Common and distinct metabolomic markers related to immune aging in Western European and East African populations.

In old age, impaired immunity causes high susceptibility to infections and cancer, higher morbidity and mortality, and poorer vaccination efficiency. Many factors, such as genetics, diet, and lifestyle, impact aging. This study aimed to investigate how immune responses change with age in healthy Dutch and Tanzanian individuals and identify common metabolites associated with an aged immune profile. We performed untargeted metabolomics from plasma to identify age-associated metabolites, and we correlated their concentrations with ex-vivo cytokine production by immune cells, DNA methylation-based epigenetic aging, and telomere length. Innate immune responses were impacted differently by age in Dutch and Tanzanian cohorts. Age-related decline in steroid hormone precursors common in both populations was associated with higher systemic inflammation and lower cytokine responses. Hippurate and 2-phenylacetamide, commonly more abundant in older individuals, were negatively correlated with cytokine responses and telomere length and positively correlated with epigenetic aging. Lastly, we identified several metabolites that might contribute to the stronger decline in innate immunity with age in Tanzanians. The shared metabolomic signatures of the two cohorts suggest common mechanisms of immune aging, revealing metabolites with potential contributions. These findings also reflect genetic or environmental effects on circulating metabolites that modulate immune responses.

Multi-omics analysis of innate and adaptive responses to BCG vaccination reveals epigenetic cell states that predict trained immunity.

Immune responses are tightly regulated yet highly variable between individuals. To investigate human population variation of trained immunity, we immunized healthy individuals with Bacillus Calmette-Guérin (BCG). This live-attenuated vaccine induces not only an adaptive immune response against tuberculosis but also triggers innate immune activation and memory that are indicative of trained immunity. We established personal immune profiles and chromatin accessibility maps over a 90-day time course of BCG vaccination in 323 individuals. Our analysis uncovered genetic and epigenetic predictors of baseline immunity and immune response. BCG vaccination enhanced the innate immune response specifically in individuals with a dormant immune state at baseline, rather than providing a general boost of innate immunity. This study advances our understanding of BCG's heterologous immune-stimulatory effects and trained immunity in humans. Furthermore, it highlights the value of epigenetic cell states for connecting immune function with genotype and the environment.

BCG vaccination induces innate immune memory in γδ T cells in humans.

Bacillus Calmette-Guérin vaccine is well known for inducing trained immunity in myeloid and natural killer cells, which can explain its cross-protective effect against heterologous infections. Although displaying functional characteristics of both adaptive and innate immunity, γδ T-cell memory has been only addressed in a pathogen-specific context. In this study, we aimed to determine whether human γδ T cells can mount trained immunity and therefore contribute to the cross-protective effect of the Bacillus Calmette-Guérin vaccine. We investigated in vivo induction of innate memory in γδ T cells by Bacillus Calmette-Guérin vaccination in healthy human volunteers by combining single-cell RNA sequencing technology with immune functional assays. The total number of γδ T cells and membrane markers of activation was not influenced by Bacillus Calmette-Guérin vaccination. In contrast, Bacillus Calmette-Guérin changed γδ T cells' transcriptional programs and increased their responsiveness to heterologous bacterial and fungal stimuli, including lipopolysaccharide and Candida albicans, as simultaneously characterized by higher tumor necrosis factor and interferon γ production, weeks after vaccination. Human γδ T cells in adults display the potential to develop a trained immunity phenotype after Bacillus Calmette-Guérin vaccination.

11-deoxycortisol positively correlates with T cell immune traits in physiological conditions.

