Innate Memory Research Articles

Safety of Zein Nanoparticles on Human Innate Immunity and Inflammation

In recent years, natural polymers have attracted great interest for the development of release systems for vaccine formulations and drug delivery. Zein, a hydrophobic proline-rich protein mixture obtained from maize, is one of the most widely used polymers, very promising for applications in tissue engineering and the parenteral delivery of bioactive agents. Still, we have a limited understanding of the interaction between zein particles and the human immune system, in particular innate immunity/inflammation, which is the first line of defense of our body. Assessing the immune safety of nanoparticles is of central importance for ensuring that nano-formulations for medical use do not cause adverse effects on human health. Here, we evaluated the capacity of zein nanoparticles to induce/modulate the innate/inflammatory response, the development of innate memory, and the macrophage polarization by using reliable in vitro systems based on human primary monocytes and monocyte-derived macrophages. We observed that zein nanoparticles do not influence any of these aspects of the innate immune/inflammatory response, suggesting its safety and its potential efficiency as a nanocarrier for drug or antigen delivery.

Innate memory against viruses.

Innate memory against viruses.

The continuous memory: A neural network with ordinary differential equations for continuous-time series analysis

Continuous-time series analysis has garnered significant research interest due to its extensive applications; however, it remains a challenging endeavor. In recent years, deep learning methods have achieved remarkable success in tasks such as time series classification and forecasting. Nevertheless, the inherent continuity of time series data has not been fully addressed, presenting a persistent obstacle to performance. Recent studies have highlighted a natural similarity between differential equations and continuous-time series, as both inherently encapsulate the concept of continuity. However, several critical issues with differential equations hinder their direct applicability to time series analysis. These issues include the elusive nature of analytical solutions, the indirect observability of the effects of differential equation parameters, and the dependence of numerical solutions on initial conditions. In this context, we propose the integration of differential equations into neural networks to serve as the continuous memory of the model. This integration imparts a continuous nature to the model, resulting in the development of an efficient deep learning architecture known as Long Short Deep Memory (LSDM). Furthermore, we analyze the characteristics of differential equations when employed as the memory component in neural networks, which leads to the proposal of a novel pre-training approach that incorporates an innate memory mechanism into these networks. Additionally, we utilize LSDM to construct a stacked architecture for processing very long time series data. The proposed model can naturally accommodate arbitrary time gaps between observations, thereby enhancing its effectiveness and suitability for continuous time series analysis tasks. Extensive experiments conducted on various real-world datasets demonstrate that the proposed model outperforms existing methods, providing a new solution to the challenges associated with continuous time series analysis.

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.

The innate memory response of macrophages to Mycobacterium tuberculosis is shaped by the nature of the antigenic stimuli.

Trained immunity (a.k.a. innate memory response) is a novel concept that has been rapidly emerging as a mechanism underpinning the non-specific immunity of innate immune cells, such as macrophages. However, the association between the nature of the stimuli and the corresponding immune correlate of trained immunity is not fully understood. Similarly, the kinetics of immunological and metabolic characteristics of macrophages upon "training" by the same antigen as primary and secondary stimuli (homologous stimulation) are not fully characterized. Furthermore, the ability of antigens such as purified protein derivative (PPD) and heat-killed-Mtb to induce trained immunity remains unknown. Similarly, the response of macrophages primed and trained by homologous stimulants to subsequent infection by pathogenic Mtb is yet to be reported. In this study, we evaluated the hypothesis that the nature of the stimuli impacts the depth and breadth of trained immunity in macrophages, which differentially affects their response to Mtb infection.

