The future clinical implications of trained immunity

ABSTRACT

Introduction

Trained Immunity (TI) refers to the long-term modulation of the innate immune response, based on previous interactions with microbes, microbial ligands, or endogenous substances. Through metabolic and epigenetic reprogramming, monocytes, macrophages, and neutrophils develop an enhanced capacity to mount innate immune responses to subsequent stimuli and this is persistent due to alterations at the myeloid progenitor compartment.

Areas covered

The purpose of this article is to review the current understanding of the TI process and to discuss its potential clinical implications in the near future. We address the evidence of TI involvement in various diseases, the currently developed new therapy, and discuss how TI may lead to new clinical tools to improve existing standards of care.

Expert opinion

The state of the art in this domain has made considerable progress, linking TI-related mechanisms in multiple immune-mediated pathologies, starting with infections to autoimmune disorders and cancers. As a relatively new area of immunology, it has seen fast progress with many of its applications ready to be investigated in clinical settings.

KEYWORDS:

• Atherosclerosis
• cancer
• inflammation
• trained immunity
• transplantation
• vaccination

1. Trained immunity

The classical innate immune response at a cellular level starts with Pattern Recognition Receptors (PRRs) [1], proteins capable of detecting various stimuli classified into Damage Associated and Pathogen Associated Molecular Patterns (DAMPs and PAMPs) [2]. Their recognition leads to the activation of pro-inflammatory pathways, such as the canonical NF-kB pathway, which induces the production of cytokines and chemokines, increased cell survival, and immune cell differentiation [3]. After an initial stimulation of certain PRRs, the functional status of innate immune cells is altered, with specific epigenetic marks and changes in the cellular metabolism, that persist even after the initial immune response is over. This will be further referred to as trained immunity (TI) and cells that possess this phenotype show a more potent immune response upon re-stimulation with the same or different pathogens [4,5] (Figure 1). Therefore, TI represents the memory of the innate immune response, characterized by a long-term effect of initial stimulations in which cells respond with a higher intensity to subsequent stimulation with DAMPs or PAMPs [6]. It is also a form of heterologous immunity strictly restricted to the innate branch of the immune response, in contrast to other adaptive mechanisms such as heterologous T-cell priming.

Figure 1. A simplified schematic representation of TI. The innate immune system (purple lines) is activated by engagement of PRRs, which is followed by downstream signaling pathways, such as the NF-kB pathway, leading to transcriptional activation of pro-inflammatory genes and release of cytokines and chemokines. On the first interaction (a) with a PAMP/DAMP, an interplay between metabolic and epigenetic changes induces the trained phenotype (blue lines). Upon the second interaction (b), a more potent cytokine response will be observed, due to the functional epigenetic reprogramming of the cell.

In the context of TI, the most extensively studied inducers are the Bacillus Calmette–Guérin (BCG) vaccine and β-glucan, isolated from Candida albicans. The BCG vaccine was extremely useful in TI research due to its widespread use against Mycobacterium tuberculosis infections, which resulted in significant amounts of clinical data and more opportunity for research in humans. Even though BCG presents a multitude of PAMPs, the TI phenotype was shown to be induced by the NOD2 receptor [7], which binds to muramyl dipeptide (MDP). The BCG-trained cells show increased aerobic glycolysis, oxidative phosphorylation, and glutaminolysis, changes induced through the Akt/mTOR/HIF1α pathway [8]. Associated epigenetic marks are represented by increased trimethylation of at the fourth lysine of the H3 histone (H3K4me3) and decreased H3K9me3 at the promoters of mTOR, HK2, PFKP, and GLS, with a synergistic effect of increased transcription for said genes, plus an increased H3K4me3 at TNFA and IL6 promoters and H3K27ac in multiple genes in AKT, EGFR, FGF, and VEGF pathways [9].

β-glucan is recognized by the Dectin-1 and Complement Receptor 3, which in turn activate the NF-kB pathway with all its associated effects [10]. A lot of similar findings regarding metabolic and epigenetic changes were described in β-glucan induced TI, such as the activation of the mTOR pathway, increased glycolysis, and H3K4me3 and H3K27Ac epigenetic marks [11].

