Divergent metabolic rewiring shapes altered innate immunity.

How are airborne allergens remembered by the immune system?

The onset and the termination of innate immune response must be tightly regulated to maintain homeostasis and prevent excessive inflammation, which can be detrimental to the organism, particularly in the context of sepsis. Endotoxin tolerance and compensatory anti-inflammatory response syndrome (CARS) describe a state of hypo-responsiveness characterized by reduced capacity of myeloid cells to respond to inflammatory stimuli, particularly those initiated by bacterial lipopolysaccharide (LPS). To achieve endotoxin tolerance, extensive reprogramming otherwise termed as “innate immune training”, is required that leads to both modifications of the intracellular components of TLR signaling and also to alterations in extracellular soluble mediators. Non-coding RNAs (ncRNAs) have been recognized as critical regulators of TLR signaling. Specifically, several microRNAs (miR-146, miR-125b, miR-98, miR-579, miR-132, let-7e and others) are induced upon TLR activation and reciprocally promote endotoxin tolerance and/or cross tolerance. Many other miRNAs have been also shown to negatively regulate TLR signaling. The long non-coding (lnc)RNAs (Mirt2, THRIL, MALAT1, lincRNA-21 and others) are also altered upon TLR activation and negatively regulate TLR signaling. Furthermore, the promotion or termination of myeloid cell tolerance is not only regulated by intracellular mediators but is also affected by other TLR-independent soluble signals that often achieve their effect via modulation of intracellular ncRNAs. In this article, we review recent evidence on the role of different ncRNAs in the context of innate immune cell tolerance and trained immunity, and evaluate their impact on immune system homeostasis.

Trained innate immunity and resistance to Mycobacterium tuberculosis infection

Will bacille Calmette-Guerin immunization arrest the COVID-19 pandemic?

Conventionally, immunity in humans has been classified as innate and adaptive, with the concept that only the latter type has an immunological memory/recall response against specific antigens or pathogens. Recently, a new concept of trained immunity (a.k.a. innate memory response) has emerged. According to this concept, innate immune cells can exhibit enhanced responsiveness to subsequent challenges, after initial stimulation with antigen/pathogen. Thus, trained immunity enables the innate immune cells to respond robustly and non-specifically through exposure or re-exposure to antigens/infections or vaccines, providing enhanced resistance to unrelated pathogens or reduced infection severity. For example, individuals vaccinated with BCG to protect against tuberculosis were also protected from malaria and SARS-CoV-2 infections. Epigenetic modifications such as histone acetylation and metabolic reprogramming (e.g. shift towards glycolysis) and their inter-linked regulations are the key factors underpinning the immune activation of trained cells. The integrated metabolic and epigenetic rewiring generates sufficient metabolic intermediates, which is crucial to meet the energy demand required to produce proinflammatory and antimicrobial responses by the trained cells. These factors also determine the efficacy and durability of trained immunity. Importantly, the signaling pathways and regulatory molecules of trained immunity can be harnessed as potential targets for developing novel intervention strategies, such as better vaccines and immunotherapies against infectious (e.g., sepsis) and non-infectious (e.g., cancer) diseases. However, aberrant inflammation caused by inappropriate onset of trained immunity can lead to severe autoimmune pathological consequences, (e.g., systemic sclerosis and granulomatosis). In this review, we provide an overview of conventional innate and adaptive immunity and summarize various mechanistic factors associated with the onset and regulation of trained immunity, focusing on immunologic, metabolic, and epigenetic changes in myeloid cells. This review underscores the transformative potential of trained immunity in immunology, paving the way for developing novel therapeutic strategies for various infectious and non-infectious diseases that leverage innate immune memory.