Chronic NOD2 stimulation by MDP confers protection against parthanatos through M2b macrophage polarization in RAW264.7 cells

Innate immune cells show enhanced responsiveness to secondary challenges after an initial non-related stimulation (Trained Innate Immunity, TII). Acute NOD2 activation by Muramyl-Dipeptide (MDP) promotes TII inducing the secretion of pro-inflammatory mediators, while a sustained MDP-stimulation down-regulates the inflammatory response, restoring tolerance. Here we characterized in-vitro the response of murine macrophages to lipopolysaccharide (LPS) challenge under NOD2-chronic stimulation. RAW264.7 cells were trained with MDP (1 μg/ml, 48 h) and challenged with LPS (5 μg/ml, 24 h). Trained cells formed multinucleated giant cells with increased phagocytosis rates compared to untrained/challenged cells. They showed a reduced mitochondrial activity and a switch to aerobic glycolysis. TNF-α, ROS and NO were upregulated in both trained and untrained cultures (MDP+, MDP- cells, p > 0.05); while IL-10, IL-6 IL-12 and MHCII were upregulated only in trained cells after LPS challenge (MDP + LPS+, p < 0.05). A slight upregulation in the expression of B7.2 was also observed in this group, although differences were not statistically significant. MDP-training induced resistance to LPS challenge (p < 0.01). The relative expression of PARP-1 was downregulated after the LPS challenge, which may contribute to the regulatory milieu and to the innate memory mechanisms exhibited by MDP-trained cells. Our results demonstrate that a sustained MDP-training polarizes murine macrophages towards a M2b profile, inhibiting parthanatos. These results may impact on the development of strategies to immunomodulate processes in which inflammation should be controlled.

Innate and germline immune memory: specificity and heritability of the ancient immune mechanisms for adaptation and survival.

The immune memory is one of the defensive strategies developed by both unicellular and multicellular organisms for ensuring their integrity and functionality. While the immune memory of the vertebrate adaptive immune system (based on somatic recombination) is antigen-specific, encompassing the generation of memory T and B cells that only recognize/react to a specific antigen epitope, the capacity of vertebrate innate cells to remember past events is a mostly non-specific mechanism of adaptation. This "innate memory" can be considered as germline-encoded because its effector tools (such as innate receptors) do not need somatic recombination for being active. Also, in several organisms the memory-related information is integrated in the genome of germline cells and can be transmitted to the progeny for several generations, but it can also be erased depending on the environmental conditions. Overall, depending on the organism, its environment and its living habits, innate immune memory appears to be a mechanism for achieving better protection and survival against repeated exposure to microbes/stressful agents present in the same environment or occurring in the same anatomical district, able to adapt to changes in the environmental cues. The anatomical and functional complexity of the organism and its lifespan drive the generation of different immune memory mechanisms, for optimal adaptation to changes in the living/environmental conditions. The concept of innate immunity being non-specific needs to be revisited, as a wealth of evidence suggests a significant degree of specificity both in the primary immune reaction and in the ensuing memory-like responses. This is clearly evident in invertebrate metazoans, in which distinct scenarios can be observed, with both non-specific (immune enhancement) or specific (immune priming) memory-like responses. In the case of mammals, there is evidence that some degree of specificity can be attained in different situations, for instance as organ-specific protection rather than microorganism-specific reaction. Thus, depending on the challenges and conditions, innate memory can be non-specific or specific, can be integrated in the germline and transmitted to the progeny or be short-lived, thereby representing an exceptionally plastic mechanism of defensive adaptation for ensuring individual and species survival.

GM-CSF receptor expression determines opposing innate memory phenotypes at different stages of myelopoiesis

