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Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation., (© 2021 by The American Society of Hematology.)

Objectives: Histone methyltransferase G9a, also known as Euchromatic Histone Lysine Methyltransferase 2 (EHMT2), mediates H3K9 methylation which is associated with transcriptional repression. It possesses immunomodulatory effects and is overexpressed in multiple types of cancer. In this study, we investigated the role of G9a in the induction of trained immunity, a de facto innate immune memory, and its effects in non-muscle-invasive bladder cancer (NMIBC) patients treated with intravesical Bacillus Calmette-Guérin (BCG)., Methods: EHMT2 expression was assessed upon induction of trained immunity by RNA sequencing and Western blotting. G9a inhibitor BIX-01294 was used to investigate the effect on trained immunity responses in vitro . Subsequent cytokine production was measured by ELISA, epigenetic modifications were measured by ChIP-qPCR, Seahorse technology was used to measure metabolic changes, and a luminescence assay was used to measure ROS release. RNA sequencing was performed on BIX-01294-treated monocytes ex vivo ., Results: The expression of EHMT2 mRNA and protein decreased in monocytes during induction of trained immunity. G9a inhibition by BIX-01294 induced trained immunity and amplified trained immunity responses evoked by various microbial ligands in vitro . This was accompanied by decreased H3K9me2 at the promoters of pro-inflammatory genes. G9a inhibition was also associated with amplified ex vivo trained immunity responses in circulating monocytes of NMIBC patients. Additionally, altered RNA expression of inflammatory genes in monocytes of NMIBC patients was observed upon ex vivo G9a inhibition. Furthermore, intravesical BCG therapy decreased H3K9me2 at the promoter of pro-inflammatory genes., Conclusion: Inhibition of G9a is important in the induction of trained immunity, and G9a may represent a novel therapeutic target in NMIBC patients., Competing Interests: MGN and LABJ are scientific founders of Trained Therapeutics Discovery., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology Inc.)

BACKGROUNDThe antituberculosis vaccine bacillus Calmette-Guérin (BCG) reduces overall infant mortality. Induction of innate immune memory, also termed trained immunity, contributes toward protection against heterologous infections. Since immune cells display oscillations in numbers and function throughout the day, we investigated the effect of BCG administration time on the induction of trained immunity.METHODSEighteen volunteers were vaccinated with BCG at 6 pm and compared with 36 age- and sex-matched volunteers vaccinated between 8 am and 9 am. Peripheral blood mononuclear cells were stimulated with Staphylococcus aureus and Mycobacterium tuberculosis before, as well as 2 weeks and 3 months after, BCG vaccination. Cytokine production was measured to assess the induction of trained immunity and adaptive responses, respectively. Additionally, the influence of vaccination time on induction of trained immunity was studied in an independent cohort of 302 individuals vaccinated between 8 am and 12 pm with BCG.RESULTSCompared with evening vaccination, morning vaccination elicited both a stronger trained immunity and adaptive immune phenotype. In a large cohort of 302 volunteers, early morning vaccination resulted in a superior cytokine production capacity compared with later morning. A cellular, rather than soluble, substrate of the circadian effect of BCG vaccination was demonstrated by the enhanced capacity to induce trained immunity in vitro in morning- compared with evening-isolated monocytes.CONCLUSIONSBCG vaccination in the morning induces stronger trained immunity and adaptive responses compared with evening vaccination. Future studies should take vaccine administration time into account when studying specific and nonspecific effects of vaccines; early morning should be the preferred moment of BCG administration.FUNDINGThe Netherlands Organization for Scientific Research, the European Research Council, and the Danish National Research Foundation.

BACKGROUNDInduction of innate immune memory, also termed trained immunity, by the antituberculosis vaccine bacillus Calmette-Guérin (BCG) contributes to protection against heterologous infections. However, the overall impact of BCG vaccination on the inflammatory status of an individual is not known; while induction of trained immunity may suggest increased inflammation, BCG vaccination has been epidemiologically associated with a reduced incidence of inflammatory and allergic diseases.METHODSWe investigated the impact of BCG (BCG-Bulgaria, InterVax) vaccination on systemic inflammation in a cohort of 303 healthy volunteers, as well as the effect of the inflammatory status on the response to vaccination. A targeted proteome platform was used to measure circulating inflammatory proteins before and after BCG vaccination, while ex vivo Mycobacterium tuberculosis- and Staphylococcus aureus-induced cytokine responses in peripheral blood mononuclear cells were used to assess trained immunity.RESULTSWhile BCG vaccination enhanced cytokine responses to restimulation, it reduced systemic inflammation. This effect was validated in 3 smaller cohorts, and was much stronger in men than in women. In addition, baseline circulating inflammatory markers were associated with ex vivo cytokine responses (trained immunity) after BCG vaccination.CONCLUSIONThe capacity of BCG to enhance microbial responsiveness while dampening systemic inflammation should be further explored for potential therapeutic applications.FUNDINGNetherlands Organization for Scientific Research, European Research Council, and the Danish National Research Foundation.

