INTRODUCTION
Immunological memory was originally thought to be a feature exclusive to B and T cells. During infection with a pathogen, effector T cells are induced and help to fight the infection. If the pathogen is cleared, most effector T cells die, but a small percentage remain in the tissues for months or years as memory T cells. This type of memory is a feature of the adaptive immune system, and the T and B cells are specific for antigens on the invading pathogen. However, recent studies have shown that innate immune cells can be “trained” or “primed” in response to various stimuli, suggesting an alternative form of immune memory, often called trained innate immunity or innate immune memory.1
Trained innate immunity has recently been shown to play a role in protective immunity against infection in humans. In 2012, Kleinnijenhuis et al demonstrated that circulating monocytes from healthy volunteers vaccinated with Bacillus Calmette-Guérin (BCG), a vaccine against Mycobacterium tuberculosis (Mtb), secreted higher concentrations of proinflammatory cytokines in response to various pathogen-associated molecular patterns (PAMPs).2 Interestingly, it has been known for some time that BCG has nonspecific protective effects against a range of malignancies, including bladder cancer, melanoma, and leukemia.3-5 Furthermore, immunization with BCG can confer nonspecific protection against infection with unrelated pathogens.6,7 Importantly, studies using RAG1-deficient mice, which lack T and B cells, have demonstrated that protection mediated by trained immunity is independent of adaptive immunity and is mediated by cells of the innate immune system.8-10
Following its first description over 10 y ago, the focus has turned to deciphering the cellular and molecular mechanism of trained innate immunity. Trained innate immunity that renders monocytes/macrophages or natural killer (NK) cells hyperresponsive to proinflammatory stimuli can be induced by bacterial, viral, and fungal components, cytokines, alarmins, and microRNAs.11 Many of the early studies focused on the capacity of BCG and β-glucan, a component of many bacterial and fungal cell walls and an agonist for Dectin-1, to induce trained innate immunity in monocytes/macrophages.
Studies in mice have generated considerable evidence of trained innate immunity in vivo. For example, injection of mice with β-glucan results in enhanced bacterial clearance and lower mortality following challenge with Staphylococcus aureus.12,13 Intriguingly, the protective effects of β-glucan were found to be independent of its receptor, Dectin-1.13,14 Similarly, treatment with the Toll-like receptor (TLR) 5-agonist flagellin protected mice against both Staphylococcus aureus and rotavirus infections.15,16 Furthermore, treatment with unmethylated cytosine-guanidine motifs, agonists for TLR9, enhanced survival of neutropenic mice in a model of Escherichia coli–induced sepsis and meningitis.17 Protection was associated with enhanced inflammatory monocytes, interleukin (IL)-12 and interferon (IFN)-γ production, and typical type-1 immune responses. Furthermore, latent infection of mice with murine gamma-herpesvirus 68 or murine cytomegalovirus confers protection against Listeria monocytogenes and Yersinia pestis.18 The protection was not antigen specific but was mediated by persistent IFN-γ production and activation of macrophages. Moreover, induction of trained innate immunity is not confined to pattern recognition receptor (PRR) signaling by PAMPs. Injection of mice with low doses of IL-1β 3 d before bacterial challenge enhanced protection against Pseudomonas aeruginosa infection.19
Trained innate immunity can also be seen in type-2 immune responses. For example, infection of mice with the parasitic nematode, Nippostrongylus brasiliensis, resulted in priming of alternatively activated neutrophils which prime long-lived effector macrophages that mediate rapid expulsion of helminths following rechallenge with the parasite.20 Interestingly, infection of humans with parasitic worms during pregnancy can result in reduced T helper (Th)1 and enhanced Th2 responses in offspring following immunization at birth with the BCG vaccine.21 This suggests that trained innate immunity is long-lived, can be maternally transferred, and can influence type-1 and type-2 arms of the immune response. We have recently provided evidence that products from a helminth pathogen that promote type-2 immune responses can induce anti-inflammatory trained innate immunity.22 Macrophages treated with total extract from Fasciola hepatica (liver fluke) had heightened production of the anti-inflammatory cytokine IL-10 but reduced production of tumor necrosis factor (TNF) following exposure to proinflammatory stimuli. Furthermore, treatment of mice with the helminth products induced anti-inflammatory trained immunity in macrophages in vivo, and this suppressed activation of pathogenic Th17 cells that mediated experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis.