AbstractInflammatory responses must be tightly coordinated with the activation of emergency myelopoiesis to produce potent myeloid cells that fight infection without causing excessive host damage. Here, we show that granulocyte-macrophage colony-stimulating factor (GM-CSF) programs myeloid-committed progenitors to produce trained macrophages (increased cytokine response), but programs the upstream noncommitted LKS+ progenitors (defined as Lin− c-Kit+ Sca-1+ cells) to produce tolerized macrophages (decreased cytokine response). In myeloid progenitors, GM-CSF strongly activates signal transducer and activator of transcription 5 (STAT5), Ras-Raf-extracellular signal regulated kinase (ERK), and Akt-mTOR signaling pathways, which are essential to establish a training program, whereas in LKS+ progenitors, GM-CSF induces NF-κB translocation to the nucleus to establish a tolerization program. These differences arise from higher GM-CSF receptor expression in myeloid progenitors compared with LKS+ cells. We demonstrate that β-catenin regulation of NF-κB nuclear translocation is central in this process. In myeloid progenitors, glycogen synthase kinase 3 (GSK3) inactivation by strong ERK and phosphatidylinositol 3 kinase (PI3K)-Akt signaling increases cytoplasmic β-catenin levels to block NF-κB nuclear translocation. In contrast, when ERK and PI3K-Akt signaling are weak, active GSK3 causes a decrease in β-catenin, allowing NF-κB nuclear translocation in LKS+ progenitors. Finally, GM-CSF-induced LKS+ tolerization takes place in several murine models of trained immunity and in human CD34+ CD38− progenitors. Our study reveals that in addition to activating myelopoiesis, GM-CSF also programs early and immediate myeloid progenitors to produce opposing immune memory phenotypes. We propose that the inflammatory response from immediate myeloid progenitors may be balanced by the tolerized phenotype of early progenitors, thus providing a mechanism for appropriate resolution of inflammation and protection against a prolonged cytokine storm.

The causes and consequences of trained immunity in myeloid cells.

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.

Interferons and epigenetic mechanisms in training, priming and tolerance of monocytes and hematopoietic progenitors.

Training and priming of innate immune cells involve preconditioning by PAMPs, DAMPs, and/or cytokines that elicits stronger induction of inflammatory genes upon secondary challenge. Previous models distinguish training and priming based upon whether immune activation returns to baseline prior to secondary challenge. Tolerance is a protective mechanism whereby potent stimuli induce refractoriness to secondary challenge. Training and priming are important for innate memory responses that protect against infection, efficacy of vaccines, and maintaining innate immune cells in a state of readiness; tolerance prevents toxicity from excessive immune activation. Dysregulation of these processes can contribute to pathogenesis of autoimmune/inflammatory conditions, post-COVID-19 hyperinflammatory states, or sepsis-associated immunoparalysis. Training, priming, and tolerance regulate similar "signature" inflammatory genes such as TNF, IL6, and IL1B and utilize overlapping epigenetic mechanisms. We review how interferons (IFNs), best known for activating JAK-STAT signaling and interferon-stimulated genes, also play a key role in regulating training, priming, and tolerance via chromatin-mediated mechanisms. We present new data on how monocyte-to-macrophage differentiation modulates IFN-γ-mediated priming, affects regulation of AP-1 and CEBP activity, and attenuates superinduction of inflammatory genes. We present a "training-priming continuum" model that integrates IFN-mediated priming into current concepts about training and tolerance and proposes a central role for STAT1 and IRF1.

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.

Invertebrate Immunity, Natural Transplantation Immunity, Somatic and Germ Cell Parasitism, and Transposon Defense.

While the vertebrate immune system consists of innate and adaptive branches, invertebrates only have innate immunity. This feature makes them an ideal model system for studying the cellular and molecular mechanisms of innate immunity sensu stricto without reciprocal interferences from adaptive immunity. Although invertebrate immunity is evolutionarily older and a precursor of vertebrate immunity, it is far from simple. Despite lacking lymphocytes and functional immunoglobulin, the invertebrate immune system has many sophisticated mechanisms and features, such as long-term immune memory, which, for decades, have been exclusively attributed to adaptive immunity. In this review, we describe the cellular and molecular aspects of invertebrate immunity, including the epigenetic foundation of innate memory, the transgenerational inheritance of immunity, genetic immunity against invading transposons, the mechanisms of self-recognition, natural transplantation, and germ/somatic cell parasitism.

BCG immunization induces CX3CR1hi effector memory T cells to provide cross-protection via IFN-γ-mediated trained immunity.

