutrophils toward an anti-tumor phenotype.Specifically, both type I interferon (IFN) signaling and reactive oxygen species (ROS) production were required for the suppression of tumor growth by trained neutrophils. 194However, whether ROS production is a result of trained granulopoiesis or functions as a reinforcing stimulus for its sustained efficacy, and if type I IFN signaling is causally linked to the upregulation of ROS in trained neutrophils, remains to be determined.Moreover, virus-trained innate immune cells can also play a beneficial role in anti-tumor immunity.A recent study showed that influenza infection in mice trains respiratory mucosal-resident alveolar macrophages (AMs) to exert long-lasting and tissuespecific anti-tumor immunity. 195Following exposure to the influenza A virus (IAV), lung resident AMs acquire trained immunity characteristics, including increased TNF and IL-6 secretion after LPS stimulation, and upregulation of glycolysis and PI3K-Akt, HIF-1, and mTOR signaling.Following IAV training, tumor-infiltrating AMs showed enhanced transcription of gene clusters related to phagocytosis, cell killing, and the ROS biosynthetic process that are closely related to the anti-tumor functions of macrophages. 195reover, resident AMs were resistant to tumor-associated immune-suppressive microenvironments.These studies altogether put forth overwhelming evidence that the modulation of innate immune training represents a unique anti-tumor strategy with long-term effects.
On the contrary, exaggerated or chronic inflammation can lead to the spread and differentiation of tumors, which ultimately causes disease progression and needs to be avoided.In this regard, innate immune cells have the potential to undergo a reprogramming process once they infiltrate into the tumor microenvironment. 196As a result, persistent inflammatory responses are established, which carry outcomes such as elevated antigen-driven lymphoproliferation, hindered apoptosis, induced mitochondrial dysfunction, and intensified oxidative stress within the tumor microenvironment that ultimately drives tumor progression.Moreover, trained immune cells repeatedly secreting inflammatory cytokines such as TNF and IL-6 have been linked to unregulated proliferation, leading to increased chemo-radioresistance and metastasis in certain types of tumors such as oral squamous cell carcinoma, as well as lung, kidney, and breast cancers. 197,198Therefore, it is crucial that the epigenetic reprogramming mechanisms that control the upregulation of the immune response after trained immunity induction are tightly regulated, so that an overly active phenotype that drives tumor progression is avoided.

| Organ transplantation
Classically, the development of therapeutic approaches to improve organ transplantation outcomes has focused on adaptive immune mechanisms.However, recent developments have provided evidence of the establishment of innate immune memory in graft rejection due to allogeneic recognition of non-self-antigens. 199Importantly, trained immunity-specific epigenetic changes do not only contribute to rejection, as they have also been reported to regulate macrophage functions and promote graft survival. 199,200Another study used an experimental transplantation mouse model to show that endogenous stimuli, namely vimentin, and HMGB1, can activate graft-infiltrating monocyte-derived cells and cause increased cytokine production during allograft rejection. 201Following short-term inhibition of training mTOR high-density lipoprotein (HDL) nanoparticles, CD8 + T cell-mediated immunity was prevented, while CD4 + regulatory T cell expansion was promoted, thus improving allograft survival. 201Consequently, trained immunity constitutes a new therapeutic option for the prevention of organ rejection.

