Immunoresponsive Gene 1 Facilitates TLR4-agonist-Induced Augmentation of Innate Antimicrobial Immunity.

Treatment with the toll-like receptor (TLR) 4 agonist monophosphoryl lipid A (MPLA) conditions innate immunocytes to respond robustly to subsequent infection, a phenotype termed innate immune memory. Our published studies show that metabolic reprogramming of macrophages is a prominent feature of the memory phenotype. We undertook studies to define the functional contributions of tricarboxylic acid (TCA) cycle reprogramming to innate immune memory. We observed that priming of wild type (WT) mice with MPLA potently facilitated accumulation of the TCA cycle metabolite itaconate at sites of infection and enhanced microbial clearance. Augmentation of itaconate accumulation and microbial clearance was ablated in immuneresponsive gene 1 (Irg1) -deficient mice. We further observed that MPLA potently induces expression of Irg1 and accumulation of itaconate in macrophages. Compared to WT macrophages, the ability of Irg1-deficient macrophages to kill Pseudomonas aeruginosa was impaired. We further observed that itaconate is directly antimicrobial against P. aeruginosa at pH 5, which is characteristic of the phagolysosome, and is facilitated by reactive oxygen species. MPLA-induced augmentation of glycolysis, oxidative phosphorylation and accumulation of the TCA cycle metabolites succinate and malate was decreased in Irg1 KO macrophages compared to WT controls. RNA sequencing revealed suppressed transcription of genes associated with phagolysosome function and increased expression of genes associated with cytokine production and chemotaxis in Irg1 deficient macrophages. This study identifies a contribution of itaconate to MPLA-induced augmentation of innate antimicrobial immunity via facilitation of microbial killing as well as impact on metabolic and transcriptional adaptations.

Antiviral innate immune memory in alveolar macrophages following SARS-CoV-2 infection ameliorates secondary influenza A virus disease.

Pathogen encounter can result in epigenetic remodeling that shapes disease caused by heterologous pathogens. Here, we examined innate immune memory in the context of commonly circulating respiratory viruses. Single-cell analyses of airway-resident immune cells in a disease-relevant murine model of SARS-CoV-2 recovery revealed epigenetic reprogramming in alveolar macrophages following infection. Post-COVID-19 human monocytes exhibited similar epigenetic signatures. In airway-resident macrophages, past SARS-CoV-2 infection increased activity of type I interferon (IFN-I)-related transcription factors and epigenetic poising of antiviral genes. Viral pattern recognition and canonical IFN-I signaling were required for the establishment of this innate immune memory and augmented secondary antiviral responses. Antiviral innate immune memory mounted by airway-resident macrophages post-SARS-CoV-2 was necessary and sufficient to ameliorate secondary disease caused by influenza A virus and curtailed hyperinflammatory dysregulation and mortality. Our findings provide insights into antiviral innate immune memory in the airway that may facilitate the development of broadly effective therapeutic strategies.

Impact of Oil-in-Water Adjuvanted ß-Glucan on Innate Immune Memory in Piglets.

The non-specific protective effects offered by the concept of "innate immune memory" might represent a promising strategy to tackle early-life threatening infections. Here we tested the potential of an in vitro selected ß-glucan in inducing trained immunity using an in vivo porcine model. We assessed the leukocyte transcriptome using blood transcriptomic module (BTM), proinflammatory cytokines, and clinical scoring after a first "training" and a second "stimulation" phase. The possible induction of innate immune memory was tested during a "stimulation" by an LPS-adjuvanted Mycoplasma hyopneumoniae vaccine (Hyogen®) one day after weaning. Following the "training", no major group differences were found, with the exception of a plasma TNF that was only induced by Adj and Adj_BG treatment. After vaccination, all groups developed similar antibody responses. A significant induction of plasma TNF and IL-1ß was found in groups that received Adj and Adj_BG. However, following vaccination, the expected early innate BTMs were only induced by the PBS group. In conclusion, the adjuvant alone, adjuvant-formulated ß-glucan, or orally applied ß-glucan were unable to enhance innate immune reactivity but rather appeared to promote innate immune tolerance. Such an immune status could have both positive and negative implications during this phase of the piglet's life.