BACKGROUND: Endogenous steroid hormones have significant effects on inflammatory and immune processes, but the immunological activities of steroidogenesis precursors remain largely unexplored. METHODS: We conducted a systematic approach to examine the association between steroid hormones profile and immune traits in a cohort of 534 healthy volunteers. Serum concentrations of steroid hormones and their precursors (cortisol, progesterone, testosterone, androstenedione, 11-deoxycortisol and 17-OH progesterone) were determined by liquid chromatography-tandem mass spectrometry. Immune traits were evaluated by quantifying cellular composition of the circulating immune system and ex vivo cytokine responses elicited by major human pathogens and microbial ligands. An independent cohort of 321 individuals was used for validation, followed by in vitro validation experiments. FINDINGS: We observed a positive association between 11-deoxycortisol and lymphoid cellular subsets numbers and function (especially IL-17 response). The association with lymphoid cellularity was validated in an independent validation cohort. In vitro experiments showed that, as compared to androstenedione and 17-OH progesterone, 11-deoxycortisol promoted T cell proliferation and Candida-induced Th17 polarization at physiologically relevant concentrations. Functionally, 11-deoxycortisol-treated T cells displayed a more activated phenotype (PD-L1high CD25high CD62Llow CD127low) in response to CD3/CD28 co-stimulation, and downregulated expression of T-bet nuclear transcription factor. INTERPRETATION: Our findings suggest a positive association between 11-deoxycortisol and T-cell function under physiological conditions. Further investigation is needed to explore the potential mechanisms and clinical implications. FUNDING: Found in acknowledgements.

Dimethyl itaconate induces long-term innate immune responses and confers protection against infection.

Itaconate is an immunomodulatory metabolite produced by immune cells under microbial stimulation and certain pro-inflammatory conditions and triggers antioxidant and anti-inflammatory responses. We show that dimethyl itaconate, a derivative of itaconate previously linked to suppression of inflammation and widely employed as an alternative to the endogenous metabolite, can induce long-term transcriptional, epigenomic, and metabolic changes, characteristic of trained immunity. Dimethyl itaconate alters glycolytic and mitochondrial energetic metabolism, ultimately leading to increased responsiveness to microbial ligand stimulation. Subsequently, mice treated with dimethyl itaconate present increased survival to infection with Staphylococcus aureus. Additionally, itaconate levels in human plasma correlate with enhanced ex vivo pro-inflammatory cytokine production. Collectively, these findings demonstrate that dimethyl itaconate displays short-term anti-inflammatory characteristics and the capacity to induce long-term trained immunity. This pro-and anti-inflammatory dichotomy of dimethyl itaconate is likely to induce complex immune responses and should be contemplated when considering itaconate derivatives in a therapeutic context.

A single-cell view on host immune transcriptional response to in vivo BCG-induced trained immunity.

Bacillus Calmette-Guérin (BCG) vaccination is a prototype model for the study of trained immunity (TI) in humans, and results in a more effective response of innate immune cells upon stimulation with heterologous stimuli. Here, we investigate the heterogeneity of TI induction by single-cell RNA sequencing of immune cells collected from 156 samples. We observe that both monocytes and CD8+ T cells show heterologous transcriptional responses to lipopolysaccharide, with an active crosstalk between these two cell types. Furthermore, the interferon-γ pathway is crucial in BCG-induced TI, and it is upregulated in functional high responders. Data-driven analyses and functional experiments reveal STAT1 to be one of the important transcription factors for TI shared in all identified monocyte subpopulations. Finally, we report the role of type I interferon-related and neutrophil-related TI transcriptional programs in patients with sepsis. These findings provide comprehensive insights into the importance of monocyte heterogeneity during TI in humans.

Plasma metabolome predicts trained immunity responses after antituberculosis BCG vaccination.