| CON CLUS I ON AND FUTURE PER S PEC TIVE S
Trained immunity is described as the functional state of the innate immune system characterized by long-term epigenetic and metabolic reprogramming of cells, resulting in robust immune responses to subsequent heterologous stimuli.Although the past decade has witnessed numerous advancements in the field of trained immunity, we are also observing key knowledge gaps that hinder our ability to fully exploit its benefits for combating disease (Figure 3).At the molecular level, we need to expand our knowledge of the immune and non-immune cell populations and tissues involved in the induction and regulation of training upon their encounter with stressful environments.This includes studying their communication patterns and unraveling the functional consequences they experience after undergoing these trained immune functional programs.The elucidation of the molecular mechanisms and cells will, therefore, establish the foundational framework for targeted therapies.Consequently, the regulation of specific trained immunity processes will be done within distinct cell populations and their progenitors, ensuring highly efficient targeting.Additionally, the influence of the host microbiota on the regulation of the trained innate immune response requires further exploration.
There are currently still key details missing regarding the underlying molecular mechanisms involved in the onset of intracellular metabolic and epigenetic changes that accompany the non-specific protection conferred by trained immunity.For example, for how long these modifications persist, as well as the exact mechanisms through which they are transmitted to daughter cells are still unknown.Furthermore, there is a need for further investigation into the interaction between DNA methylation and histone modifications that take place following initial stimulation to elucidate their role in regulating the trained innate immune response.Host genetic variability remains an important challenge for the establishment of an efficient trained immune response. 202These polymorphisms can severely impact gene regulation changing the expression of noncoding RNA.Future studies ought to explore the diversity of these functional non-coding elements involved in this process.This analysis is pivotal for accurately predicting how various disease-related factors will impact inflammatory processes, such as the strength of innate immune memory or responses to vaccination.It is now understood that both post-transcriptional and post-translational modifications play key roles in the induction and regulation of the inflammatory response.Yet, a more in-depth understanding of their regulators, targets, expression profiles, molecular interactions, and biological functions in disease pathogenesis is essential for the future development of innovative diagnostic markers and therapeutic targets for disease.
Further investigation also needs to be geared towards understanding the transgenerational transmission of innate immune memory traits.Sudden epigenetic and metabolic rewiring of innate immune cells may occur early in life and contribute to the development of allergies or autoimmune diseases.It is hypothesized that modifications occurring without altering the DNA sequence are more likely to be inherited.[205][206] Interestingly, emerging evidence suggests that the non-specific protection against reinfection achieved through cellular, developmental, transcriptional, and epigenetic reprogramming following trained immunity generation in mice can be passed on to subsequent generations, 207 and research should be done to explore whether similar processes take place in humans.Additional research into the implications of transgenerational transfer of trained immunity is crucial for a comprehensive understanding of disease pathology, including infectious diseases, as well as conditions characterized by long-term reprogramming of innate immune responses, such as atherosclerosis, diabetes, and cancer.
Moreover, this research may have broader implications, including the potential development of a mass vaccination program that involves the induction of herd immunity.
An important line of future research involves the underlying mechanisms that induce and modulate trained immunity and their importance in the pathogenesis of immune-mediated disorders.As we have previously described in detail, the generation of a trained innate immune response can be highly beneficial in boosting the immune response to protect against infections or in the case of diseases characterized by an impaired host defense mechanism, such as post-sepsis-induced immune paralysis or cancer.However, excessive trained immunity activation carries a maladaptive effect, contributing to the pathogenesis of chronic inflammatory diseases such as atherosclerosis, rheumatic diseases, and neurodegenerative disorders.Further research into the correlation between the various stimuli and disease pathologies, along with understanding the associated intracellular metabolic and epigenetic changes that cause the heightened inflammatory response upon rechallenge, is essential to maximize the therapeutic potential of trained immunity. 208rthermore, gaining insight into the connection between trained immunity and the processes of immune senescence and clonal hematopoiesis is also pivotal for gaining a better understanding of the mechanisms of immune diseases.Based on these concepts, novel therapeutic applications that employ trained immunity have already started to be developed.Some examples include novel vaccine formulations that are either based on the induction of trained immunity 209 or combine it with the generation of adaptive immune memory 210 ; the development of compounds that induce training administered in combination with other treatments 211,212 ; and the use of trained immunity-promoting nanobiologics for treatment of tumors. 213ere is limited information available regarding the impacts of various classes of drugs known to influence immune responses, metabolic processes, or epigenetic enzymes that have the potential to modulate trained immunity.Nevertheless, these central mechanisms underlying trained immunity have been suggested to represent ideal therapeutic targets in the fight for combating immune-related diseases. 208,214,215We have previously outlined the epigenetic enzymes KDM5 and Set7 as important regulators of trained immunity.As such, these two enzymes represent promising targets for preventing trained innate immune activation in autoinflammatory diseases.Additionally, bromodomain and extraterminal domain inhibitors (BETis) have started to become increasingly recognized for their therapeutic potential in inflammation by disrupting the recognition of acetylated histone marks associated with trained immunity establishment. 216For example, iBET151 was shown to exhibit efficacy in suppressing immune responses and preventing trained immunity induction. 217Therefore, the reversal of the trained immune phenotype through the removal of histone marks represents an attractive therapeutic target for diseases characterized by an overly activated immune response.Conversely, the targeting metabolic changes also represents an attractive target in trained immunity for future therapeutic strategies.Rapamycin-loaded HDL nanoparticles have already been proven effective in targeting the Akt-mTOR pathway, inhibiting trained immunity induced by allograft transplantation and preventing allograft rejection. 201e new generation of therapies and vaccines against immuneassociated diseases that are already in development combines the generation of classical adaptive immune memory and trained immunity.Furthermore, there is ongoing research into various other technologies, such as BCG-based nanoparticles and β-glucan capsules that have started to gain popularity due to their ability to be used to modulate specific features such as custom drug release properties. 218,219A final point that future studies should not overlook is the elucidation of the specific duration of the trained immune phenotype

F I G U R E 3
Future perspectives in the field of trained immunity.(A) At the molecular level, in-depth exploration into the distinct cell populations and their progenitors, and the specific organs involved in the initiation of trained immunity is essential.(B) Major advances are needed for a deeper understanding of the underlying molecular mechanisms that influence the efficacy of training.Additionally, investigations into the influence of the host microbiota, as well as the impacts of diverse classes of drugs on the efficacy of the trained immune response are warranted.(C) Current trends indicate the promise of various proteins as diagnostic markers and as prime targets for targeted therapies that employ innate immune training.Notable examples include targeted drugs, vaccines, and nanobiologics, which demonstrate potential in modulating the induction of trained immunity for the treatment of diseases characterized by either overly active or defective immune responses.CMP, common myeloid progenitor; DCs, dendritic cells; GMP, granulocyte-macrophage progenitor; HSC, hematopoietic stem cell; ILCs, innate lymphoid cells; MPP, multipotent progenitor; NK, natural killer; TI, trained immunity.Image created with BioRe nder.com, with permission.