Trained innate immunity: concept, nomenclature and future perspectives.

During the past decade, compelling evidence has accumulated demonstrating that innate immune cells can mount adaptive characteristics, leading to long-term changes in their function. This de-facto innate immune memory has been termed trained immunity. Trained immunity is mediated through extensive metabolic rewiring and epigenetic modifications, and has important effects in human diseases. While the upregulation of trained immunity by certain vaccines provides heterologous protection against infections, the inappropriate activation of trained immunity by endogenous stimuli contributes to the pathogenesis of inflammatory and neurodegenerative disorders. Development of vaccines that can induce both classical adaptive immunity and trained immunity may lead to a new generation of vaccines with increased efficacy. Activation of trained immunity can also lead to novel strategies for the treatment of cancer, while modulation of trained immunity can provide new approaches for the treatment of inflammatory diseases.

Repeated LPS induces training and tolerance of microglial responses across brain regions.

BACKGROUND: Neuroinflammation is involved in the pathogenesis of almost every central nervous system disorder. As the brain's innate immune cells, microglia fine tune their activity to a dynamic brain environment. Previous studies have shown that repeated bouts of peripheral inflammation can trigger long-term changes in microglial gene expression and function, a form of innate immune memory. METHODS AND RESULTS: In this study, we used multiple low-dose lipopolysaccharide (LPS) injections in adult mice to study the acute cytokine, transcriptomic, and microglia morphological changes that contribute to the formation of immune memory in the frontal cortex, hippocampus, and striatum, as well as the long-term effects of these changes on behavior. Training and tolerance of gene expression was shared across regions, and we identified 3 unique clusters of DEGs (2xLPS-sensitive, 4xLPS-sensitive, LPS-decreased) enriched for different biological functions. 2xLPS-sensitive DEG promoters were enriched for binding sites for IRF and NFkB family transcription factors, two key regulators of innate immune memory. We quantified shifts in microglia morphological populations and found that while the proportion of ramified and rod-like microglia mostly remained consistent within brain regions and sexes with LPS treatment, there was a shift from ameboid towards hypertrophic morphological states across immune memory states and a dynamic emergence and resolution of events of microglia aligning end-to-end with repeated LPS. CONCLUSIONS: Together, findings support the dynamic regulation of microglia during the formation of immune memories in the brain and support future work to exploit this model in brain disease contexts.

[Infection stress and a driver mutation interact to promote transformation to hematological malignancies].

Myelodysplastic syndrome (MDS) is a refractory cancer that arises from hematopoietic stem cells and predominantly affects elderly adults. In addition to driver gene mutations, which are also found in clonal hematopoiesis in healthy elderly people, systemic inflammation caused by infection or collagen disease has long been known as an extracellular factor in the pathogenesis of MDS. Wild-type HSCs have an "innate immune memory" that functions in response to infection and inflammatory stress, and my colleagues and I used an infection stress model to demonstrate that the innate immune response by the TLR-TRIF-PLK-ELF1 pathway is similarly critical in impairment of hematopoiesis and dysregulation of chromatin in MDS stem cells. This revealed that not only are MDS stem cells expanded by the TRAF6-NF-kB pathway, the innate immune response is also involved in generating MDS stem cells. In this review, I will present research findings related to "innate immune memory," one of the pathogenic mechanisms of blood cancer, and discuss future directions for basic pathological research and potential therapeutic development.

Induction of trained immunity in broiler chickens following delivery of oligodeoxynucleotide containing CpG motifs to protect against Escherichia coli septicemia.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) can promote antimicrobial immunity in chickens by enriching immune compartments and activating immune cells. Innate memory, or trained immunity, has been demonstrated in humans and mice, featuring the absence of specificity to the initial stimulus and subsequently cross-protection against pathogens. We hypothesize that CpG-ODN can induce trained immunity in chickens. We delivered single or multiple administrations of CpG-ODN to birds and mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis of peripheral blood mononuclear cells were quantified using Seahorse XFp. Next, chickens were administered with CpG-ODN twice at 1 and 4 day of age and challenged with Escherichia coli at 27 days of age. The CpG-ODN administered groups had significantly higher mitochondrial OXPHOS until 21 days of age while cellular glycolysis gradually declined by 14 days of age. The group administered with CpG-ODN twice at 1 and 4 days of age had significantly higher survival, lower clinical score and bacterial load following challenge with E. coli at 27 d of age. This study demonstrated the induction of trained immunity in broiler chickens following administration of CpG-ODN twice during the first 4 days of age to protect birds against E. coli septicemia at 27 days of age.