The currently proposed mechanism for H3K4me3 involves the WDR5/MLL1 methyltransferase complex guided by immune priming long non-coding RNAs (IPLs) [12]. Following BCG vaccination, the levels of IPLs UMLILO and IPL-IL1, which direct H3K4me3 at IL-8 and IL-1β promoters, were increased in human neutrophils. The cells with a higher expression of IPLs also had an increased activity [13]. As for other methylation marks, the Lysine Methyltransferase Set7, which induces H3K4me1, was identified as a key driver of TI, and several polymorphisms in the SETD7 gene were associated with TI response change [14]. Another Methyltransferase that plays a significant role in TI response is G9a, responsible for H3K9 methylation, which is associated with transcriptional downregulation of the methylated region. G9a is downregulated in trained monocytes and its pharmacological inhibition promoted the TI phenotype [15].

The current knowledge about metabolic and epigenetic changes in TI were extensively reviewed previously [16–18]. In brief, the rate of metabolic processes in trained cells is changed in order to meet the new energetic requirements. More importantly, the accumulation of certain metabolites induces specific epigenetic changes in TI. In turn, these epigenetic programs lead to altered expression of metabolic enzymes, ultimately maintaining the same metabolic circuits. An example of such an interaction is the accumulation TCA cycle metabolites, fumarate, succinate, and malate, which lead to the inhibition of α-ketoglutarate-dependent demethylases. In trained cells, one of such demethylases – KDM5 – is inhibited, a process that is directly linked to increased H3K4me3 [19]. The accumulation of Acetyl-CoA due to increased glycolysis can increase the activity of acetyltransferases [20]. In trained cells, the increased level of nicotinamide adenine dinucleotide (NAD+) stimulates the activity of sirtuins, a group of enzymes with important effects on immune responses [21]. Additionally, the previously mentioned Set7 methyltransferase is necessary for the accumulation of TCA cycle metabolites.

The TI phenotype was also confirmed to be induced by PAMPs and DAMPs such as LPS, C. albicans [4], poly I:C, oxidized LDL, Lipoprotein A, and others [22–24]. Even though the trained phenotype was established in these cases, the pathways involved and the metabolic changes can be different. For example, in β-glucan induced TI, oxidative phosphorylation is reduced, unlike the case with BCG. Similarly, Reactive Oxygen Species (ROS) production is increased in BCG and oxLDL training, while unchanged upon stimulation with β-glucan [22]. However, stimuli such as LPS [25], C. albicans [26], or poly I:C [27] can also induce immune tolerance, a phenomenon that can be viewed as the opposite of training. The shift between training and tolerance was shown to be dose-dependent [23]. These dual effects of stimuli may also be time dependent: despite a broad association of sepsis with post-septic immunosuppression because of innate immune tolerance, in a polymicrobial sepsis mouse model, bone marrow monocytes had a TI phenotype and depicted higher innate immune responses at 3 months after sepsis [28]. An interesting finding is that while trained cells can accumulate lactate, it was shown to posses anti-inflammatory effects on PBMCs, Monocytes [29] and Macrophages [30] through histone lactylation. This highlights that the mechanisms of immune training and tolerance are interlinked in a complex interaction between metabolic and epigenetic factors.

Some effects of the TI protection were confirmed to last from 3 months up to 1 year [31], and a nonspecific protective effect of the BCG vaccine against other infections potentially even lasts up to 5 years [32]. The presence of a long-term effect is due to the involvement of Hematopoietic Stem Cells (HSCs), a finding initially confirmed in mice as β-glucan was able to induce a shift toward myelopoiesis and metabolic changes in HSCs [33]. A later study confirmed similar findings in BCG-vaccinated healthy volunteers, highlighting the importance of HNF1A and HNF1B as Transcription Factors in BCG-induced TI and proposed S100A12, CCL20, and CCL23 as predictive serum biomarkers for TI [34]. Of note, the capacity to develop TI was observed in other cell types, such as fibroblasts, microglial cells [35], epithelial [36], and even smooth muscle cells [37], most likely due to their expression of PRRs.

The TI effect can play dual roles in the pathophysiology of immune-mediated diseases, the understanding of which is likely to bring new avenues for personalized medicine approaches in the future. On the one hand, harnessing TI to prevent or treat infections and immunodeficiencies or to promote anti-tumor processes is a strategy for TI-deficient states. On the other hand, the hyperinflammatory states that are associated with TI contribute to autoimmune and autoinflammatory diseases (Figure 2). Subject to future research findings, the knowledge on TI will provide us with tools to help in the prevention and therapy of immune-mediated diseases where TI is either defective or exacerbated. In the following sections, we will review the evidence that supports the implications of TI in clinical situations and outline future directions of development toward practical clinical tools based on TI.