The antituberculosis vaccine Bacillus Calmette-Guérin (BCG) induces nonspecific protection against heterologous infections, at least partly through induction of innate immune memory (trained immunity). The amplitude of the response to BCG is variable, but the factors that influence this response are poorly understood. Metabolites, either released by cells or absorbed from the gut, are known to influence immune responses, but whether they impact BCG responses is not known. We vaccinated 325 healthy individuals with BCG, and collected blood before, 2 weeks and 3 months after vaccination, to assess the influence of circulating metabolites on the immune responses induced by BCG. Circulating metabolite concentrations after BCG vaccination were found to have a more pronounced impact on trained immunity responses, such as the increase in IL-1ß and TNF-α production upon Staphylococcus aureus stimulation, than on specific adaptive immune memory, assessed as IFN-γ production in response to Mycobacterium tuberculosis. Circulating metabolites at baseline were able to predict trained immunity responses at 3 months after vaccination and enrichment analysis based on the metabolites positively associated with trained immunity revealed enrichment of the tricarboxylic acid (TCA) cycle and glutamine metabolism, both of which were previously found to be important for trained immunity. Several new metabolic pathways that influence trained immunity were identified, among which taurine metabolism associated with BCG-induced trained immunity, a finding validated in functional experiments. In conclusion, circulating metabolites are important factors influencing BCG-induced trained immunity in humans. Modulation of metabolic pathways may be a novel strategy to improve vaccine and trained immunity responses.

The Genetic Risk for COVID-19 Severity Is Associated With Defective Immune Responses.

Recent genome-wide association studies (GWASs) of COVID-19 patients of European ancestry have identified genetic loci significantly associated with disease severity. Here, we employed the detailed clinical, immunological and multi-omics dataset of the Human Functional Genomics Project (HFGP) to explore the physiological significance of the host genetic variants that influence susceptibility to severe COVID-19. A genomics investigation intersected with functional characterization of individuals with high genetic risk for severe COVID-19 susceptibility identified several major patterns: i. a large impact of genetically determined innate immune responses in COVID-19, with ii. increased susceptibility for severe disease in individuals with defective cytokine production; iii. genetic susceptibility related to ABO blood groups is probably mediated through the von Willebrand factor (VWF) and endothelial dysfunction. We further validated these identified associations at transcript and protein levels by using independent disease cohorts. These insights allow a physiological understanding of genetic susceptibility to severe COVID-19, and indicate pathways that could be targeted for prevention and therapy.

Immune modulatory effects of progesterone on oxLDL-induced trained immunity in monocytes.

Atherosclerotic cardiovascular diseases (CVD) are among the leading causes of death in the world. Monocyte-derived macrophages are key players in the pathophysiology of atherosclerosis. Innate immune memory following exposure of monocytes to atherogenic compounds, such as oxidized low-density lipoproteins (oxLDL), termed trained immunity, can contribute to atherogenesis. The current study aimed to elucidate intracellular mechanisms of oxLDL-induced trained immunity. Using untargeted intracellular metabolomics in isolated human primary monocytes, we show that oxLDL-induced trained immunity results in alterations in the balance of intracellular steroid hormones in monocytes. This was reflected by a decrease in extracellular progesterone concentrations following LPS stimulation. To understand the potential effects of steroid hormones on trained immunity, monocytes were costimulated with oxLDL and the steroid hormones progesterone, hydrocortisone, dexamethasone, ß-estradiol, and dihydrotestosterone. Progesterone showed a unique ability to attenuate the enhanced TNFα and IL-6 production following oxLDL-induced trained immunity. Single nucleotide polymorphisms in the nuclear glucocorticoid, progesterone, and mineralocorticoid receptor were shown to correlate with ex vivo oxLDL-induced trained immunity in 243 healthy volunteers. Pharmacologic inhibition experiments revealed that progesterone exerts the suppression of TNFα in trained immunity via the nuclear glucocorticoid and mineralocorticoid receptors. Our data show that progesterone has a unique ability to suppress oxLDL-induced trained immunity. We hypothesize that this effect might contribute to the lower incidence of CVD in premenopausal women.

An integrative genomics approach identifies KDM4 as a modulator of trained immunity.