Collectively, the emerging data suggest that innate immune cells can be modified to be more proinflammatory or anti-inflammatory depending on the stimulus. This suggests that trained immunity is distinct from activation through PRRs or tolerance to PAMPs but, as discussed next, involves epigenetic modification of innate immune cells. Furthermore, although most of the studies on the mechanism of trained immunity have used short-term training approaches, a few including those that examined the effects of BCG vaccination and the effects of helminth parasite in vivo are more longer term. This is consistent with the suggestion that trained immunity is a distinct process from transient activation of innate immune cells.
Mechanisms of Trained Innate Immunity
Although antigen-specific memory in T and B cells is maintained by the genetic restructuring of specific loci in T- and B-cell receptors, the exact mechanism of trained innate immunity is still unclear. It appears that nonspecific memory or trained innate immunity involves metabolic and epigenetic changes in cells of the innate immune system.9,23
Studies on epigenetics, heritable changes in gene expression, as the basis for trained innate immunity have focused on chromatin remodeling, which enables more robust transcription through easier access of specific loci on the genome by transcription factors. Histone modifications, including acetylation and methylation, can weaken or tighten the bond between histones and DNA, modifying access to transcription factors that are essential to metabolism and activation of innate immune cells.24 Histone modifications influence trained innate immunity through epigenetic regulation of genes coding for inflammatory or anti-inflammatory cytokines and other molecules. For example, infection of mice with Candida albicans or treatment with β-glucan, which resulted in enhanced cytokine production by monocytes, resulted in stable changes in histone trimethylation at H3K4 in various promoter regions of genes coding for inflammatory cytokines and chemokines. Quintin et al25 found H3K4me3 at promoters for myeloid differentiation primary response 88 (MyD88), TNF, IL-6, and various TLRs and C-type lectin receptors. Further analysis revealed changes in positive histone regulatory marks for H3K4me1 and H3K27ac, resulting in the enhanced ability to produce proinflammatory cytokines.26 These epigenetic modifications appear consistently in the heightened response to inflammatory stimuli associated with trained innate immunity induced by a variety of different mediators (Figure 1). Furthermore, we have reported that helminth product-induced anti-inflammatory trained innate immunity is reversed by pretreatment of macrophages with a histone methyltransferase inhibitor.22
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FIGURE 1.: Molecular mechanisms of peripheral trained innate immunity. Transient exposure of innate immune cells to certain proinflammatory stimuli from bacteria, viruses, or fungi or endogenous danger molecules, such as HMGB1, induces metabolic activation and epigenetic reprogramming. After the initial training signal has dissipated, the innate immune cell returns to basal levels of activation. Upon secondary challenge with the same or a novel stimulus, chromatin marks allow for faster and more robust responses to challenge. In proinflammatory peripheral trained immunity, this results in increased proinflammatory cytokine secretion and enhanced MHC-II and costimulatory molecule expression on the surface of the cells. The resulting trained response, which is distinct from activation or differentiation, confers on the cells an improved capacity to kill pathogens and present antigens to T cells. Note: stimulation of innate immune cells with other mediators, such as helminth products, can also result in anti-inflammatory trained innate immunity (not shown; see
Figure 2). BCG, Bacillus Calmette-Guérin; HMGB1, high mobility group box 1; IFN, interferon; IL, interleukin; MHC, major histocompatibility complex; mTOR, mechanistic target of rapamycin; TLR, toll-like receptor; TNF, tumor necrosis factor.