The lack of defined correlates of protection hampers development of vaccines against tuberculosis (TB). In vitro mycobacterial outgrowth assays are thought to better capture the complexity of the human host/Mycobacterium tuberculosis (Mtb) interaction. Here, we used a mycobacterial growth inhibition assay (MGIA) based on peripheral blood mononuclear cells to investigate the capacity to control outgrowth of bacille Calmette-Guérin (BCG). Interestingly, strong control of BCG outgrowth was observed almost exclusively in individuals with recent exposure to Mtb, but not in (long-term) latent TB infection, and only modestly in BCG vaccinees. Mechanistically, control of mycobacterial outgrowth strongly correlated with the presence of a CD14dim monocyte population, but also required the presence of T cells. The nonclassical monocytes produced CXCL10, and CXCR3 receptor blockade inhibited the capacity to control BCG outgrowth. Expression of CXCR3 splice variants was altered in recently Mtb-exposed individuals. Cytokines previously associated with trained immunity were detected in MGIA supernatants, and CXCL9, CXCL10, and CXCL11 represent new markers of trained immunity. These data indicate that CXCR3 ligands are associated with trained immunity and are critical factors in controlling mycobacterial outgrowth. In conclusion, control of mycobacterial outgrowth early after exposure to Mtb is the result of trained immunity mediated by a CXCL10-producing nonclassical CD14dim monocyte subset.

In recent years, it has become appreciated that immune cells have different metabolic profiles depending on their activation status. During sepsis, circulating leukocytes go through a hyperinflammatory state, which can be accompanied or followed by defective antimicrobial defenses (also described as immune tolerance or paralysis). In this review, the modulation of different cellular metabolic pathways during sepsis in monocytes and macrophages will be discussed. Glycolysis is studied extensively in sepsis and is up-regulated in hyperinflammatory cells, whereas in immune tolerance, it is often down-regulated. Few data are available on other metabolic pathways in immune cells from patients with sepsis. The pentose phosphate pathway is up-regulated during acute hyperinflammatory responses, whereas fatty acid β-oxidation is increased later during sepsis and is associated with an anti-inflammatory (M2) phenotype of macrophages. Within the amino acid metabolism we will discuss the most studied metabolites. Collectively, these data argue that exploration of the immunometabolic pathways in sepsis is an important area of research, and the targeting of metabolic pathways may represent a promising novel strategy as a therapy of sepsis., (© Society for Leukocyte Biology.)

Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment in non-medullary thyroid cancer (TC), the most common endocrine malignancy. However, little is known regarding the regulation of their function in TC. Transcriptome analysis in a model of TC-induced macrophages identified increased inflammatory characteristics and rewiring of cell metabolism as key functional changes. This functional reprogramming was partly mediated by TC-derived lactate that induced upregulation of cytokine production through an AKT1/mTOR-dependent increase in aerobic glycolysis. This led to epigenetic modifications at the level of histone methylation, and subsequently long-term functional changes. Immunohistochemistry assessment validated the increase in glycolysis enzymes and lactate receptor in TAMs in tissue samples from patients with TC. In conclusion, Akt/mTOR-dependent glycolysis mediates TC-induced reprogramming of TAMs and inflammation, and this may represent a novel therapeutic target in TC.

BCG vaccination is associated with a reduced mortality from nonmycobacterial infections. This is likely to be mediated by a combination of innate-immune memory ("trained immunity") and heterologous effects on adaptive immunity. As such, BCG could be used to boost host immunity but not in immunocompromised hosts, as it is a live, attenuated vaccine. Therefore, we assessed whether killed γBCG has similar potentiating effects. In an in vitro model of trained immunity, human monocytes were incubated with γBCG for 24 h and restimulated after 6 d. Cytokine production and the role of pattern recognition receptors and histone methylation markers were assessed. The in vivo effects of γBCG vaccination were studied in a proof-of-principle trial in 15 healthy volunteers. γBCG induced trained immunity in vitro via the NOD2 receptor pathway and up-regulation of H3K4me3 histone methylation. However, these effects were less strong than those induced by live BCG. γBCG vaccination in volunteers had only minimal effects on innate immunity, whereas a significant increase in heterologous Th1/Th17 immunity was observed. Our results indicate that γBCG induces long-term training of innate immunity in vitro. In vivo, γBCG induces mainly heterologous effects on the adaptive-immune system, whereas effects on innate cytokine production are limited., (© Society for Leukocyte Biology.)

An increasing body of evidence shows that the innate immune system has adaptive characteristics that involve a heterologous memory of past insults. Both experimental models and proof-of-principle clinical trials show that innate immune cells, such as monocytes, macrophages, and NK cells, can provide protection against certain infections in vaccination models independently of lymphocytes. This process is regulated through epigenetic reprogramming of innate immune cells and has been termed "trained immunity." It has been hypothesized that induction of trained immunity is responsible for the protective, nonspecific effects induced by vaccines, such as BCG, measles vaccination, and other whole-microorganism vaccines. In this review, we will present the mechanisms of trained immunity responsible for the long-lasting effects of vaccines on the innate immune system., (© Society for Leukocyte Biology.)

Epidemiologic studies suggest that VAS has long-lasting immunomodulatory effects. We hypothesized that ATRA inhibits inflammatory cytokines in a model of trained immunity in monocytes by inducing epigenetic reprogramming through histone modifications. We used an previously described in vitro model of trained immunity, in which adherent monocytes of healthy volunteers were incubated for 24 h with BCG in the presence or absence of ATRA. After washing the cells, they were incubated for an additional 6 d in culture medium and restimulated with microbial ligands, and cytokine production was assessed. ATRA inhibited cytokine responses upon restimulation of monocytes, and this effect was exerted through increased expression of SUV39H2, a histone methyltransferase that induces the inhibitory mark H3K9me3. H3K9me3 at promoter sites of several cytokines was up-regulated by ATRA, and inhibition of SUV39H2 restored cytokine production. In addition to H3K9me3, the stimulatory histone mark H3K4me3 was down-regulated by ATRA at several promoter locations of cytokine genes. Therefore, we can conclude that ATRA inhibits cytokine production in models of direct stimulation or BCG-induced trained immunity and that these effects are mediated by histone modifications., (© Society for Leukocyte Biology.)

Background: Vitamin A deficiency (VAD) is associated with increased mortality. To prevent VAD, WHO recommends high-dose vitamin A supplementation (VAS) every 4-6 months for children aged between 6 months and 5 years of age in countries at risk of VAD. The policy is based on randomized clinical trials (RCTs) conducted in the late 1980s and early 1990s. Recent RCTs indicate that the policy may have ceased to be beneficial. In addition, RCTs attempting to extend the benefits to younger children have yielded conflicting results. Stratified analyses suggest that whereas some subgroups benefit more than expected from VAS, other subgroups may experience negative effects., Methods and Results: We reviewed the potential modifiers of the effect of VAS. The variable effect of VAS was not explained by underlying differences in VAD. Rather, the effect may depend on the sex of the child, the vaccine status and previous supplementation with vitamin A. Vitamin A is known to affect the Th1/Th2 balance and, in addition, recent evidence suggests that vitamin A may also induce epigenetic changes leading to down-regulation of the innate immune response. Thus VAS protects against VAD but has also important and long-lasting immunological effects, and the effect of providing VAS may vary depending on the state of the immune system., Conclusions: To design optimal VAS programmes which target those who benefit and avoid those harmed, more studies are needed. Work is ongoing to define whether neonatal VAS should be considered in subgroups. In the most recent RCT in older children, VAS doubled the mortality for males but halved mortality for females. Hence, we urgently need to re-assess the effect of VAS on older children in large-scale RCTs powered to study effect modification by sex and other potential effect modifiers, and with nested immunological studies., (© The Author 2015. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Bacille Calmette-Guérin (BCG) vaccine exerts nonspecific immunostimulatory effects and may therefore represent a novel therapeutic option to treat sepsis-induced immunoparalysis. We investigated whether BCG vaccination modulates the systemic innate immune response in humans in vivo during experimental endotoxemia. We used inactivated gamma-irradiated BCG vaccine because of the potential risk of disseminated disease with the live vaccine in immunoparalyzed patients. In a randomized double-blind placebo-controlled study, healthy male volunteers were vaccinated with gamma-irradiated BCG (n = 10) or placebo (n = 10) and received 1 ng/kg lipopolysaccharide (LPS) intravenously on day 5 after vaccination to assess the in vivo immune response. Peripheral blood mononuclear cells were stimulated with various related and unrelated pathogens 5, 8 to 10, and 25 to 35 days after vaccination to assess ex vivo immune responses. BCG vaccination resulted in a scar in 90% of vaccinated subjects. LPS administration elicited a profound systemic immune response, characterized by increased levels of pro- and anti-inflammatory cytokines, hemodynamic changes, and flu-like symptoms. However, BCG modulated neither this in vivo immune response, nor ex vivo leukocyte responses at any time point. In conclusion, gamma-irradiated BCG is unlikely to represent an effective treatment option to restore immunocompetence in patients with sepsis-induced immunoparalysis. This trial is registered with NCT02085590.

TREM-1 is an important signaling receptor expressed on neutrophils and monocytes that plays an important role in systemic infections. Here, we review the intracellular signaling pathways that mediate the immunological effects of TREM-1. Because of the absence of signaling motifs, TREM-1 constitutively associates with DAP12 for induction of intracellular signals. After phosphorylation of DAP12, production of chemokines and cytokines is induced. Moreover, TREM-1 also modulates signaling pathways induced by known classes of PRRs, such as TLRs and NLRs. The exact mechanisms through which TREM-1 influences TLR and NLR pathways are still largely elusive.