Innate immune memory after brain injury drives inflammatory cardiac dysfunction.

The medical burden of stroke extends beyond the brain injury itself and is largely determined by chronic comorbidities that develop secondarily. We hypothesized that these comorbidities might share a common immunological cause, yet chronic effects post-stroke on systemic immunity are underexplored. Here, we identify myeloid innate immune memory as a cause of remote organ dysfunction after stroke. Single-cell sequencing revealed persistent pro-inflammatory changes in monocytes/macrophages in multiple organs up to 3 months after brain injury, notably in the heart, leading to cardiac fibrosis and dysfunction in both mice and stroke patients. IL-1ß was identified as a key driver of epigenetic changes in innate immune memory. These changes could be transplanted to naive mice, inducing cardiac dysfunction. By neutralizing post-stroke IL-1ß or blocking pro-inflammatory monocyte trafficking with a CCR2/5 inhibitor, we prevented post-stroke cardiac dysfunction. Such immune-targeted therapies could potentially prevent various IL-1ß-mediated comorbidities, offering a framework for secondary prevention immunotherapy.

Long-Lasting Enhanced Cytokine Responses Following SARS-CoV-2 BNT162b2 mRNA Vaccination.

The mRNA vaccine against COVID-19 protects against severe disease by the induction of robust humoral and cellular responses. Recent studies have shown the capacity of some vaccines to induce enduring non-specific innate immune responses by the induction of trained immunity, augmenting protection against unrelated pathogens. This study aimed to assess whether the mRNA vaccine BNT162b2 can induce lasting non-specific immune responses in myeloid cells following a three-dose vaccination scheme. In a sample size consisting of 20 healthy individuals from Romania, we assessed inflammatory proteins using the Olink® Target 96 Inflammation panel, as well as ex vivo cytokine responses following stimulations with unrelated PRR ligands. We assessed the vaccine-induced non-specific systemic inflammation and functional adaptations of myeloid cells. Our results revealed the induction of a stimulus- and cytokine-dependent innate immune memory phenotype that became apparent after the booster dose and was maintained eight months later in the absence of systemic inflammation.

High-throughput screen identifies non inflammatory small molecule inducers of trained immunity.

Trained immunity is characterized by epigenetic and metabolic reprogramming in response to specific stimuli. This rewiring can result in increased cytokine and effector responses to pathogenic challenges, providing nonspecific protection against disease. It may also improve immune responses to established immunotherapeutics and vaccines. Despite its promise for next-generation therapeutic design, most current understanding and experimentation is conducted with complex and heterogeneous biologically derived molecules, such as ß-glucan or the Bacillus Calmette-Guérin (BCG) vaccine. This limited collection of training compounds also limits the study of the genes most involved in training responses as each molecule has both training and nontraining effects. Small molecules with tunable pharmacokinetics and delivery modalities would both assist in the study of trained immunity and its future applications. To identify small molecule inducers of trained immunity, we screened a library of 2,000 drugs and drug-like compounds. Identification of well-defined compounds can improve our understanding of innate immune memory and broaden the scope of its clinical applications. We identified over two dozen small molecules in several chemical classes that induce a training phenotype in the absence of initial immune activation-a current limitation of reported inducers of training. A surprising result was the identification of glucocorticoids, traditionally considered immunosuppressive, providing an unprecedented link between glucocorticoids and trained innate immunity. We chose seven of these top candidates to characterize and establish training activity in vivo. In this work, we expand the number of compounds known to induce trained immunity, creating alternative avenues for studying and applying innate immune training.

Endotoxin tolerance and trained immunity: breaking down immunological memory barriers.

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.

Induction of trained immunity using ß-glucan and its protective responses in Nile tilapia, Oreochromis niloticus.