After a century of using the Bacillus Calmette-Guérin (BCG) vaccine, our understanding of its ability to provide protection against homologous (Mycobacterium tuberculosis) or heterologous (for example, influenza virus) infections remains limited. Here we show that systemic (intravenous) BCG vaccination provides significant protection against subsequent influenza A virus infection in mice. We further demonstrate that the BCG-mediated cross-protection against influenza A virus is largely due to the enrichment of conventional CD4+ effector CX3CR1hi memory αβ T cells in the circulation and lung parenchyma. Importantly, pulmonary CX3CR1hi T cells limit early viral infection in an antigen-independent manner via potent interferon-γ production, which subsequently enhances long-term antimicrobial activity of alveolar macrophages. These results offer insight into the unknown mechanism by which BCG has persistently displayed broad protection against non-tuberculosis infections via cross-talk between adaptive and innate memory responses.

Increase in the incidence of invasive bacterial infections following the COVID-19 pandemic: potential links with decreased herd trained immunity - a novel concept in medicine.

A global increase in the incidence of various infectious diseases has been observed since the end of the COVID‑19 pandemic. This may be due to 2 independent phenomena. One of them is impaired immunity of long‑COVID patients. The second (major) one is associated with long‑term isolation of many people during the global pandemic‑related lockdown, resulting in an extreme reduction of exposure to natural environmental human microbiota. This, in turn, led to a silencing state of the body's defense systems, including a decline of the prepandemic trained immunity (innate memory), which only persists for weeks to months after exposure to a pathogen. This decrease in the performance of trained immunity may be especially important for morbidity of infectious diseases without currently available vaccines, such as invasive group A Streptococcus pyogenes (GAS) infections, primarily streptococcal toxic shock syndrome. This review discusses data that support an important role of trained macrophages in host defense, and demonstrates the potential clinical implications of β‑glucan, the major inducer of trained macrophages, for prophylactic and therapeutic use in individuals with impaired personal innate immunity. Altogether, it might be speculated that trained innate immunity within an entire population can lead to the development of herd trained immunity (HTI), a newly‑coined medical term. HTI can supplement classic, antigen‑specific herd immunity (memory B and T cells), and it plays a key role in preventing the spread of various infectious diseases, including invasive GAS infections. Unfortunately, the global HTI has been overthrown during the COVID‑19 pandemic; however, it should be restored shortly.

Current Understanding of Bacillus Calmette-Guérin-Mediated Trained Immunity and Its Perspectives for Controlling Intracellular Infections.

The bacillus Calmette-Guérin (BCG) is an attenuated bacterium derived from virulent Mycobacterium bovis. It is the only licensed vaccine used for preventing severe forms of tuberculosis in children. Besides its specific effects against tuberculosis, BCG administration is also associated with beneficial non-specific effects (NSEs) following heterologous stimuli in humans and mice. The NSEs from BCG could be related to both adaptive and innate immune responses. The latter is also known as trained immunity (TI), a recently described biological feature of innate cells that enables functional improvement based on metabolic and epigenetic reprogramming. Currently, the mechanisms related to BCG-mediated TI are the focus of intense research, but many gaps are still in need of elucidation. This review discusses the present understanding of TI induced by BCG, exploring signaling pathways that are crucial to a trained phenotype in hematopoietic stem cells and monocytes/macrophages lineage. It focuses on BCG-mediated TI mechanisms, including the metabolic-epigenetic axis and the inflammasome pathway in these cells against intracellular pathogens. Moreover, this study explores the TI in different immune cell types, its ability to protect against various intracellular infections, and the integration of trained innate memory with adaptive memory to shape next-generation vaccines.

Fatty acid desaturation and lipoxygenase pathways support trained immunity

Infections and vaccines can induce enhanced long-term responses in innate immune cells, establishing an innate immunological memory termed trained immunity. Here, we show that monocytes with a trained immunity phenotype, due to exposure to the Bacillus Calmette-Guérin (BCG) vaccine, are characterized by an increased biosynthesis of different lipid mediators (LM) derived from long-chain polyunsaturated fatty acids (PUFA). Pharmacological and genetic approaches show that long-chain PUFA synthesis and lipoxygenase-derived LM are essential for the BCG-induced trained immunity responses of human monocytes. Furthermore, products of 12-lipoxygenase activity increase in monocytes of healthy individuals after BCG vaccination. Grasping the underscoring lipid metabolic pathways contributes to our understanding of trained immunity and may help to identify therapeutic tools and targets for the modulation of innate immune responses.