Innate immune cells are able to build memory characteristics via a process termed "trained immunity." Host factors that influence the magnitude of the individual trained immunity response remain largely unknown. Using an integrative genomics approach, our study aimed to prioritize and understand the role of specific genes in trained immunity responses. In vitro-induced trained immunity responses were assessed in two independent population-based cohorts of healthy individuals, the 300 Bacillus Calmette-Guérin (300BCG; n = 267) and 200 Functional Genomics (200FG; n = 110) cohorts from the Human Functional Genomics Project. Genetic loci that influence cytokine responses upon trained immunity were identified by conducting a meta-analysis of QTLs identified in the 300BCG and 200FG cohorts. From the identified QTL loci, we functionally validated the role of PI3K-Akt signaling pathway and two genes that belong to the family of Siglec receptors (Siglec-5 and Siglec-14). Furthermore, we identified the H3K9 histone demethylases of the KDM4 family as major regulators of trained immunity responses. These data pinpoint an important role of metabolic and epigenetic processes in the regulation of trained immunity responses, and these findings may open new avenues for vaccine design and therapeutic interventions.

The influence of the gut microbiome on BCG-induced trained immunity.

BACKGROUND: The bacillus Calmette-Guérin (BCG) vaccine protects against tuberculosis and heterologous infections but elicits high inter-individual variation in specific and nonspecific, or trained, immune responses. While the gut microbiome is increasingly recognized as an important modulator of vaccine responses and immunity in general, its potential role in BCG-induced protection is largely unknown. RESULTS: Stool and blood were collected from 321 healthy adults before BCG vaccination, followed by blood sampling after 2 weeks and 3 months. Metagenomics based on de novo genome assembly reveals 43 immunomodulatory taxa. The nonspecific, trained immune response is detected by altered production of cytokines IL-6, IL-1ß, and TNF-α upon ex vivo blood restimulation with Staphylococcus aureus and negatively correlates with abundance of Roseburia. The specific response, measured by IFN-γ production upon Mycobacterium tuberculosis stimulation, is associated positively with Ruminococcus and Eggerthella lenta. The identified immunomodulatory taxa also have the strongest effects on circulating metabolites, with Roseburia affecting phenylalanine metabolism. This is corroborated by abundances of relevant enzymes, suggesting alternate phenylalanine metabolism modules are activated in a Roseburia species-dependent manner. CONCLUSIONS: Variability in cytokine production after BCG vaccination is associated with the abundance of microbial genomes, which in turn affect or produce metabolites in circulation. Roseburia is found to alter both trained immune responses and phenylalanine metabolism, revealing microbes and microbial products that may alter BCG-induced immunity. Together, our findings contribute to the understanding of specific and trained immune responses after BCG vaccination.

The Immunological Factors Predisposing to Severe Covid-19 Are Already Present in Healthy Elderly and Men.

Male sex and old age are risk factors for COVID-19 severity, but the underlying causes are unknown. A possible explanation for this might be the differences in immunological profiles in males and the elderly before the infection. With this in mind, we analyzed the abundance of circulating proteins and immune populations associated with severe COVID-19 in 2 healthy cohorts. Besides, given the seasonal profile of COVID-19, the seasonal response against SARS-CoV-2 could also be different in the elderly and males. Therefore, PBMCs of female, male, young, and old subjects in different seasons of the year were stimulated with heat-inactivated SARS-CoV-2 to investigate the season-dependent anti-SARS-CoV-2 immune response. We found that several T cell subsets, which are known to be depleted in severe COVID-19 patients, were intrinsically less abundant in men and older individuals. Plasma proteins increasing with disease severity, including HGF, IL-8, and MCP-1, were more abundant in the elderly and males. Upon in vitro SARS-CoV-2 stimulation, the elderly produced significantly more IL-1RA and had a dysregulated IFNγ response with lower production in the fall compared with young individuals. Our results suggest that the immune characteristics of severe COVID-19, described by a differential abundance of immune cells and circulating inflammatory proteins, are intrinsically present in healthy men and the elderly. This might explain the susceptibility of men and the elderly to SARS-CoV-2 infection.

oxLDL-Induced Trained Immunity Is Dependent on Mitochondrial Metabolic Reprogramming.