Vaccines such as Vaccinia or BCG have non-specific effects conferring protection against other diseases than their target infection, which are likely partly mediated through induction of innate immune memory (trained immunity). MVA85A, a recombinant strain of modified Vaccinia Ankara (MVA), has been suggested as an alternative vaccine against tuberculosis, but its capacity to induce positive or negative non-specific immune effects has not been studied. This study assesses whether Vaccinia and MVA are able to induce trained innate immunity in monocytes. Human primary monocytes were primed in an in vitro model with Vaccinia or MVA for 1day, after which the stimulus was washed off and the cells were rechallenged with unrelated microbial ligands after 1week. Heterologous cytokine responses were assessed and the capacity of MVA to induce epigenetic changes at the level of cytokine genes was investigated using chromatin immunoprecipitation and pharmacological inhibitors. Monocytes trained with Vaccinia showed significantly increased IL-6 and TNF-α production to stimulation with non-related stimuli, compared to non-trained monocytes. In contrast, monocytes primed with MVA showed significant decreased heterologous IL-6 and TNF-α responses, an effect which was abrogated by the addition of a histone methyltransferase inhibitor. No effects on H3K4me3 were observed after priming with MVA. It can be thus concluded thatVaccinia induces trained immunity in vitro, whereas MVA induces innate immune tolerance. This suggests the induction of trained immunity as an immunological mechanism involved in the non-specific effects of Vaccinia vaccination and points to a possible explanation for the lack of effect of MVA85A against tuberculosis.

For decades, innate immune cells were considered unsophisticated first responders, lacking the adaptive memory of their T and B cell counterparts. However, mounting evidence demonstrates the surprising complexity of innate immunity. Beyond quickly deploying specialized cells and initiating inflammation, two fascinating phenomena - endotoxin tolerance (ET) and trained immunity (TI) - have emerged. ET, characterized by reduced inflammatory response upon repeated exposure, protects against excessive inflammation. Conversely, TI leads to an enhanced response after initial priming, allowing the innate system to mount stronger defences against subsequent challenges. Although seemingly distinct, these phenomena may share underlying mechanisms and functional implications, blurring the lines between them. This review will delve into ET and TI, dissecting their similarities, differences, and the remaining questions that warrant further investigation.

Recent research has increasingly highlighted the adaptive characteristics of the innate immune system, revealing its capacity for a heterologous memory of previous infections. Allergen-specific immunotherapy (AIT) has demonstrated that innate immune cells, such as monocytes, macrophages, and natural killer (NK) cells, can provide protection against specific diseases even in the absence of lymphocyte support. The mechanisms underlying innate host defense and the immunological memory of adaptive immunity differ significantly in terms of cellular populations and molecular pathways. Prototypical innate immune cells, including NK cells and monocytes/macrophages, contribute to the sustained heightened state of innate immunity known as "trained immunity," which enhances resistance to secondary infections. Trained immunity is typically initiated through the engagement of pattern recognition receptors (PRRs) by microbial structures, suggesting that vaccines designed to induce trained immunity should incorporate appropriate PRR ligands. This approach not only offers protection against reinfection in a manner independent of T and B cells but also promotes nonspecific epigenetic reprogramming that enhances immune responses. For instance, Bacillus Calmette-Guérin (BCG) vaccination has been linked to long-lasting immune modifications associated with a non-specific immune response to various infections, characterized by heterologous T helper 1 (Th1) and Th17 responses. Emerging evidence indicates that heat-killed Mycobacterium manresensis can induce trained immunity in vitro, although its effectiveness in vivo remains to be fully established. This highlights the potential of novel strategies in vaccine development, particularly through the lens of trained immunity. The concept of trained immunity-based vaccines (TIbV) presents a paradigm shift in immunization strategies, as these vaccines can elicit broad-spectrum protection against a variety of pathogens. By leveraging the principles of trained immunity, TIbV can enhance both innate and adaptive immune responses, potentially improving the efficacy of conventional vaccines and offering new avenues for immunotherapy.The integration of trained innate immunity into vaccine development holds significant promise for enhancing immune protection against infectious diseases. By harnessing the principles of trained immunity, these innovative vaccines can enhance innate immune responses, potentially improving protection against a wide range of infectious diseases and contributing to better public health outcomes.