Cellular metabolism has been shown to play a crucial role in the development of trained innate immunity. Although there is yet to be a centrally defined mechanism for induction of trained innate immunity, several factors have been established, including mechanistic target of rapamycin (mTOR) and a preference for glycolysis. For example, induction of glycolysis has been shown to be essential for β-glucan–induced trained innate immunity through the Akt-mTOR-HIF-1α pathway.27 Further evidence for activation of mTOR and a push toward aerobic glycolysis was provided by analysis of monocytes from BCG-vaccinated patients.28 β-glucan training of mice also leads to the enhancement of cholesterol biosynthesis and glycolysis in bone marrow (BM) myeloid progenitor populations.29 Additionally, there is a tight link between metabolic and epigenetic changes that lead to trained innate immunity.30 Indeed, blockade of glycolysis results in a lack of epigenetic changes and no trained immunity.27,28 Furthermore, there is evidence of a nuanced interplay between metabolic intermediates and epigenetics in trained innate immunity. For example, the metabolic intermediate, acetyl coenzyme A, is increased in trained monocytes and is required for histone acetylation at loci responsible for transcription of glycolytic enzymes.9 In addition, fumarate, a byproduct of the tricarboxylic acid cycle, modifies epigenetic modifications leading to trained innate immunity by downregulating histone demethylases.31
Role of Hematopoietic Stem Cells in Trained Innate Immunity
Although there is now convincing evidence that innate immune cells can be trained to respond differently following reexposure to a pathogen or its products, innate immune cells are short-lived, especially monocytes, with circulating lifespan of <1 wk.32,33 However, the beneficial nonspecific effects of BCG-induced trained innate immunity can last for months to years.2,34 A potential explanation for this paradox comes in the form of hematopoietic stem cells (HSCs), which are long-lived, self-renewing cells of the BM that can differentiate into every cell of the innate and adaptive immune systems. Consequently, recent research on trained innate immunity has focused on the role of HSCs and their possible modulation by proinflammatory or anti-inflammatory mediators (Figure 2). This phenomenon is known as central trained innate immunity, whereas peripheral trained innate immunity refers to training of circulating and tissue-resident mature innate immune cells.
FIGURE 2.: Anti-inflammatory and proinflammatory central trained innate immunity. Exposure of HSCs to anti-inflammatory and proinflammatory stimuli induces metabolic and epigenetic remodeling. These modifications lead to the differentiation and development of innate immune cells with altered phenotypes. Administration of helminth products and other factors leads to altered HSC differentiation into anti-inflammatory myeloid cells, which respond to infection and PRR stimulation with enhanced anti-inflammatory cytokine production, reduced ability to activate T cells, and a proengraftment phenotype. HSCs stimulated with BCG, for example, induce differentiation into myeloid cells with enhanced microbial killing capacity and proinflammatory cytokine secretion upon restimulation or infection. Modification of HSCs through anti-inflammatory and proinflammatory products results in long-lived memory in myeloid precursors, including a bias toward myeloid differentiation after further challenge. BCG, Bacillus Calmette-Guérin; GMP, granulocyte-monocyte progenitors; HSC, hematopoietic stem cell; LT-HSC, long-term hematopoietic stem cell; MPP, multipotent progenitor; PGE2, prostaglandin E2; PRR, pattern recognition receptor.
HSCs express various PRRs and cytokine receptors, enabling them to respond directly and indirectly to inflammation via changes to the BM microenvironment. As an example, macrophages derived from HSCs that have been directly stimulated with PAM3CSK4, a TLR2 agonist, or lipopolysaccharide (LPS), a TLR4 agonist, secreted significantly less proinflammatory cytokines IL-6 and TNF in response to restimulation with either TLR2 or TLR4 agonists.35 In contrast, HSCs treated with Candida albicans resulted in the development of macrophages that secreted significantly higher concentrations of IL-6 and TNF in response to restimulation with PAM3CSK4 or LPS. Treatment of mice with BCG significantly expands the HSC subpopulation of multipotent progenitors (MPPs) and modifies their transcriptional activity to induce genes associated with cell cycle and replication, and imprints a distinct bias toward the myeloid lineage via upregulation of C/EBP-α and IRF8.36 Furthermore, macrophages derived from mice treated with BCG mediate enhanced protection against Mtb challenge through enhanced antimicrobial elimination, and upregulated expression of Il1b, Tnf, and Ifng. It has been recently demonstrated that treatment of mice with β-glucan similarly expands myeloid progenitors, including MPPs and granulocyte-monocyte progenitors, in the BM. This effect persisted up to 4 wk posttreatment, and the increased myelopoiesis from HSCs resulted in enhanced response to secondary stimulation with LPS and conferred protection against chemotherapy-induced myelosuppression.29 The expansion of HSCs and protection mediated by β-glucan was dependent on IL-1β signaling in the BM, with significantly increased granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-1β in the BM extracellular fluid. Recent studies showed that mice treated with LPS also expanded MPPs and long-term HSCs, biasing their transcriptional landscape toward a myeloid lineage, which conferred long-lasting protection against infection with Pseudomonas aeruginosa.37 It has recently been demonstrated that LPS-mediated septic shock in mice was associated with enhanced myeloid-biased HSCs and trained BM-resident monocytes, which secreted higher concentrations of IL-6 and TNF in response to rechallenge with LPS 12 wks later.38 Therefore, although the early data demonstrated that trained innate immunity plays a critical role in the immune response to infection and autoimmune disease, there is emerging evidence that it may play a role in inflammation associated with cancer and transplantation.