Routing Innate and Adaptive Immune response against M. tuberculosis and boosting Mycobacterium bovis Bacillus Calmette Güerin (BCG) vaccine immunity through prime boost protocols

Tuberculosis (Tb) is still a global health problem, especially in developing countries. Several factors contribute to this, among them the increasing multidrug resistance strains, the dangerous liaisons with other intracellular pathogens, such as HIV, and more recently, SARS-CoV2 pandemics. There are many aspects that remain to understand the bacterial molecular mechanism of pathogenicity and the immune response induced by the interaction of M. tuberculosis (MTb) with the host. The official and current vaccine based on the attenuated Mycobacterium bovis Bacillus Calmette Guerin (BCG) is protective against several forms of Tb meningitis and Miliary TB or disseminated disease in young children. However, it fails to protect young and adult individuals. There are several new promising candidates for vaccines to replace or boost BCG-induced immunity. Several evidences exist from humans and mice on the role of the trained innate memory of monocytes and NK cells, on the second encounter with the same mycobacterial pathogen or other respiratory pathogens. This type of immune response is nonspecific and independent of the T and B cells. Thus, BCG vaccination is a double immunogen that activate specific immune responses and is also able to stimulate nonspecific immune responses. Here, it is outlined the host immunity against MTb, the potential of BCG vaccination and prime boost protocols for routing innate and adaptive immune responses in TB.

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.

In Vivo Zymosan Treatment Induces IL15-Secreting Macrophages and KLRG1-Expressing NK Cells in Mice.

Beta-glucan (β-glucan) is a natural polysaccharide produced by fungi, bacteria, and plants. Although it has been reported that β-glucan enhances innate immune memory responses, it is unclear whether different types of β-glucans display similar immune effects. To address this issue, we employed zymosan (β-1,3-glycosidic linkage) and pustulan (β-1,6-glycosidic linkage) to investigate their in vivo effects on innate memory immune responses. We examined the changes of innate memory-related markers in macrophages and natural killer (NK) cells, two immune cell types that display innate memory characteristics, at two different time points (16 h and 7 days) after β-glucan stimulation. We found that short-term (16 h) zymosan treatment significantly induced macrophages to upregulate IL15 production and increased surface IL15Rα expression on NK cells. In addition, long-term (7 days) zymosan treatment significantly induced macrophages to upregulate the expression of innate memory-related markers (e.g., TNFα, HIF1α, and mTOR) and induced NK cells to express enhanced levels of KLRG1, known as an innate memory-like marker. Our results provide support that zymosan can be an effective adjuvant to promote innate memory immune responses, providing a bridge between innate and adaptive immune cells to enhance various immune responses such as those directed against tumors.

Lsd1 safeguards T-cell development via suppressing endogenous retroelements and interferon responses.

The histone demethylase Lsd1 has been shown to play multiple essential roles in mammalian biology. However, its physiological functions in thymocyte development remain elusive. We observed that the specific deletion of Lsd1 in thymocytes caused significant thymic atrophy and reduced peripheral T cell populations with impaired proliferation capacity. Single-cell RNA sequencing combined with strand-specific total RNA-seq and ChIP-seq analysis revealed that ablation of Lsd1 led to the aberrant derepression of endogenous retroelements, which resulted in a viral mimicry state and activated the interferon pathway. Furthermore, the deletion of Lsd1 blocked the programmed sequential down-regulation of CD8 expression at the DP→CD4+CD8lo stage and induced an innate memory phenotype in both thymic and peripheral T cells. Single-cell TCR sequencing revealed the kinetics of TCR recombination in the mouse thymus. However, the preactivation state after Lsd1 deletion neither disturbed the timeline of TCR rearrangement nor reshaped the TCR repertoire of SP cells. Overall, our study provides new insight into the function of Lsd1 as an important maintainer of endogenous retroelement homeostasis in early T-cell development.