The human toll-like receptor 4 (TLR4) pathway is activated in response to lipopolysaccharide (LPS), and subsequent signal transductions lead to the production of cytokines such as tumor necrosis factor-α (TNF-α) by innate immune cells. Defects in innate immune response may contribute to the overproduction of TNF-α leading to systemic inflammation and diseases. Thus, the innate immune response needs to be tightly regulated by elaborate mechanisms to control its onset and termination. LPS tolerance is a state of hyporesponsiveness to subsequent LPS challenge and is achieved by monocytic cells after prolonged exposure to LPS. In this report, kinetics of endotoxin-responsive microRNAs expression analysis revealed a unique pattern of gradual increase for miR-146a starting 4 h after LPS stimulation in THP-1 cells and continued up to 35-fold over 24 h. Conversely, TNF-α increased up to 4 h and then decreased gradually implicating a negative correlation with miR-146a progression. The characteristic up-regulation of miR-146a toward subsequent LPS challenge in THP-1 cells was studied. Strikingly, microRNA expression analysis during the tolerized state of THP-1 cells showed only miR-146a overexpression suggesting its important role in LPS tolerance. In addition, LPS tolerance was dependent on a LPS-priming dose and associated miR-146a up-regulation. LPS-tolerized cells were observed to regain responsiveness in TNF-α production 22 h after LPS removal correlating with a decrease in miR-146a level. Transfection of miR-146a into THP-1 cells mimicked LPS priming, whereas transfection of miR-146a inhibitor largely abolished LPS tolerance. Thus our studies demonstrated that miR-146a is critical for the in vitro monocytic cell-based endotoxin tolerance.

Gestational age-dependent immune intolerance at the maternal-fetal interface might be a contributing factor to placental pathology and adverse pregnancy outcomes. Although the intrauterine setting is highly choreographed and considered to be a protective environment for the fetus, unscheduled inflammation might overwhelm the intrauterine milieu to cause a cascade of events leading to adverse pregnancy outcomes. The old paradigm of a sterile intrauterine microenvironment has been challenged, and altered microflora has been detected in gestational tissues and amniotic fluid in the absence of induction of significant inflammation. Is there a role for endotoxin tolerance at the maternal-fetal interface? Endotoxin tolerance is a phenomenon in which tissues or cells exposed to the bacterial product, particularly lipopolysaccharide, become less responsive to subsequent exposures accompanied by decreased expression of pro-inflammatory mediators. This could also be related to trained or experienced immunity that leads to the successful outcome of subsequent pregnancies. Adaptation to endotoxin tolerance or trained immunity might be critical in preventing rejection of the fetus by the maternal immune system and protecting the fetus from excessive maternal inflammatory responses to infectious agents; however, to date, the exact mechanisms contributing to the establishment and maintenance of tolerance at the maternal-fetal interface remain incompletely understood. There is now extensive evidence suggesting that microRNAs (miRNAs) play important roles in the maintenance of a healthy pregnancy. miRNAs not only circulate freely in extracellular fluids but are also packaged within extracellular vesicles (EVs) produced by various cells and tissues. The placenta is a known, abundant, and transient source of EVs; therefore, our proposed model suggests that repeated exposure to infectious agents induces a tolerant phenotype at the maternal-fetal interface mediated by specific miRNAs mostly contained within placental EVs. We hypothesize that impaired endotoxin tolerance or failed trained immunity at the maternal-fetal interface will result in a pathological inflammatory response contributing to early or late pregnancy maladies.

Gene-selective epigenetic reprogramming and shifts in cellular bioenergetics develop when Toll-like receptors (TLR) recognize and respond to systemic life-threatening infections. Using a human monocyte cell model of endotoxin tolerance and human leukocytes from acute systemic inflammation with sepsis, we report that energy sensor sirtuin 1 (SIRT1) coordinates the epigenetic and bioenergy shifts. After TLR4 signaling, SIRT1 rapidly accumulated at the promoters of TNF-α and IL-1β, but not IκBα; SIRT1 promoter binding was dependent on its co-factor, NAD(+). During this initial process, SIRT1 deacetylated RelA/p65 lysine 310 and nucleosomal histone H4 lysine 16 to promote termination of NFκB-dependent transcription. SIRT1 then remained promoter bound and recruited de novo induced RelB, which directed assembly of the mature transcription repressor complex that generates endotoxin tolerance. SIRT1 also promoted de novo expression of RelB. During sustained endotoxin tolerance, nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme for endogenous production of NAD(+), and SIRT1 expression increased. The elevation of SIRT1 required protein stabilization and enhanced translation. To support the coordination of bioenergetics in human sepsis, we observed elevated NAD(+) levels concomitant with SIRT1 and RelB accumulation at the TNF-α promoter of endotoxin tolerant sepsis blood leukocytes. We conclude that TLR4 stimulation and human sepsis activate pathways that couple NAD(+) and its sensor SIRT1 with epigenetic reprogramming.