Trained Innate Immunity in Solid Organ Transplantation
Organ transplantation is a life-saving procedure that extends and improves the quality of life for patients with end-stage organ failure. In 2015, according to the Global Observatory on Donation and Transplantation, over 125 000 solid organ transplantations were performed around the world, primarily in North America and Europe. The success of organ transplantation is dependent on establishing immune tolerance in the recipient to the donor organ. The adaptive immune system plays a key role in graft rejection by responding to alloantigens of the graft. Transgenic mice lacking T and B lymphocytes are resistant to transplantation rejection.39,40 Immediate rejection of allografts can now largely be overcome by treatment with potent immunosuppressive drugs, such as corticosteroids, calcineurin inhibitors, and mTOR inhibitors. However, although the widespread use of immunosuppressive drugs has improved the first-year outcome of many patients, the long-term (5–10 y) success of transplantations has progressed little over the past few decades.41-43
Activation of adaptive immune responses is driven by innate immune cells that act as antigen-presenting cells and respond to various PAMPs and damage-associated molecular patterns (DAMPs), which enhance major histocompatibility complex (MHC) and costimulatory molecule expression and produce T cell–polarizing cytokines. Therefore, an understanding of how the innate immune system directs T-cell responses that mediate graft rejection or tolerance may be key to improving treatment approaches for graft rejection and outcomes for transplant patients. Innate immune cell activation in the donor organ plays a central role in graft rejection and tolerance. Resident and monocyte-derived macrophages and dendritic cells (DCs) can mediate immune response to transplanted organs. Infiltrating monocytes rapidly respond to the transplant and mature into DCs and macrophages, which either suppress or enhance alloreactive T-cell responses. In kidney and liver transplant patients, elevated numbers of circulating monocytes correlate with poor graft function.44,45 Furthermore, macrophages constitute up to 60% of the immune cell infiltrates in biopsies from severe kidney rejection.46
DAMPs, such as heat shock protein 60, heat shock protein 70, surfactant protein-A, and high mobility group box 1 (HMGB1) are released during transplantation and induce proinflammatory responses in innate immune cells.47,48 DAMP release during transplantation is associated with the surgery or ischemic/reperfusion injury (IRI), where the lack of oxygen and subsequent reintroduction of blood flow causes tissue damage. During IRI, there is significant release of multiple DAMPs, including TLR4 agonist HMGB1. HMGB1-TLR4 and inflammasome activation may contribute to enhanced inflammatory responses in low-quality kidneys and may affect transplant outcomes.49 In a model of renal transplantation, mice deficient in TLR4 or its signaling adaptor molecule MyD88 have diminished tissue damage postreperfusion.50 Concomitantly with the release of DAMPs, brain death in organ donors results in the release of a plethora of inflammatory cytokines, including IL-6, IL-8, and TNF, which can be found throughout the donor plasma and organ.51,52