Figure 2. Implications of TI in disease and possible TI-based interventions. In some pathology, the immune response is skewed – for example in Autoimmune disorders or Atherosclerosis, a therapeutic goal would be to generally reduce the reactivity of the immune system. Immunosuppression is a useful tool in transplantation in order to avoid rejection. A boost in immunity is favorable in cancer and potentially immunodeficient states. In the case of vaccination, TI usually acts as a nonspecific protective effect, but immunomodulation can also be an important tool to combat specific diseases.

2. Vaccination

An important body of research addressing the clinical relevance of TI is directed toward the development and improvement of vaccines. By inducing TI, it is possible to ensure a stronger innate immune response that will offer a better protection against the targeted pathogen and cross-protection against other infections. The heterologous effects of vaccines have been previously reviewed [38,39], with most data available for the BCG vaccine.

Besides the expected effect against tuberculosis, the BCG vaccine was shown to possess a protective role against C. albicans [40], S. aureus [41], malaria [42], as well as viral infections like herpes [43], yellow fever [9], HPV [44] and a general protection against acute upper [45] and lower respiratory tract infections [46]. These effects can be explained by both innate and adaptive heterologous immunity; however, the cross-protective effect of BCG was observed in T- and B-cell-deficient mice [7], confirming TI as a key player. An unexpected effect of the BCG vaccination, which highlights its immunomodulatory effects and the complexity of immune interactions, is the protection against Type I Diabetes and Multiple Sclerosis, currently explained by TNF induced autoreactive T cell death [47].

The use of BCG as a vector for recombinant vaccines started over 30 years ago and has shown great promise in multiple studies. Its use presents high interest due to its good safety profile and low manufacturing cost. VPM1002 is a recombinant BCG vaccine obtained by replacing the C gene with hly, encoding listeriolysin. It is a potential candidate to replace its predecessor as an improvement, and is currently in a phase 3 clinical trial for Tuberculosis prevention in infants (NCT04351685). Another recombinant vaccine that presents high interest is MTBVAC, a live attenuated M. tuberculosis vaccine, obtained through deletion of the phoP and fadD26 genes. MTBVAC is also in phase 3 trials for TB treatment (NCT04975178), and initial studies suggest that it has a significantly higher efficacy compared to BCG [48]. When tested for TI effects, MTBVAC induced training in human monocytes, with higher pro-inflammatory cytokine release upon re-stimulation, metabolic reprogramming, H3K4me3 at TNFΑ and IL6 gene promoters, and in mouse models, cross-protection against S. pneumoniae was confirmed [49].

The development of rBCG vaccines is an approach currently used in respiratory viral infections such as human Respiratory Syncytial Virus (hRSV) and Metapneumovirus (hMPV). After multiple unsuccessful attempts in the development of vaccines against hRSV, the rBCG vaccines that express the nucleoprotein (N) or the M2-1 protein have shown good results. rBCG-N-hRSV passed a phase 1 clinical trial, showing an adequate immune response and no safety issues reported 30 days post-vaccination [50]. The rBCG-P (hMPV phosphoprotein) vaccine is still in preclinical phase and was shown to induce protective immunity in mice [51]. In HIV research, multiple strains of rBCG and combinations were developed that produce a CD8 + T immune response in mice. A recombinant vaccine based on MTBVAC to combat HIV-1 has also been reported [52].

In 2020, the idea that the BCG vaccine might have a protective role against COVID-19 pandemic was proposed [53]. At the same time, another group showed that after accounting for several confounders, the epidemiological data does not support this hypothesis [54]. A definitive answer regarding this question is expected soon as multiple clinical trials studying BCG as a prevention measure against COVID-19 are approaching their final phase (NCT04348370, NCT04327206, NCT04328441, NCT04379336, NCT04417335). VPM1002 is similarly investigated for COVID-19 prevention (NCT04439045, NCT04387409, NCT04435379). A recent study finds that influenza vaccination has immunomodulatory/suppressive effects and changes the response to SARS-CoV-2 and other viral stimuli [55]. Whether this effect plays a positive or negative role in the severity of Covid-19 is still a subject for discussion and confidently asserting that it is a challenge. The idea that the immune response in coronavirus infections is pathologically upregulated is also supported by the fact that MERS-CoV infected endothelial cells show signs of trained immunity, such as increased proinflammatory cytokine expression, downregulated OXPHOS, accumulation of ROS, and epigenetic reprogramming [56].

The TI model can also explain the efficacy of multibacterial formulas and bacterial lysates in nonspecific respiratory tract infections (RTIs). MV-130, a heat-inactivated bacterial mix, was confirmed to induce TI in monocytes and HSCs, through the activation of the mTOR pathway and provide cross-protection against viral infections in mice [57], including SARS-CoV-2 [58]. The bacterial lysate, OM-85 has similarly shown TI cross-protection properties against murine coronavirus, triggering a stronger type 1 IFN response [59]. Multiple lysate formulas are available, with a considerable number displaying nonspecific protection in RTIs [60].

As research regarding TI continues, it becomes clearer that evaluating and formulating guidelines for vaccine use that also account for nonspecific effects and cross-protection from other pathogens is important. This approach allows to significantly improve existing vaccines by considering their immunomodulatory effects, and for diseases in which an efficient vaccine has not yet been developed, the nonspecific protection developed by innate immune training can become a temporary solution. As reviewed elsewhere [61], the addition of an ‘amplifier,’ which represents an epigenetic and/or metabolic modulator, to the classic vaccine formulas could increase their efficacy. The vaccines that contain TI inducers are also referred to as Trained Immunity-based Vaccines [62].

3. Cancer

Intravesical BCG vaccine administration was used to treat non-muscle-invasive bladder cancer (NIBC) since the 1960s [63]. The intravesical application of BCG is seen as the first immunotherapy of cancer. It is currently part of the standard of care in the treatment and recurrence prevention of stage I and II NIBC, alongside gemcitabine, which has similar effectiveness [64,65]. The primary mechanism driving this involves important local effects of BCG on the urothelium and, more specifically, the tumor microenvironment. BCG possesses tropism to fibronectin [66], found mostly on damaged urothelium, where the cancer is located to produce an immune response. The resolution of the carcinoma requires the involvement of local macrophages, which are activated through the MyD88/NF-kB signaling pathway [67]. The resulting cytokine release is later followed by recruitment of CD8+ T cells, NK cells, neutrophils, macrophages, and other cell types [68]. Besides the local interactions, important systemic effects suggestive of TI have been noted even since the 90s, when a pro-inflammatory phenotype was observed in blood circulating monocytes after BCG instillations [69]. Furthermore, the second and following instillations elicit a stronger immune response with a higher release of urinary IL-1, IL-2, IL-6, and TNF [70]. The currently proposed TI mechanism in BCG immunotherapy is that the systemic training effects will result in a trained population of circulating monocytes and macrophages, following an increased release of cytokines that will facilitate the activation of T cells [71].

TI is strongly linked to autophagy, since β-glucan training induces transcription of autophagy-related genes and the inhibition of autophagy blocks trained immunity in vitro in response to BCG or β-glucan [72]. When examining SNPs in autophagy-related genes, rs3759601 in ATG2B was shown to change the TI response without affecting the initial immune response, and more importantly, the SNP correlates with NIBC progression and recurrence [72]. This evidence shows that TI plays a role in BCG therapy and an ongoing clinical study (NTC03091660) set to investigate the outcome of intradermal BCG priming before the intravesical therapy should provide more information regarding the importance of the systemic TI effect.

In support of the TI-induced protection against cancer, it can also be mentioned that BCG immunization reduces the risk of lung cancer [73], leukemia [74], and lymphomas [75], and intralesional BCG administration is also used for a long time as therapy in stage III–IV for melanomas. Multiple trials testing the use of BCG as an adjuvant for chemotherapy or tumor cell vaccines have shown good results [76].

A field that currently shows great promise is nanobiologic therapy. It allows for more precise targeting of specific tissues in order to avoid unwanted side-effects. Multiple methods to deliver BCG with nanoparticles were explored. One of them is the use of BCG cell wall skeleton encapsulated in lipid particles (BCG-CWS) [77], a drug that was also proposed as an adjuvant for other types of cancer [78]. MTP10-HDL is derived from muramyl dipeptide, capable of inducing TI, with apolipoprotein A1 as a carrier molecule, which is used for hematopoietic stem cells, multipotent progenitors, and myeloid progenitors targeting. MTP10-HDL was able to successfully induce TI in innate immune cells and inhibit tumor growth in a mouse melanoma model [79].

4. Inflammatory and autoimmune disorders

The fact that TI can lead to inflammatory consequences has best been illustrated to date in the context of atherosclerosis. Older evidence in rodents already showed that BCG administration is positively correlated to atherosclerosis [80], while in vitro training of monocytes with beta-glucan induced transcriptional activation of proinflammatory and proatherogenic genes [4]. After the discovery that oxLDL can train human primary monocytes, the role of TI in the development of atherosclerosis received more attention. Known hallmarks of TI were identified in monocytes such as the activation of the mTOR pathway [81] or characteristic epigenetic and metabolic changes [82]. Many studies show that a high-fat diet, which is a known risk factor for atherosclerosis, induces a pro-inflammatory phenotype in monocytes and HSCs [83], which plays an important role in the progression of atherosclerotic lesions [84,85]. Similarly, an increased level of glucose in diabetes, a pathology often associated with atherosclerosis, leads to increased glycolysis and a pro-inflammatory phenotype in monocytes, an effect driven by Runt-related transcription factor (RUNX) 1 [86]. As it was already mentioned, the involvement of HSCs increases the effective duration of immune cell training and this was confirmed in the context of atherosclerosis. When bone marrow from diabetic mice was transplanted to atherosclerosis prone normoglycemic ones, the progression of plaques was significantly accelerated [87].

In addition to the implications for atherosclerosis and cardiovascular diseases, TI can predispose and aggravate other inflammatory and autoimmune diseases as well. Current circumstantial evidence has already pointed out several features of innate immune responses in autoinflammatory and autoimmune conditions, which are shared with trained immunity, also reviewed by Arts et al. [88]. Several such examples are outlined below.

Rheumatoid arthritis (RA), the most common autoimmune disease, is characterized by circulating autoantibodies and synovitis with progressive cartilage and bone damage. Anti-parietal cell antibodies often precede clinical manifestations and were shown to induce TI phenotype in monocytes [89]. The hyperinflammatory status of circulating monocytes was established in several studies, which confirmed an increased cytokine response and hypermetabolic status with increased glycolysis induced by STAT3 [90,91]. In contrast, another study reported that H3K4me3 was not increased at TNFA and IL6 promoters in RA patients [92]. The targeting of P13K/Akt/mTOR pathway in RA is already a topic of scientific research for some time. Rapamycin was tested in humans and combined with conventional RA therapy reduces the disease activity [93] and its combination in nanoparticles also showed good results in RA mice models [94].

In Systemic Lupus Erythematosus (SLE), besides the well-known presence of autoantibodies and autoreactive T cell activity, more and more evidence indicates the involvement of innate immunity as a major player in the complex interactions characteristic for this disease [95]. Even though the link between SLE and TI has not been formally established, certain similar features point in that direction, such as the mTOR pathway activation [96] or the epigenetic profile of monocytes from patients with SLE compared to trained monocytes (enrichment in H3K4Me3 [97] and H4 acetylation [98]). In addition, functional reprogramming at the HSCs level has been reported based on transcriptomic assessment of bone marrow samples in patients with SLE and controls, suggestive of myeloid skewing similarly to a TI signature [99].

Gout is an inflammatory disease caused by deposits of Monosodium Urate (MSU) crystals in joints and is usually preceded by increased serum urate concentrations. MSU is a DAMP that triggers an immune response dependent on the NLRP3 inflammasome activation [100]. Interestingly, urate was reported to depict antitumoral properties [101] and was proposed as an adjuvant for the BCG vaccine [102]. Soluble urate was also shown to alter the transcription and proinflammatory cytokine production profile in primary human peripheral mononuclear cells [103,104] by activating the Akt/mTOR pathway [105]. Conversely, conflicting results evoking anti-inflammatory properties of soluble urate were reported in monocytes [106], which could be explained with differences in methodology.

5. Transplantation

Pharmacological immunosuppression is an important tool that greatly improved the outcomes of organ transplantation, but its long-term use is associated with major adverse effects. Because of that, multiple alternatives are currently being explored. Targeting TI is an important avenue to explore, due to the fact that macrophages play a crucial role in allograft rejection by initiating the CD4+ T cell response [107].

As previously mentioned, precise targeting of immune cells is possible with nanobiologics, so multiple attempts at developing immunosuppressants were made using this technology. These include H3K27 demethylase inhibitor GSK-J4 or inhibition of ROS production with diethyl malonate, while the most promising avenue to explore was mTOR pathway inhibition with rapamycin [108]. In 2018, a combination of mTORi-HDL and TRAF6i-HDL was developed and tested on mice with promising results, inhibiting TI in macrophages, and reducing organ transplant rejection [109].