Following brief exposure to endogenous atherogenic particles, such as oxidized low-density lipoprotein (oxLDL), monocytes/macrophages can adopt a long-term pro-inflammatory phenotype, which is called trained immunity. This mechanism might contribute to the chronic low-grade inflammation that characterizes atherosclerosis. In this study, we aim to elucidate immunometabolic pathways that drive oxLDL-induced trained immunity. Primary isolated human monocytes were exposed to oxLDL for 24 h, and after five days stimulated with LPS to measure the cytokine production capacity. RNA-sequencing revealed broad increases in genes enriched in mitochondrial pathways after 24 h of oxLDL exposure. Further omics profiling of oxLDL-trained macrophages via intracellular metabolomics showed an enrichment for tricarboxylic acid (TCA) cycle metabolites. Single cell analysis revealed that oxLDL-trained macrophages contain larger mitochondria, potentially likely linked to increased oxidative phosphorylation (OXPHOS) activity. Co-incubation with pharmacological blockers of OXPHOS inhibited oxLDL-induced trained immunity. The relevance of OXPHOS was confirmed in a cohort of 243 healthy subjects showing that genetic variation in genes coding for enzymes relevant to OXPHOS correlated with the capacity of monocytes to be trained with oxLDL. Interestingly, OXPHOS appears to play an important role in the increased cytokine hyperresponsiveness by oxLDL-trained macrophages. The TCA-cycle can also be fuelled by glutamine and free fatty acids, and pharmacological blockade of these pathways could prevent oxLDL-induced trained immunity. This study demonstrates that the mitochondria of oxLDL-trained macrophages undergo changes to their function and form with OXPHOS being an important mechanism for trained immunity, which could unveil novel pharmacological targets to prevent atherogenesis.

Integration of metabolomics, genomics, and immune phenotypes reveals the causal roles of metabolites in disease.

BACKGROUND: Recent studies highlight the role of metabolites in immune diseases, but it remains unknown how much of this effect is driven by genetic and non-genetic host factors. RESULT: We systematically investigate circulating metabolites in a cohort of 500 healthy subjects (500FG) in whom immune function and activity are deeply measured and whose genetics are profiled. Our data reveal that several major metabolic pathways, including the alanine/glutamate pathway and the arachidonic acid pathway, have a strong impact on cytokine production in response to ex vivo stimulation. We also examine the genetic regulation of metabolites associated with immune phenotypes through genome-wide association analysis and identify 29 significant loci, including eight novel independent loci. Of these, one locus (rs174584-FADS2) associated with arachidonic acid metabolism is causally associated with Crohn's disease, suggesting it is a potential therapeutic target. CONCLUSION: This study provides a comprehensive map of the integration between the blood metabolome and immune phenotypes, reveals novel genetic factors that regulate blood metabolite concentrations, and proposes an integrative approach for identifying new disease treatment targets.

The role of sirtuin 1 on the induction of trained immunity.

Sirtuin 1 (SIRT1) has been described to modify immune responses by modulation of gene transcription. As transcriptional reprogramming is the molecular substrate of trained immunity, a de facto innate immune memory, we investigated the role of SIRT1 in the induction of trained immunity. We identified various SIRT1 genetic single nucleotide polymorphisms affecting innate and adaptive cytokine production of human peripheral blood mononuclear cells (PBMCs) in response to various stimuli on the one hand, and in vitro induction of trained immunity on the other hand. Furthermore, inhibition of SIRT1 upregulated pro-inflammatory innate cytokine production upon stimulation of PBMCs. However, inhibition of SIRT1 in vitro had no effect on cytokine responses upon induction of trained immunity, while activation of SIRT1 mildly modified trained immunity responses. In conclusion, SIRT1 modifies innate cytokine production by PBMCs in response to various microbes, but has only a secondary role for BCG and ß-glucan-induced trained immunity responses.

Glutathione Metabolism Contributes to the Induction of Trained Immunity.

The innate immune system displays heterologous memory characteristics, which are characterized by stronger responses to a secondary challenge. This phenomenon termed trained immunity relies on epigenetic and metabolic rewiring of innate immune cells. As reactive oxygen species (ROS) production has been associated with the trained immunity phenotype, we hypothesized that the increased ROS levels and the main intracellular redox molecule glutathione play a role in the induction of trained immunity. Here we show that pharmacological inhibition of ROS in an in vitro model of trained immunity did not influence cell responsiveness; the modulation of glutathione levels reduced pro-inflammatory cytokine production in human monocytes. Single nucleotide polymorphisms (SNPs) in genes involved in glutathione metabolism were found to be associated with changes in pro-inflammatory cytokine production capacity upon trained immunity. Also, plasma glutathione concentrations were positively associated with ex vivo IL-1ß production, a biomarker of trained immunity, produced by monocytes of BCG-vaccinated individuals. In conclusion, glutathione metabolism is involved in the induction of trained immunity, and future studies are warranted to explore its functional consequences in human diseases.

In vitro induction of trained immunity in adherent human monocytes.

A growing number of studies show that innate immune cells can undergo functional reprogramming, facilitating a faster and enhanced response to heterologous secondary stimuli. This concept has been termed "trained immunity." We outline here a protocol to recapitulate this in vitro using adherent monocytes from consecutive isolation of peripheral blood mononuclear cells. The induction of trained immunity and the associated functional reprogramming of monocytes is described in detail using ß-glucan (from Candida albicans) and Bacillus Calmette-Guérin as examples. For complete details on the use and execution of this protocol, please refer to Repnik et al. (2003) and Bekkering et al. (2016).

A modular approach toward producing nanotherapeutics targeting the innate immune system.

Immunotherapies controlling the adaptive immune system are firmly established, but regulating the innate immune system remains much less explored. The intrinsic interactions between nanoparticles and phagocytic myeloid cells make these materials especially suited for engaging the innate immune system. However, developing nanotherapeutics is an elaborate process. Here, we demonstrate a modular approach that facilitates efficiently incorporating a broad variety of drugs in a nanobiologic platform. Using a microfluidic formulation strategy, we produced apolipoprotein A1-based nanobiologics with favorable innate immune system-engaging properties as evaluated by in vivo screening. Subsequently, rapamycin and three small-molecule inhibitors were derivatized with lipophilic promoieties, ensuring their seamless incorporation and efficient retention in nanobiologics. A short regimen of intravenously administered rapamycin-loaded nanobiologics (mTORi-NBs) significantly prolonged allograft survival in a heart transplantation mouse model. Last, we studied mTORi-NB biodistribution in nonhuman primates by PET/MR imaging and evaluated its safety, paving the way for clinical translation.

Lysine methyltransferase G9a is an important modulator of trained immunity.

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.

Thyrotrophin and thyroxine support immune homeostasis in humans.

The endocrine and the immune systems interact by sharing receptors for hormones and cytokines, cross-control and feedback mechanisms. To date, no comprehensive study has assessed the impact of thyroid hormones on immune homeostasis. By studying immune phenotype (cell populations, antibody concentrations, circulating cytokines, adipokines and acute-phase proteins, monocyte-platelet interactions and cytokine production capacity) in two large independent cohorts of healthy volunteers of Western European descent from the Human Functional Genomics Project (500FG and 300BCG cohorts), we identified a crucial role of the thyroid hormone thyroxin (T4) and thyroid-stimulating hormone (TSH) on the homeostasis of lymphocyte populations. TSH concentrations were strongly associated with multiple populations of both effector and regulatory T cells, whereas B-cell populations were significantly associated with free T4 (fT4). In contrast, fT4 and TSH had little impact on myeloid cell populations and cytokine production capacity. Mendelian randomization further supported the role of fT4 for lymphocyte homeostasis. Subsequently, using a genomics approach, we identified genetic variants that influence both fT4 and TSH concentrations and immune responses, and gene set enrichment pathway analysis showed enrichment of fT4-affected gene expression in B-cell function pathways, including the CD40 pathway, further supporting the importance of fT4 in the regulation of B-cell function. In conclusion, we show that thyroid function controls the homeostasis of the lymphoid cell compartment. These findings improve our understanding of the immune responses and open the door for exploring and understanding the role of thyroid hormones in the lymphocyte function during disease.