Together with the increasing interest in the role of innate immune responses in transplantation, there is emerging evidence to suggest that trained innate immunity is a factor in transplant rejection and tolerance.53 For example, it has been reported that infection with viruses, including cytomegalovirus, herpes viruses, hepatitis B and C, and adenoviruses, is associated with graft rejection.54,55 Conversely, studies in mice have shown that infection with helminth parasites enhances long-term graft survival in models of heart and kidney allograft transplantations.56,57 Furthermore, fibrosis associated with allograft rejection can be intensified by the enhanced proinflammatory effects of trained innate immune cells. Macrophages from mice treated with BCG were found to secrete significantly higher concentrations of profibrotic cytokines and exacerbated immune-mediated fibrosis in a mouse model of systemic sclerosis.58,59 Moreover, Schreurs et al60 found that macrophages derived from peripheral blood mononuclear cells of lung transplant patients preferentially differentiate into profibrotic alternatively activated macrophages, potentially contributing to chronic lung allograft dysfunction.60
Interestingly, it has been reported that NK cells, monocytes, and macrophages can respond directly to alloantigens and nonself MHC molecules, thereby initiating allograft rejection.61–64 For example, donor-derived signal regulatory protein alpha binding to CD47 on monocytes promotes rapid differentiation into DCs, which produce IL-12 and prime alloreactive T cells that mediate graft rejection.65 Recently, it was demonstrated that macrophages and NK cells could produce memory-like immune responses to allografts.66 NK cells in mice deficient of T and B cells were found to bind directly to alloantigens and develop a memory-like phenotype and promote graft rejection. However, although macrophages are capable of sensing allo-specific epitopes, memory to and rejection of allografts required both allo-specific priming and CD40 signaling through CD4 T cells. Recently, inflammatory Ly6Chigh monocytes have been shown to play a role in myeloid cell memory to allografts. A study by Dai et al67 showed that monocytes/macrophages could acquire memory specific for MHC class I. In mice lacking lymphoid populations, A-type paired immunoglobulin-like receptors expressed on Ly6Chigh monocytes and macrophages recognized allo-MHC-I on the surface of allograft splenocytes. Blocking the interaction between A-type paired immunoglobulin-like receptor and MHC-I inhibited the memory response and suppressed rejection of kidney and heart allografts.
Innate immune cells that are trained may mediate their effects from modification of adaptive immune responses by influencing the differentiation of naive T cells into effector and memory T cells. However, there is evidence that trained innate immunity may influence transplant rejection in the absence of T cells. Using RAG−/− mice, which are deficient in B and T lymphocytes, Zecher et al68 found that monocytes developed a memory phenotype after immunization with allogeneic splenocytes, which resulted in enhanced proinflammatory response to allogenic skin grafts up to 4 wk posttreatment. Further evidence of a role for monocyte training in transplant rejection came from studies by Braza et al,48 who found that HMGB1 and vimentin were capable of inducing epigenetic and metabolic modifications in transplant-infiltrating monocytes and macrophages. The trained myeloid cells had significantly enhanced TNF and IL-6 secretion in response to LPS restimulation ex vivo, an effect that was lost in Dectin-1– or TLR4-deficient mice. Furthermore, through inhibition of myeloid-specific mTOR using a nanobiologic inhibited this allo-training, which allowed expansion of regulatory macrophage populations, leading to regulatory T-cell infiltration that promoted long-term tolerance to heart transplants.48 Importantly, while the nanobiologic was concentrated in myeloid cells in the periphery, it also inhibited mTOR in multiple HSC populations, implicating central trained innate immunity in transplant tolerance and rejection.
Innate immune cells not only are important in acute rejection but also are found differentially regulated in long-term outcomes of patients. For example, Dos Santos et al found that the success of renal transplants is associated with the extent of monocyte infiltration at 1 y posttransplantation. Indeed, patients with fewer graft-infiltrating monocytes and macrophages had significantly higher rates of graft survival (90%) compared with those with elevated innate cell infiltration (58%).69 Recent studies have suggested that trained innate immunity can extend beyond the lifespan of a macrophage or monocyte through epigenetic and metabolic reprogramming of myeloid precursor HSC populations. There is also some evidence to suggest that this may extend to a role in allograft rejection and tolerance. For example, delayed graft function 1 y after kidney transplantation was more likely to occur in patients with circulating monocytes that exhibited lower expression of TLR4.70 Furthermore, Lorenz et al71 found that solid organ transplant recipients who had previously received an HSC transplant had a trend toward lower incidence of graft-versus-host disease (GVHD) when the HSC donor had at least 1 TLR4 polymorphism.71 Activation through TLR4 or other PRRs could also enhance trained immunity. It has been demonstrated that bacterial components and endogenous danger molecules, such as HMGB1, that bind to PRRs on myeloid cells can promote production of proinflammatory cytokines that enhance alloreactive T-cell responses against host tissue.72
Clearly, there is emerging evidence of a role for trained innate immunity in graft tolerance or rejection following solid organ transplantation. Although trained innate immune cells may mediate their effect locally in the graft, the induction of these cells may take place in the periphery. Therefore, manipulation of trained innate immunity may provide novel treatment approaches to ensure graft acceptance.
Trained Innate Immunity in HSC Transplantation
Transplantation of HSCs is an effective therapeutic approach for a number of chronic diseases, including primary immunodeficiency diseases, hematological cancers, and autoimmune diseases, such as multiple sclerosis.73 Many HSC transplants use autologous HSCs reintroduced to the body after chemotherapy or radiation therapy.74 However, allogeneic HSC transplantation has the advantage of graft-versus-leukemia response in treating hematological cancers.75 However, allogeneic HSC transplantation is associated with the risks of developing GVHD. Conversely, the possibility of disease relapse or development of a secondary autoimmune disease is a common adverse effect of autologous HSC transplantation.73 In contrast to solid organ transplant, long-term survival beyond 2 y is relatively high (~85%) post HSC transplantation; however, the short-term response to the graft is the key determinant in HSC transplantation tolerance.76 This has led to the search for novel approaches for suppressing immune response early in HSC transplantation.
The innate immune system is intricately linked to the BM microenvironment, and therefore, it has a major influence on the success of HSC transplantation. Importantly, activation of the innate immune response plays a central role in initiating GVHD following allogeneic HSC transplantation. NK cells play a role in HSC transplant engraftment or rejection. Indeed, in mouse models of HSC transplantation, NK cells recognized nonself MHC molecules and mediated rejection of donor HSCs.77–79 Innate immune cell activation and allo-specific Th1 responses were significantly attenuated, and development of lethal GVHD was reduced in mice when BM donors or recipients lacked TLR4.80 BM-resident macrophages play a key role in maintaining homeostasis of HSC in the normal and diseased BM microenvironment. As such, BM macrophage dysfunction during allo-HSC transplantation is essential for improving HSC engraftment. Classically activated macrophages are enhanced in the BM of patients with poor graft function after allogeneic HSC transplantation.81 Furthermore, macrophages from the BM of patients with poor graft function secreted more TNF and IL-12 when cocultured with HSCs ex vivo. HSCs cocultured with macrophages from patients with poor graft function had enhanced reactive oxygen species production and apoptosis, resulting in a reduced ability to differentiate into all cell types of the hematopoietic system.
Although central training of HSCs is a recently recognized aspect of trained innate immunity, the priming of HSCs to differentiate into cells of the innate and adaptive immune system that provide engraftment may be an attractive therapeutic approach to enhance the success of HSC transplantation. It appears that HSC or myeloid progenitors can undergo modification and give rise to macrophages with features consistent with trained innate immunity. There is some evidence that these effects on HSCs can last for months. Injection of mice with Mtb induced epigenetic modifications in HSCs, which persisted for up to 1 y.82 Furthermore, human HSCs exhibited changes that persisted for at least 90 d after vaccination with BCG.83,84
HSCs express receptors for growth factors, chemokines, cytokines, and neurotransmitters. Indeed, a common method for isolating HSCs for transplantations involves the injection of granulocyte colony-stimulating factor (G-CSF) to donors, which induces the mobilization of HSCs into the peripheral blood for relatively pain-free extraction.75 However, in addition to mobilization of HSCs, G-CSF can promote differentiation and mobilization of innate immune cells. For example, G-CSF induces production and mobilization of myeloid-derived suppressor cells (MDSCs) in the transplant donor.85 These MDSCs are capable of inhibiting T-cell responses, and recipients of HSC grafts that contained higher numbers of MDSC were less likely to develop acute GVHD. G-CSF has been shown to modify BM allografts, resulting in enhanced engraftment when compared with nonprimed BM.86 Additionally, various studies have demonstrated that genetic manipulation of HSCs to express autoantigens or alloantigens ex vivo before transplantation improves engraftment due to clonal deletion of recipient antigen-specific T cells.87–89 These data indicate that modification of HSCs before transplantation could potentiate tolerance and minimize the possibility of GVHD. Therefore, central innate immune training may be an attractive approach for enhancing transferred graft tolerance by modifying the collection and treatment of donor HSCs before transplantation. Studies in mice demonstrated that a single intraperitoneal injection of flagellin, an agonist for TLR5 and NOD-like receptor 4, induced proliferation in HSCs and that transplantation of HSCs from flagellin-treated mice significantly improved survival in irradiated recipient mice.90 Furthermore, the flagellin-modified HSCs were shown to be myeloid lineage biased, providing further evidence that training of HSCs could provide a novel method of enhancing graft tolerance through induction of trained innate immunity.
Recent studies have demonstrated that ex vivo modulated HSCs can generate modified innate immune cells. Stimulation of HSCs for 24 h with various PAMPs resulted in the generation and differentiation of antigen-presenting cells with enhanced proinflammatory cytokine production and capacity to promote CD4 T-cell proliferation and IFN-γ and IL-17 production.91 Evidence that the findings on trained innate immunity in mice can be extrapolated to humans was provided by the demonstration that ex vivo stimulation of peripheral blood HSCs with epigenetic inhibitors, such as histone deacetylase inhibitor trichostatin A, enhanced proliferation, and improved engraftment.92,93 Indeed, the epigenetic modification of HSCs for transplantation is currently being assessed in several clinical trials (NCT03164057, NCT03871296).
Anti-inflammatory Trained Immunity in HSC Transplantation
The identification of methods for training HSCs to produce tolerogenic or anti-inflammatory innate immune cells could facilitate induction of host tolerance and prevention of graft rejection. Epidemiological studies suggested that HSC transplantation from donors with genetic variations in TLR4, which confer lower reactivity to TLR4 ligands, is associated with reduced incidence of fatal infections and improved 5-year survivals.94 There is some evidence that prostaglandin E2 (PGE2) may be capable of having a training effect on HSCs. Ex vivo stimulation of HSCs with PGE2 enhanced their homing to the BM, improved engraftment, and reduced the inflammatory response of macrophages and DCs derived from the transplanted HSCs.95–97 Furthermore, early clinical trials have indicated that ex vivo treatment of umbilical cord blood HSCs with PGE2 before transplantation enhances engraftment.98
The products of helminth parasites also have potential for inducing tolerogenic responses in HSCs before transplantation. BM-derived macrophages and DCs from mice treated with phosphorylcholine-containing filarial nematode glycoprotein ES-62 had reduced responsiveness to stimulation with LPS.99 Because monocytes and DCs activate T cells that mediate graft rejection, strategies that inhibit development or maturation of DCs may be beneficial in HSC transplantation, especially in the treatment of autoimmune disease. We have found that products of the helminth parasite Fasciola hepatica can modify HSCs, giving rise to myeloid cells that are more anti-inflammatory following stimulation with PAMPs.100 Furthermore, these anti-inflammatory macrophages derived from helminth-imprinted HSCs suppressed antigen presentation and T-cell responses that mediate an autoimmune disease of the central nervous system.
Current strategies for improving HSC transplantation revolve around expanding the number of HSCs either in vivo or ex vivo with various growth factors (G-CSF, GM-CSF, stem cell factor) and antibody-directed blockade of C-X-C chemokine receptor type 4 (CXCR4; Plerixafor).75 G-CSF and GM-CSF alone have already been identified to confer trained innate immunity to HSCs, which differentiate into proinflammatory myeloid cells with improved responses to infection.29,86 Novel approaches to train HSCs to further enhance engraftment, fight infection/cancer, and promote long-term survival are the logical next step in HSC transplantation research (Figure 3).