Emerging and re-emerging diseases in fish cause drastic economic losses in the aquaculture sector. To combat the impact of disease outbreaks and prevent the emergence of infections in culture systems, understanding the advanced strategies for protecting fish against infections is inevitable in fish health research. Therefore, the present study aimed to evaluate the induction of trained immunity and its protective efficacy against Streptococcus agalactiae in tilapia. For this, Nile tilapia and the Tilapia head kidney macrophage primary culture were primed using ß-glucan @200 µg/10 g body weight and 10 µg/mL respectively. Expression profiles of the markers of trained immunity and production of metabolites were monitored at different time points, post-priming and training, which depicted enhanced responsiveness. Higher lactate and lactate dehydrogenase (LDH) production in vitro suggests heightened glycolysis induced by priming of the cells using ß-glucan. A survival rate of 60% was observed in ß-glucan trained fish post challenge with virulent S. agalactiae at an LD50 of 2.6 × 107 cfu/ml, providing valuable insights into promising strategies of trained immunity for combating infections in fish.

The potency of hematopoietic stem cell reprogramming for changing immune tone.

Innate immune memory endows innate immune cells with antigen independent heightened responsiveness to subsequent challenges. The durability of this response can be mediated by inflammation induced epigenetic and metabolic reprogramming in hematopoietic stem and progenitor cells (HSPCs) that are maintained through differentiation to mature immune progeny. Understanding the mechanisms and extent of trained immunity induction by pathogens and vaccines, such as BCG, in HSPC remains a critical area of exploration with important implications for health and disease. Here we review these concepts and present new analysis to highlight how inflammatory reprogramming of HSPC can potently alter immune tone, including to enhance specific anti-tumor responses. New findings in the field pave the way for novel HSPC targeting therapeutic strategies in cancer and other contexts of immune modulation. Future studies are expected to unravel diverse and extensive effects of infections, vaccines, microbiota, and sterile inflammation on hematopoietic progenitor cells and begin to illuminate the broad spectrum of immunologic tuning that can be established through altering HSPC phenotypes. The purpose of this review is to draw attention to emerging and speculative topics in this field where we posit that focused study of HSPC in the framework of trained immunity holds significant promise.

Discovering adaptive features of innate immune memory.

Conventionally, it was thought that innate immunity operated through a simple system of nonspecific responses to an insult. However, this perspective now seems overly simplistic. It has become evident that intricate cooperation and networking among various cells, receptors, signaling pathways, and protein complexes are essential for regulating and defining the overall activation status of the immune response, where the distinction between innate and adaptive immunity becomes ambiguous. Given the evolutionary timeline of vertebrates and the success of plants and invertebrates which depend solely on innate immunity, immune memory cannot be considered an innovation of only the lymphoid lineage. Indeed, the evolutionary innate immune memory program is a conserved mechanism whereby innate immune cells can induce a heightened response to a secondary stimulus due to metabolic and epigenetic reprogramming. Importantly, the longevity of this memory phenotype can be attributed to the reprogramming of self-renewing hematopoietic stem cells (HSCs) in the bone marrow, which is subsequently transmitted to lineage-committed innate immune cells. HSCs reside within a complex regulated network of immune and stromal cells that govern their two primary functions: self-renewal and differentiation. In this review, we delve into the emerging cellular and molecular mechanisms as well as metabolic pathways of innate memory in HSCs, which harbor substantial therapeutic promise.

Proton therapy induces a local microglial neuroimmune response.

BACKGROUND AND PURPOSE: Although proton therapy is increasingly being used in the treatment of paediatric and adult brain tumours, there are still uncertainties surrounding the biological effect of protons on the normal brain. Microglia, the brain-resident macrophages, have been shown to play a role in the development of radiation-induced neurotoxicity. However, their molecular and hence functional response to proton irradiation remains unknown. This study investigates the effect of protons on microglia by comparing the effect of photons and protons as well as the influence of age and different irradiated volumes. MATERIALS AND METHODS: Rats were irradiated with 14 Gy to the whole brain with photons (X-rays), plateau protons, spread-out Bragg peak (SOBP) protons or to 50 % anterior, or 50 % posterior brain sub-volumes with plateau protons. RNA sequencing, validation of microglial priming gene expression using qPCR and high-content imaging analysis of microglial morphology were performed in the cortex at 12 weeks post irradiation. RESULTS: Photons and plateau protons induced a shared transcriptomic response associated with neuroinflammation. This response was associated with a similar microglial priming gene expression signature and distribution of microglial morphologies. Expression of the priming gene signature was less pronounced in juvenile rats compared to adults and slightly increased in rats irradiated with SOBP protons. High-precision partial brain irradiation with protons induced a local microglial priming response and morphological changes. CONCLUSION: Overall, our data indicate that the brain responds in a similar manner to photons and plateau protons with a shared local upregulation of microglial priming-associated genes, potentially enhancing the immune response to subsequent inflammatory challenges.