6. Future clinical implications of trained immunity

To summarize, TI represents the adaptation of innate immune cells upon encounter with a stimulus that elicits an innate response and also leads to persistent metabolic and epigenetic alterations. This facilitates a faster and stronger inflammatory response to a subsequent challenge with either the same or a different stimulus. The long-term persistence of TI is ensured by the presence and the mitotic heritability of these epigenetic changes at the level of progenitor cells. Different stimuli may rewire cells to different epigenetic and transcriptional programs, which may variably impact the host, depending on their own susceptibility or underlying pathology. In the coming years, the further characterization of these trained immunity features will probably represent an important scientific goal before developing clinically valid and useful TI-based strategies. Relevant open questions to be clarified include which stimuli can lead to TI, how fast is TI induced or erased, how long does TI last, and how is TI passed on during cell division or trans generationally. Fundamental knowledge in this field is, on the one hand, likely to inform the development of clinical tools contributing to personalized preventive strategies and risk assessment, while, on the other, lead to therapeutic developments aimed at either inducing or inhibiting trained immunity.

Therapeutically, TI can be targeted for inhibition at several levels – PRRs, mTOR pathway, metabolic, and epigenetic levels. Rapamycin is in use as an immunosuppressor since 1999, and the development of nanobiologics will likely lead to a new generation of drugs in this class. Precise targeting of myeloid cells is expected to reduce the toxic systemic effects of mTOR inhibitors. In a similar fashion, but with the opposite effect, the MTP10-HDL nanobiologic is a candidate drug to enhance the immune response. These two drugs may become important opposing therapies aimed at modulating trained immunity. Moreover, epigenetic marks could be targeted through enzymes, such as methyltransferases and demethylases. An example of such drugs is 3-Deazaneplanocin A, which can inhibit H3K27me3 with effects that are in line with our expectations, such as inducing apoptosis [110], reduction of cellular growth, and migration [111]. These might prove an important alternative to explore but are most likely to be inferior, due to off-target secondary effects. The alternative epigenetic approach is to target IPLs that participate in the setting of specific histone marks associated with TI.

The immune boosters may improve the clinical outcomes in all types of cancer and allow more therapeutic options. For example, the use of general anesthesia is avoided in oncologic surgeries when possible due to its immunosuppressive effect [112,113] and canceling this effect presents high clinical interest. Immunotherapy is an avenue that is currently widely explored in cancer and, if nanobiologics prove successful in humans, they will become a feasible solution that might partially replace chemotherapy. A general boost in immunity could also prove useful in elderly as prophylaxis [114].

Along the same line, it can be envisioned that the vaccination process will be impacted by TI-based approaches, as more clinical data on the efficiency of recombinant vaccines and immune boosters will be released. Various TI inducing DAMPs and PAMPs can be used as adjuvants, offering a major improvement over existing vaccines.

Assessing the TI status in patients can provide useful information to guide medical decisions. The identification of reliable biomarkers for TI may potentially help stratify patients into risk groups for strong or weak immune responses in the context of different pathologies. Feasible combinations of biomarkers may include cytokines, chemokines, or other inflammatory proteins (such as S100A12, CCL20, and CCL23 [34]), as well as relevant IPLs (UMLILO and IPL-IL1 [13]) and quantification of specific histone mark enrichment at relevant gene regions (H3K4Me3).

Assessing the TI status can be useful in order to establish if it is a good target for medication and further monitor the patient’s response. In inflammatory disorders, signs of TI often precede clinical symptoms [83,90]; therefore, a TI dysregulation can be indicative of a disease onset or increased susceptibility.

In addition to markers of TI, predictive models for the cytokine production capacity have been reported, and genetic variation was shown to be the main determinant [115]. Several polymorphisms that influence the immune response and some that are specifically associated to TI have been previously mentioned [14]. This suggests that integrating genetic susceptibility variants into stratification tools for people that may show variable TI phenotypes may prove useful for risk management strategies.

In conclusion, the data summarized in this review illustrate the involvement of TI in important pathophysiological processes and show the significant advances in targeting TI to alter disease outcome. Further knowledge into TI mechanisms and sources of variability will make it increasingly likely to translate this knowledge into clinically relevant tools for prevention and therapeutics.

7. Expert opinion

Over a decade ago, the concept of trained immunity was introduced and expanded our understanding of immunological memory. The TI phenotype is linked to immunometabolic and epigenetic changes that determine the transcriptional program of trained cells upon reinfection or subsequent challenge with sterile stimuli. These changes are passed on from Hematopoietic Stem Cells to circulating monocytes and macrophages, ensuring a systemic and relatively long-lasting effect. It is still difficult to determine the extent to which an in vivo innate immune response is the result of TI, and this is the subject of current research. Some degree of immunomodulation due to TI processes is most likely part of every immune response since hosts encounter pathogens at every step, with every interaction triggering an immune response, which in turn influences subsequent responses. The molecular markers of TI are seen in many cell types, and the involved pathways are mostly metabolic, part of the normal function of any cell. From this perspective, TI can also be seen as an evolutionarily acquired additional effect of the accelerated metabolism driven by enhanced glycolysis in immune cells. Therefore, it is important to recognize that many known pathways are interlinked and can drive additional, including long-term effects. The metabolic changes seen in TI, might also have additional roles for nonimmune cells. Understanding the epigenetic interactions that drive phenomena like TI is an important step for research in the fields of biology and medicine.

In the coming years, one focus will be implementing the approaches to modulate TI for precise and immediate gains in the existing therapeutic strategy: induction of TI in fighting infection or cancer and limitation of TI in inflammatory or autoimmune diseases. The most obvious and most likely fastest implementation is the addition of nonspecific immune boosters to existing vaccine formulas.

In our opinion, the end goals of research in this field are heavily tied into personalized medicine. This will generally involve two directions – firstly, the creation of necessary tools to assess the TI signatures or susceptibility, and secondly, the development of a new generation of immunomodulatory drugs used independently or in combination with other medication to adjust immune responses.

With regard to the former, we envisage that genetic and non-genetic factors that are associated with TI will contribute to the prediction of individuals at risk of developing heightened TI states or individuals who will have a lower degree of developing TI in response to certain stimuli. For the latter, nanobiologic drugs targeting myeloid cells already show great potential since TI involves important metabolic pathways, and avoiding off-target side-effects is of key importance. More therapeutic options to explore are drugs that influence chromatin accessibility. Further advancement into epigenetic-based therapeutic modulation of TI will likely be brought by RNA-based approaches that can specifically target certain immune priming long non-coding RNAs that orchestrate epigenetic regulation at specific loci.

In the coming years, we expect that TI signatures associated with more stimuli and more disease phenotypes will be characterized allowing us to refine our search for specific modulation approaches. As the results of important clinical studies will be released, therapeutic development will move into the next phases, while trained immunity-based vaccines and adjuvants will be an important preventive tool for limiting infection, especially in situations where no specific vaccines are available.

Article highlights

• Trained innate immunity refers to long-term modifications of the immune cell reactivity through epigenetic and metabolic changes. It is induced by pattern recognition through mTOR signaling and leads to enhanced subsequent innate immune responses.

• Therapeutic strategies targeting TI can lead to the development of new classes of immunostimulant and immunosuppressive drugs, useful in various clinical settings. The use of TI-based drugs has already been studied in the context of cancer, rheumatoid arthritis, and transplantology.

• Existing vaccines and non-specific immune boosters can lead to heterologous protection, which is explained within the TI framework. It may also contribute to tumor cell removal by increasing the activity of T cells, which is reflected in the effectiveness of BCG in different types of cancer.

• Maladaptive activation of the pathways involved in TI is proposed to be another part of the pathophysiology of autoimmune and inflammatory disorders. The effectiveness of mTOR inhibitors in these pathologies can be partially explained by the inhibition of TI. More efficient strategies that target TI may prove useful as chronic anti-inflammatory medication.

• The use of HDL nanoparticles allows for precise targeting of hematopoietic stem cells. Drugs that both activate and suppress TI, such as MTP10-HDL and mTORi-HDL, show great promise in animal studies.

Declaration of interest

L Joosten declares that he is the scientific founder of Trained Therapeutix Discovery (TTxD) and owns two patents related to trained immunity. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This work is supported by a Competitiveness Operational Program Grant of the Romanian Ministry of European Funds (HINT, ID P_37_762; MySMIS 103587). T.O.C. is supported by a grant from the Ministry of Research, Innovation and Digitization, CNCS/CCCDI – UEFISCDI, project number PD-2019-0802, within PNCDI III (Romania).