Signaling pathway
Up to now, several signaling pathways associated with trained innate immunity have been discovered. Here, we will discuss two well-understood pathways led by the dectin-1 receptor and NOD2 (nucleotide-binding oligomerization domain-containing protein 2) receptor. Commonly, β-glucan-induced trained innate immunity is mediated by dectin-1/Akt/mTOR (mechanistic target of rapamycin)/HIF1α (hypoxia-inducible factor 1α) pathway, while MDP and BCG drive trained innate immunity through NOD/NF-κB (nuclear factor kappa-B) pathway [
12,
20,
21].
Dectin-1 is a transmembrane receptor that can recognize β1,3-linked and β1,6-linked glucans via a lectin-like carbohydrate recognition domain [
22]. β-Glucan is a polysaccharide component of fungal cell walls. When encounters with β-glucan, dectin-1 actives Akt, mTOR, and HIF1α successively to achieve the purpose of training monocytes. Intriguingly, the interaction between HIF1α and glycolysis leads to a forward loop, in which succinate activates HIF1α [
23] and HIF1α promotes glycolysis by elevating pyruvate, increasing glucose consumption and lactic acid production in turn [
20]. Using the inhibitors of the mTOR pathway, rapamycin or metformin, nullify the cytokine production [
24,
25], and ascorbate (HIF1α inhibitor) can also abrogate trained innate immunity in a dose-dependent manner [
20].
Researches have shown the emerging roles of NLRs in antiviral innate immune signaling pathways [
26]. BCG and MDP (a common motif contained in all bacteria) can agonist NOD2, an intracellular sensor that belongs to the nod-like receptor (NLR) family [
27]. NOD2 activation and signaling through nuclear factor kappa-B (NF-κB) stimulates epigenetic rewriting of macrophages like H3K4me, H3K18ac, and H3K27ac and induces trained innate immunity (showed in Fig.
1). NOD2-dependent activation of the NF-κB proinflammatory cascade of IFNs, TNF-α, IL-6, and IL-1β plays a role in resisting subsequent stimuli [
12,
21]. When induced by BCG, there is no enhanced response in cells with NOD2 deficiency [
12,
21]. The ubiquitination of RIP2 promotes the transcription of NF-κB. Thus, the inhibition of Rip2 kinase can impair training. Studies also demonstrated that Butyrate could suppress the activation of macrophages by preventing the acetylation of histones [
12,
28].
Cellular metabolism has emerged as a major biological node in maintaining cell homeostasis, proliferation, and cell-specific functions by providing energy and macromolecular building blocks [
29]. Arts et al. assess network-level metabolome and transcriptome in primary human isolated monocytes induced by β-Glucan. The data identify several indispensable metabolic pathways contributing to trained innate immunity, such as glycolysis, glutaminolysis, and cholesterol synthesis [
25]. When inhibiting one of these pathways, there is a significant decrease in the production of cytokines. In these pathways, many intermediate metabolites serve as a bridge between metabolic and epigenetic processes by acting as substrates or cofactors to regulate epigenetic enzyme activity [
30]. Examples are abundant. Acetyl-CoA is the substrate for histone deacetylase. Fumarate and α-ketoglutarate are cofactors for histone lysine-specific demethylase 5 (KDM5) and JMJD3 (also known as KDM6), respectively [
30]. The metabolic pathway is a critical mediator of epigenetic reprogramming dependent on trained innate immunity. It is well established that metabolic rewriting of innate immune cells will regulate the plasticity and epigenomic reprogramming in the context of trained innate immunity [
31]. Here, we will briefly introduce major metabolites engaged in these processes.
The upregulation of glycolysis is mediated by dectin-1/Akt/mTOR/HIF1α pathway in monocytes trained with a high concentration of β-glucan [
20], marked by a shift from oxidative phosphorylation to aerobic glycolysis [
24]. Glycolysis provides pyruvate that goes into a tricarboxylic acid cycle (TCA) by converting to acetyl-CoA, an acetyl donor for histone acetyltransferases. Genes involved in glycolysis are epigenetically modified with activating histone marks. With the upregulating of acetyl-CoA in the context of trained innate immunity, acetylation of the genes of hexokinase and lactate dehydrogenase can promote glycolysis in turn [
32]. However, 2-DG, an inhibitor acting on hexokinase, can block the training process of innate immunity, leading to suppressed IL-1β [
33]. In β-glucan-activated monocytes, the accumulation of fumarate can apply the inhibitory effect to the activity of KDM5, inducing the enrichment of H3K4me3 on promoters, thus exerting influences on epigenetic reprogramming and increasing production of proinflammatory cytokines to subsequent stimulation [
25]. Glutaminolysis results in the accumulation of α-ketoglutarate, which replenishes the TCA cycle. In LPS-activated macrophages, the production of α-ketoglutarate facilitates endotoxin tolerance through Jmjd3-dependent regulations and contributes to the M2-promoting mechanism [
34]. In the same setting of macrophages, succinate is strongly upregulated to develop an enhanced proinflammatory response through HIF1α/IL-1β pathway [
23]. The α-ketoglutarate/succinate ratio is the decisive factor in the transition from the M2 phenotype to the M1 phenotype, with a low ratio promoting inflammation [
34].
Glycolysis can promote cholesterol synthesis, thus leading to the intracellular accumulation of an essential metabolite, mevalonate. It induces and amplifies trained innate immunity by increasing the expression of H3K4me3 on IL-6 and TNFα promoters through the IGF1R-Akt-mTOR pathway. Inhibition of the cholesterol synthesis pathway by 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor (HMG-CoAi) like fluvastatin prevented the induction of training for β-glucan and BCG, indicating that the cholesterol synthesis pathway is essential for trained innate immunity [
33]. Further evidence showed that patients with deleterious mutations in mevalonate kinase carried endogenous trained innate immunity phenotypes in monocytes, with increased upregulation of glycolysis, epigenetic changes, and inflammatory cytokines production [
33].
Itaconate is another essential TCA cycle metabolite derived from cis-aconitate with IRG1 protein responsible for its generation, and its concentration is highly upregulated in LPS-activated macrophages [
35]. An anti-inflammatory effector induces immune tolerance by inhibiting mitochondrial metabolism and cytokines such as IL-6 and IL-12 [
36]. Three mechanisms account for the pronounced tolerizing effects. The major one is the inhibition of succinate dehydrogenase-mediated oxidation of succinate to fumarate, resulting in the elevated expression of KDM5 and decreasing of H3K4me3, which can lead to a state of immunoparalysis deleterious to the host as a result of increased susceptibility toward secondary infections [
37]. Secondly, it supports the activity of the NRF2, an anti-inflammatory transcription factor, which limits further inflammatory gene expression of IL-1β and downregulates the IFN response. [
38]. Thirdly, it selectively regulates secondary transcriptional responses to TLR stimulation by inhibition of LPS-mediated IκB induction via a key mediator, ATF3 [
36]. However, β-glucan can offset this immune tolerance by inhibiting IRG1. The counterbalance induced by β-glucan enhances succinate dehydrogenase activity by downregulating the itaconate production, thus elevating the fumarate accumulation, leading to a series of subsequent activation of trained innate immunity [
35]. Itaconate is a crucial regulatory node between tolerance and trained immunity and could be employed as a therapeutic tool in sepsis or cancers.
Epigenetic remodeling
Epigenetic remodeling means heritable alternation in gene function without any change in DNA sequence, leading to a shift in phenotype, which is just the way how trained innate immunity allows cells like monocytes and macrophages to respond more or less strongly upon secondary challenge. Current studies have shown that epigenetic reprogramming can occur in histone modification, non-coding RNA, and DNA methylation. Histone modification refers to an enzymatic modification such as methylation, acetylation, and phosphorylation. Several genetic markers are associated with trained innate immunity: H3K4me3, marking active promoters, H3K4me1, observed at the enhancers, and H3K27ac, characterized in both promoters and enhancers [
20,
39]. Histone modifications can induce a proinflammatory phenotype in cells, but how do they play a role in trained innate immunity upon secondary stimulation? When the native myeloid cells are first stimulated, they are rapidly activated and upregulate gene transcription, associated with activating histone modification, characterized by a shift from high DNA methylation to low DNA methylation and an absence of histone modifications to an active expression of H3K4me3 and H3K27ac. Then, when stimuli are removed, these depositions of chromatin marks still partially rest in histones like H3K4me1, which is a notable feature since these quiescent myeloid cells can enhance gene expression and lead to a more robust cytokine production with re-stimulation. Based on this mechanism, it is worthy of attention to epigenetic enzymes (histone methyltransferase; histone demethylase; histone acetyltransferase; histone deacetylase) that can write or erase epigenetic markers. It is known that KDM5 and Lysine methyltransferase (Set7) play a key role in β-glucan-induced trained immunity. When the monocytes are exposed to β-glucan, the expression of KDM5 is downregulated by the accumulation of fumarate, followed by the persistence of H3K4me3 activity on the enhancers of specific metabolic enzymes [
25]. While histone methyltransferase Set7 is enhanced, activating the deposition of histone modification H3K4me1 and opening the chromatin needed for the production of proinflammatory cytokines upon re-stimulation [
40].
A recent study found that immune gene-priming lncRNAs, UMLILO underlies the molecular basis of trained innate immunity. In addition to the connection between β-glucan and the dectin-1/AKT/mTOR pathway, β-glucan can also activate NFAT signaling by triggering calcium influx via dectin-1, which permits NFAT to translocate into the nucleus. The increased level of NFAT on the UMLILO promoter results in an upregulation of its transcription. Finally, WDR5–MLL1 complex is recruited by UMLILO to certain promoters, such as IL-8, CXCL1, CXCL2, and CXCL3, to facilitate H3K4me3 expression on target genes [
41]. As such, lncRNAs play a role as transporters for methyltransferases in the chromatin that contains trainable genes to link epigenetic and metabolic changes in trained innate immunity. Notwithstanding, how to modify the innate immune response genes by regulating UMLILO activity and how other stimuli other than β-glucan induce trained innate immunity through lncRNAs to warrant further studies.
As for DNA methylation, relevant research is scarce, and there needs to be an investigation into the function of DNA methylation in the development of trained innate immunity. A study among BCG-vaccinated subjects revealed that DNA methylation patterns on promoters of inflammatory genes were lost in a wide range of responders characterized by containment of
Mycobacterium tuberculosis replication compared with non-responders [
42]. Another study indicated that DNA methylation changes are restricted to a small scope of the genomic region compared with histone modification. And the majority of DMR appears at distal elements marked by H3K4me1. DNA methylation changes can be utilized as a biomarker for LPS-induced tolerance in macrophages [
15], an exciting new field awaiting exploration.