Candida albicans N-Linked Mannans Potentiate the Induction of Trained Immunity via Dectin-2.

The interaction between the Candida albicans cell wall and pattern recognition receptors is crucial for the initiation of host immune responses, which, ultimately, contribute to the clearance of this pathogenic fungus. In the present study, we investigate the ability of C. albicans mannans to modulate immune response and induce innate immune memory (also termed trained immunity). Using mutants of C. albicans that are defective in or lack mannosyl residues, we show that alterations in the mannosylation of the C. albicans cell wall affect the innate cytokine response and strongly reduce the secretion of T-cell-derived cytokines. Subsequently, we demonstrate that the branching of N-linked mannan, but not O-linked mannan, is essential to potentiate the induction of trained immunity, a process mediated by dectin 2. In conclusion, N-linked mannan is needed, in addition to ß-glucans, for an effective induction of trained immunity by C. albicans.

Trained innate immunity modulates osteoblast and osteoclast differentiation.

Macrophages are key regulators in bone repair and regeneration. Recent studies have shown that long-term epigenetic changes and metabolic shifts occur during specific immune training of macrophages that affect their functional state, resulting in heightened (trained) or reduced (tolerant) responses upon exposure to a second stimulus. This is known as innate immune memory. Here, we study the impact of macrophages' memory trait on osteoblast differentiation of human mesenchymal stromal cells (hMSCs) and osteoclast differentiation. An in vitro trained immunity protocol of monocyte-derived macrophages was employed using inactivated Candida albicans and Bacillus Calmette-Guérin (BCG) to induce a 'trained' state and Pam3CSK4 (PAM) and Lipopolysaccharides (LPS) to induce a 'tolerance' state. Macrophages were subsequently cocultured with hMSCs undergoing osteogenic differentiation during either resting (unstimulated) or inflammatory conditions (restimulated with LPS). Alkaline phosphatase activity, mineralization, and cytokine levels (TNF, IL-6, oncostatin M and SDF-1α) were measured. In addition, macrophages underwent osteoclast differentiation. Our findings show that trained and tolerized macrophages induced opposing results. Under resting conditions, BCG-trained macrophages enhanced ALP levels (threefold), while under inflammatory conditions this was found in the LPS-tolerized macrophages (fourfold). Coculture of hMSCs with trained macrophages showed mineralization while tolerized macrophages inhibited the process under both resting and inflammatory conditions. While osteoclast differentiation was not affected in trained-macrophages, this ability was significantly loss in tolerized ones. This study further confirms the intricate cross talk between immune cells and bone cells, highlighting the need to consider this interaction in the development of personalized approaches for bone regenerative medicine.

Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis.

Monocytes can develop an exhausted memory state characterized by reduced differentiation, pathogenic inflammation, and immune suppression that drives immune dysregulation during sepsis. Chromatin alterations, notably via histone modifications, underlie innate immune memory, but the contribution of DNA methylation remains poorly understood. Using an ex vivo sepsis model, we show altered DNA methylation throughout the genome of exhausted monocytes, including genes implicated in immune dysregulation during sepsis and COVID-19 infection (e.g., Plac8). These changes are recapitulated in septic mice induced by cecal slurry injection. Methylation profiles developed in septic mice are maintained during ex vivo culture, supporting the involvement of DNA methylation in stable monocyte exhaustion memory. Methylome reprogramming is driven in part by Wnt signaling inhibition in exhausted monocytes and can be reversed with DNA methyltransferase inhibitors, Wnt agonists, or immune training molecules. Our study demonstrates the significance of altered DNA methylation in the maintenance of stable monocyte exhaustion memory.