Memory formation, guided by microbial ligands, has been reported for innate immune cells. Epigenetic imprinting plays an important role herein, involving histone modification after pathogen-/danger-associated molecular patterns (PAMPs/DAMPs) recognition by pattern recognition receptors (PRRs). Such “trained immunity” affects not only the nominal target pathogen, yet also non-related targets that may be encountered later in life. The concept of trained innate immunity warrants further exploration in cancer and how these insights can be implemented in immunotherapeutic approaches. In this review, we discuss our current understanding of innate immune memory and we reference new findings in this field, highlighting the observations of trained immunity in monocytic and natural killer cells. We also provide a brief overview of trained immunity in non-immune cells, such as stromal cells and fibroblasts. Finally, we present possible strategies based on trained innate immunity that may help to devise host-directed immunotherapies focusing on cancer, with possible extension to infectious diseases.

Rheumatic diseases include a variety of autoimmune and autoinflammatory conditions that are characterised by musculoskeletal involvement and systemic disease. Both innate and adaptive immunity can contribute to the complex inflammatory processes that take part in the pathogenesis of these debilitating disorders. Over the past decade, studies have led to a paradigm-shift around the concept of immune memory, generating the knowledge that cells of the innate immune system can develop a de facto memory mediated by epigenetic reprograming and metabolic changes (trained immunity). Here we provide an overview of current data that describe features of trained immunity in rheumatic diseases. We link evidence on inflammatory mediators and cytokine production, immunometabolism and epigenetic regulation of immunological programs, and outline the fact that trained immunity could play mechanistic roles in rheumatic diseases such as gout, rheumatoid arthritis, systemic lupus erythematosus or systemic sclerosis. This review describes recent findings in several important rheumatic disorders and emphasizes changes in the functional program of innate immune cells that are reminiscent of a trained immune phenotype. Further assessment of trained immunity in rheumatic disease can provide targetable mechanisms that could potentially alter the disease symptomatology and evolution.

Background and Aims Allograft rejection is largely mediated by adaptive immune cells. Although innate immune cells are also involved, their role is less clear. We hypothesize that suppression of innate immune responses, particularly trained innate immunity, can contribute to graft tolerance in transplantation. Trained immunity refers to the long-term epigenetic and metabolic reprogramming of innate immune cells, which potentiates responses to secondary stimuli. Here, we studied the effect of blocking CD40-TRAF6 signaling in myeloid cells on trained immunity induction using in vitro stimulations, ChIP-sequencing and metabolic analyses. We evaluated the effect of myeloid CD40-TRAF6 inhibition on T cell responses in mixed lymphocyte reactions (MLR) and the effect of CD40-TRAF6 inhibitor (TRAF6i) nanobiologic (NB) treatment on allograft rejection in a murine heterotopic heart transplantation model. Method Human peripheral blood mononuclear cells (PBMCs) were stimulated for 24 h with heat-killed Candida albicans (HKCA), a well-described inducer of trained immunity, in presence or absence of TRAF6i, or RPMI medium (control) followed by 5 days of rest. Interleukin-6 (IL-6) and tumor necrosis factor (TNF) production upon restimulation with lipopolysaccharide (LPS) was assessed using ELISA. We performed ChIP-sequencing and metabolic analyses with Seahorse assays at day 6. To investigate the effect of TRAF6i on T cell responses to allogeneic stimuli, we performed 7-day MLR with HKCA- or HKCA+TRAF6i trained monocytes and CellTrace Violet-labeled naïve T cells. Proliferation and FOXP3 expression of naïve T cells was measured by flow cytometry. The effect of TRAF6i-NB on graft survival was assessed by injection of myeloid-directed TRAF6i-NB 0, 2 and 5 days post-transplantation in C57BL/6J mice heterotopically transplanted with BALB/c hearts, that did or did not receive pre-operative CTLA4-Ig treatment. Results TRAF6i treatment inhibited IL-6 and TNF production upon LPS restimulation of HKCA-treated PBMCs in vitro (Fig. 1A). ChIP-sequencing analysis and Seahorse technology respectively revealed that the epigenetic changes (Fig. 1B) and metabolic alterations (Fig. 1C) underlying trained immunity were prevented by TRAF6i. Induction of trained immunity in monocytes reduced differentiation of naïve T cells to FOXP3+CD4+ T cells upon allogeneic stimulation, which was partially reversed by TRAF6i treatment of monocytes (Fig. 1D). Treatment with TRAF6i-NB, combined with CTLA4-Ig, induced >100 days graft survival in 5 out of 6 C57BL/6J mice heterotopically transplanted with BALB/c hearts, while one graft was rejected at 99 days post-transplantation (Fig. 1E). Conclusion Inhibition of TRAF6 prevents trained immunity in monocytes at the functional, epigenetic and metabolic level. We show in vitro that inhibiting trained immunity has the effect of modulating T cell responses to allogeneic stimuli towards FOXP3+CD4+ T cell differentiation. Using a mouse heart transplant model, we show that the combination of TRAF6i and CTLA4-Ig treatment prevents allograft rejection in vivo. This study identifies CD40-TRAF6 signaling as a novel target to inhibit trained immunity and to promote allograft tolerance.