FIGURE 3.: Current and potential methods of treatment of HSCs to improve engraftment. Current and potential therapeutic avenues using central trained innate immunity to improve HSC engraftment include: (A) mobilization and expansion of donor HSCs to the circulation for isolation, (B) ex vivo expansion, and (C) recipient therapeutic intervention. CXCR4, C-X-C chemokine receptor type 4; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; HSC, hematopoietic stem cell; mTOR, mechanistic target of rapamycin; PGE2, prostaglandin E2; SCF, stem cell factor.
Strategies That Target Trained Immunity for Improving Transplantation
Recent advances in immunosuppressive therapies have helped to promote short-term allograft tolerance. However, long-term engraftment rates have remained very low for decades.41,101 Long-term acceptance of transplanted organs requires a tolerogenic adaptive immune response that must be initiated by similarly tolerogenic innate immune cells, and recent studies have highlighted the essential role for NK cells, macrophages, monocytes, and DCs.48,81,102–104 During transplantation, innate immune cells are activated by DAMPs, PAMPs, nonself MHC-I, and inflammatory cytokines. Evidence is emerging that many of these immune modulators can also induce trained innate immunity. Suppressing the proinflammatory effects of trained innate immunity may provide a novel method for enhancing graft tolerance. In contrast, enhancement of proinflammatory trained innate immunity could be beneficial in promoting graft-versus-leukemia responses in HSC transplantation.
Evidence from animal models and clinical trials has indicated that targeting innate immune cells is a viable approach to improve transplant outcomes. Several current immunosuppressive therapies used in transplant patients are metabolic inhibitors that target innate immune cells. These drugs include sirolimus (rapamycin) and everolimus, which inhibit mTOR, a major protein in the metabolic reprogramming required for proinflammatory trained innate immunity. Although mTOR is broadly expressed in many cell types, innate immune cells are probably key therapeutic targets for mTOR inhibitors. Studies in mice on allogeneic transplantation of pancreatic islets showed that treatment of mice with a combination of G-CSF and rapamycin enhanced transplant infiltration of alternatively activated macrophages that promoted activation of regulatory T cells, thereby enhancing graft survival.102 Clodronate-mediated depletion of macrophages within 2 wk of transplantation resulted in graft rejection, providing further evidence of the importance of initiating a robust innate immune response in graft survival. Indeed, administration of an mTOR inhibiting nanobiologic with specificity for myeloid cells, and their progenitors inhibited the induction of proinflammatory trained macrophages in vivo and promoted the survival of mice after heart allograft transplantation.48 A number of drugs in widespread use to treat graft rejection modulate metabolism in immune cells that play a key role in trained innate immunity.105–107 Therefore, there is a need to fully understand the extent to which these inhibitors play a role in innate immune cell-mediated transplantation rejection and tolerance.
The concept of trained innate immunity, although only recently recognized, has already changed our understanding of immunological memory and how the immune system can be primed nonspecifically by pathogens or their products. Innate immune cells and their progenitors are new targets for novel vaccination and therapeutic approaches for infectious diseases and cancer. Recent studies that have identified a role for proinflammatory trained innate immunity in the pathogenesis of both graft rejection and tolerance have highlighted the potential for targeting the HSC precursors of innate immune cells in transplantation. A number of inflammatory mediators known to play a role in trained innate immunity, including cytokines, alarmins, and alloantigens, such as nonself MHC-I, are released following solid organ transplantation.48 Understanding the role of these mediators in induction of peripheral and central trained innate immunity should help in the application of trained responses in transplantation. Furthermore, the longevity of such effects and the role of HSCs in mediating life-long alterations to innate immune cells are key unexplored aspects. Therapeutic strategies that target metabolic, epigenetic, and signaling processes involved in the induction of either proinflammatory or anti-inflammatory trained innate immunity may provide novel approaches for long-lasting acceptance of HSC and solid organ transplants.
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