Introduction Obesity is the most prevalent modifiable risk factor for atherosclerotic cardiovascular disease and is characterized as a chronic inflammatory disease. Cells of the innate immune system, especially monocytes and macrophages, play a pivotal role in the various stages of atherosclerosis, although it still remains elusive why the strong inflammatory response persists in time. We recently showed that cells of the innate immune system, such as monocytes, can adopt a long-term immunological memory. Upon brief stimulation with atherogenic stimuli, monocytes demonstrate an enhanced long-term pro-inflammatory and pro-atherogenic phenotype. This is termed trained immunity and is mediated via epigenetic and metabolic reprogramming. The clinical relevance of these findings was verified in patients with symptomatic atherosclerosis, in which circulating monocytes showed a trained immune phenotype. Purpose As various adipose tissue-related particles, including pro-inflammatory cytokines and fatty acids, are capable of inducing trained immunity in vitro, we hypothesized that adipose tissue from obese subjects might induce training in peripheral monocytes, thereby contributing to the increased risk of atherosclerotic CVD in these patients. In line with this hypothesis, it is unclear whether chronic inflammation sustains after a previous period of obesity despite significant weight loss. Methods We obtained blood from 25 patients with obesity before and 6 months after bariatric surgery. Monocyte subsets and activation phenotype were studied using flow cytometry. Cytokine production capacity of isolated PBMCs was studied after ex vivo stimulation with several infectious and metabolic stimuli and we characterized isolated monocytes using transcriptomics. Next, we obtained visceral (VAT) and subcutaneous adipose tissue (SAT) biopsies from 10 patients. Using our established in vitro model for trained immunity, we co-incubated healthy human monocytes with the adipose tissue biopsies for 24 hours in a trans-well set-up. After 24 hours, the adipose tissue was removed and monocytes were rested. On day 6, the cells were re-stimulated for 24 hours with a second stimulus and cytokine production and the transcriptome of the macrophages was analyzed. Results Both SAT and VAT obtained from patients with obesity can induce a long-term memory in healthy human monocytes, as demonstrated by an increased cytokine production capacity 6 days after co-incubation. Interestingly, VAT induced a higher cytokine response compared to SAT. Analysis of the inflammatory phenotype of peripheral cells before and after bariatric surgery is currently ongoing. Conclusions Adipose tissue-secreted metabolites, particularly secreted by VAT, have the potential to induce persistent innate immune cell activation. Our further analyses will show whether the secretion of these molecules and the activation of the innate immune system persists upon weight loss. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation