Long-term histone lactylation connects metabolic and epigenetic rewiring in innate immune memory.

Mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via mTORC2/HK1-mediated metabolic rewiring

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel human coronavirus12, has infected close to 22 million people and killed about 0.77 million people in more than 200 countries (as of August 18, 2020)3. Given the fact that SARS-CoV-2 poses an unprecedented threat in terms of transmission and mortality, the World Health Organization has geared up efforts to control, contain and prevent coronavirus disease 2019 (COVID-19). The development of a vaccine has a high attrition rate and involves linear steps of clinical trial and evaluation. For those systems that have been tested on humans previously, parallel testing can involve both animals and phase I human trials4. Although some of the potential COVID-19 vaccine candidates have made it through phase I and II clinical trials, mass availability of COVID-19 vaccine could only be possible by 20215. Repurposing of the existing drugs and development of vaccines are thus feasible options to protect people from the severity of the COVID-19 pandemic. Host immunity plays a crucial role in the elimination of viruses and prevents disease progression. Strategies to boost the same, especially during incubation and non-severe phase of SARS-CoV-2 infection, can be a viable option to check disease severity. Bacille Calmette-Guerin (BCG) induces non-specific protection against a range of bacteria and viruses6. Therefore, it is worth exploring the immunostimulatory and protective potential of BCG against SARS-CoV-2 infection. BCG is a live attenuated strain of Mycobacterium bovis widely used as a vaccine for the prevention of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb). Intradermal/subcutaneous delivery of BCG vaccine protects against disseminated forms of TB in children and provides variable protection against pulmonary TB in adults78. Recent reports suggest that the efficacy of BCG can be improved by selective delipidation of the outer membrane or alternative route of delivery910. BCG immunization elicits non-specific immunological outcomes that prevent general infections and reduce mortality caused by unrelated pathogens6, besides amplifying responses to other paediatric vaccines11. BCG has long been employed as an immunotherapeutic agent or adjuvant for preventing recurrence and progression of bladder cancer12. A recent report13 suggests that the potential of BCG in preventing SARS-CoV-2 infection may be validated among hyper-susceptible population subset such as the front line healthcare personnel and the elderly people. Clinical trials to test the efficacy of BCG in boosting the immune system against SARS-CoV-2, have been initiated in the Netherlands (NCT04417335), Australia (NCT04327206) and Columbia (NCT04362124) (http://www.clinicaltrials.gov). Host pattern recognition receptors (PRRs), expressed by innate immune cells, interact with pathogen-associated molecular patterns (PAMPs) of viruses and initiate innate immune response against viral pathogens. Many of the PAMPs are common among different species of bacteria and viruses. Multiple families of PRRs such as NOD-like receptors, RIG-l-like receptors and toll-like receptors detect viral proteins, which in turn induce interferons and cytokines, aiding in the elimination of the virus. It is tempting to speculate that immune response against BCG may cross-recognize SARS-CoV-2-associated PAMPs and confer immunity against this infection. Prior immunization with BCG or re-vaccination with BCG in adults can induce 'trained immunity' (innate immune memory), a term used to describe the possible mechanism(s) underlying heterologous protection induced by BCG against non-mycobacterial antigens. The trained immunity is a kind of conditioning of the innate immune cells, mainly monocytes/macrophages/natural killer cells, to undergo specific epigenetic changes (including de-methylation/de-acetylation) in the genes associated with mounting a robust and non-specific immune response14, resulting in a heightened recall response by primed innate immune cells upon a second encounter to the same/different/broad range of unrelated microbial PAMPs. The degree of immunological response has been correlated with the chromatin accessibility at the genome regions controlling immune response15. Regulatory RNA species, including long non-coding RNAs and microRNAs, have also emerged as the regulators of epigenetic reprogramming of innate immune cells, besides substantial rewiring of host metabolic landscape with a predominant shift from oxidative phosphorylation to glycolysis14. In humans, limited documentation exists on the impact of unintended protective effects of BCG against viral infections. BCG-immunized mice exhibited higher resistance to influenza viral infection challenge as compared to the unvaccinated mice16. Arts et al17 reported significantly low levels of viraemia in BCG-vaccinated human volunteers challenged with experimental yellow fever virus (attenuated strain), and the BCG-induced protection correlated with increased production of interleukin (IL)-1β. Consistent with these studies, BCG re-vaccination induced innate and adaptive immune responses in adult TB patients18 also suggest its utility to renew response against mycobacteria and other unrelated pathogens possibly via induction of trained immunity. MTBVAC, a live attenuated Mtb vaccine strain, provides long-term non-specific immunological effect on innate trained immunity in addition to adaptive immune response19. Kleinnijenhuis et al20 have demonstrated that the levels of pro-inflammatory cytokines such as IL-1β, tumour necrosis factor-α and IL-6 remain elevated up to one year post-BCG vaccination and subsequently waned. In line with previous reports showing beneficial non-specific immune potentiating effects of BCG vaccine, one can logically speculate that BCG vaccination (or re-vaccination in countries with universal BCG immunization) may lead to improved clinical outcomes in viral or other respiratory tract infections, including SARS-CoV-2. A recent ecological study has suggested that countries where BCG is part of the immunization schedule, the extent of mortality and morbidity due to COVID-19 is significantly reduced, and may be able to contain the spread of SARS-CoV-2 better than those countries which did not have BCG vaccination21. The differences in the number of SARS-CoV-2-infected cases between countries adhering to the universal BCG vaccination policy and those where universal BCG vaccination is not a policy were evaluated, based on the data of COVID-19 cases across the globe (https://www.coronavirus.jhu.edu/map.html) and BCG vaccination status from BCG World Atlas database22. It was observed that in countries where BCG was a part of the immunization schedule, COVID-19 cases/100,000 population were almost similar to those where BCG was not a part of universal vaccination schedule (Figure A). However, the cause-specific death rate/100,000 population was significantly lower in countries having BCG immunization policy, compared to those where a universal BCG vaccination policy did not exist (Figure B). This suggests that BCG vaccination-induced non-specific immunity may be associated with the mitigation of disease severity in COVID-19-pandemic areas.Figure: Scatter plot showing differences in the number of confirmed cases of SARS-CoV-2 infection (A) and cause-specific death rate (B) per 100,000 population between countries having universal BCG vaccination policy (BCG+ countries) and countries that do not have a universal BCG vaccination policy (BCG− countries). The description of the different colour and symbols is as follows: European countries (sky blue square), Asian countries (red circle), Middle East countries (yellow square), African countries (brown diamond), North American countries (purple upright triangle), South American countries (green inverted triangle) and Australia (grey hexagon). The countries represented in the graph are those with >3000 confirmed COVID-19 cases, as of July 9, 2020. The significance of the two data sets was tested using unpaired, non-parametric Student's t test using GraphPad Prism software version 6.7 (San Diego, CA, USA) and is shown on the top of the plot (ns, non-significant; *** P<0.001).Source: http://www.bcgatlas.org; http://www.gisanddata.maps.arcgis.com.We hypothesized that prior BCG vaccination status was associated with the extent of the COVID-19 epidemic. Because COVID-19 cases started or peaked at different time periods and the infection or death rate stabilized differently among various countries, it posed a limitation to the current study. Our results, therefore, are a pointer rather than final conclusion about the role of BCG in arresting the COVID-19 pandemic. To have a more definitive picture, the association of BCG vaccination with the protection or recovery from the SARS-CoV-2 infection must be reinforced with the data available at the individual level. A more comprehensive comparison of BCG-vaccinated individuals on the basis of age at the time of vaccination or re-vaccination may provide critical evidence and logical conclusion for ascertaining the use of BCG in the prevention of COVID-19. A significant amount of work has been done to engineer BCG to enhance its immune-boosting and protective properties. VPM1002, a genetically engineered BCG, exhibits improved immunogenicity23, has completed phase I clinical trial in Germany (NCT00749034) and phase II clinical trial in South Africa (NCT01479972) and is currently in phase III clinical trials in India (NCT03152903) for assessing the prevalence of TB recurrence in drug-treated individuals. A separate phase III trial of VPM1002 (NCT04387409) has also been initiated to assess healthcare professionals' absenteeism during the COVID-19 pandemic in Germany (Table). VPM1002 was engineered to survive within the phagosome (unlike BCG), and was equipped with listeriolysin (from Listeria) to perforate phagosomal membrane. VPM1002 has also been reported to prevent recurrence of bladder tumours, highlighting its non-specific benefits. Another recombinant BCG strain overexpressing STING (stimulator of interferon genes)-agonist has shown significantly augmented pro-inflammatory cytokine response and protective efficacy in mice and guinea pigs challenged with Mtb24.Table: Role of bacille Calmette-Guerin (BCG), recombinant BCG, attenuated Mycobacterium tuberculosis (Mtb) or M. indicus pranii (MIP) as immunotherapy and possible intervention against COVID-19Alternatively, immunogenic components of BCG, such as muramyl dipeptide, can also be tested as an adjunct therapy for immune stimulation in those at high-risk to be affected with COVID-19. In addition to being broadly protective, safe and immunogenic, BCG is cost-effective, easy to produce in bulk and, therefore, may be suitable in terms of both availability and affordability. However, before exploiting BCG-induced training of innate immune responses against infections by unrelated pathogens, several potentially confounding factors such as host genetic polymorphisms, endemicity to other viral/bacterial infections and route of immunization need to be examined. Of particular note is the association of BCG vaccination, with some adverse effects such as formation of abscess and lymphadenitis and local cutaneous inflammation2829. Intravesical BCG therapy for the treatment of non-invasive bladder cancer has resulted in short period of fever and discomfort in majority of the patients30. Clinical trials for testing the efficacy of BCG, administered (or re-administration) through a conventional or alternate route910, against SARS-CoV-2 can be initiated as an interim intervention against COVID-19. The pros and cons of diverting the stock of BCG as a temporary measure for non-specific protection till actual vaccines for COVID-19 are commercially available must be deliberated upon, as it should not limit the supply of BCG for the people in TB-infected endemic regions. Therefore, it is important to explore agents similar to BCG that can act as immunomodulator. In this regard, it is equally tempting to suggest another mycobacteria discovered in India, Mycobacterium indicus pranii (MIP)31, earlier known as Mw. MIP has been found to be a strong immunomodulator with proven utility as an adjunct therapy for leprosy treatment25, category II TB26 in humans and in inducing tumour regression27, and possibly functions by invoking trained immunity (Table).

Innate immune cells experience long lasting metabolic and epigenetic changes after an encounter with specific stimuli. This facilitates enhanced immune responses upon secondary exposition to both the same and unrelated pathogens, a process termed trained immunity. Trained immunity-based vaccines (TIbV) are vaccines able to induce innate immune memory, thus conferring heterologous protection against a broad range of pathogens. While trained immunity has been well documented in the context of infections and multiple immune-mediated diseases, the role of innate immune memory and its contribution to the initiation and maintenance of chronic allergic diseases remains poorly understood. Over the last years, different studies attempting to uncover the role of trained immunity in allergy have emerged. Exposition to environmental factors impacting allergy development such as allergens or viruses induces the reprogramming of innate immune cells to acquire a more pro-inflammatory phenotype in the context of asthma or food allergy. Several studies have convincingly demonstrated that prevention of viral infections using TIbV contributes to reduce wheezing attacks in children, which represent a high-risk factor for asthma development later in life. Innate immune cells trained with specific stimuli might also acquire anti-inflammatory features and promote tolerance, which may have important implications for chronic inflammatory diseases such as allergies. Recent findings showed that allergoid-mannan conjugates, which are next generation vaccines for allergen-specific immunotherapy (AIT), are able to reprogram monocytes into tolerogenic dendritic cells by mechanisms depending on metabolic and epigenetic rewiring. A better understanding of the underlying mechanisms of trained immunity in allergy will pave the way for the design of novel trained immunity-based allergen vaccines as potential alternative strategies for the prevention and treatment of allergic diseases.

How are airborne allergens remembered by the immune system?

Vaccine development against tuberculosis (TB) is based on the induction of adaptive immune responses endowed with long-term memory against mycobacterial antigens. Memory B and T cells initiate a rapid and robust immune response upon encounter with Mycobacterium tuberculosis, thus achieving long-lasting protection against infection. Recent studies have shown, however, that innate immune cell populations such as myeloid cells and NK cells also undergo functional adaptation after infection or vaccination, a de facto innate immune memory that is also termed trained immunity. Experimental and epidemiological data have shown that induction of trained immunity contributes to the beneficial heterologous effects of vaccines such as bacille Calmette-Guérin (BCG), the licensed TB vaccine. Moreover, increasing evidence argues that trained immunity also contributes to the anti-TB effects of BCG vaccination. An interaction among immunological signals, metabolic rewiring, and epigenetic reprogramming underlies the molecular mechanisms mediating trained immunity in myeloid cells and their bone marrow progenitors. Future studies are warranted to explore the untapped potential of trained immunity to develop a future generation of TB vaccines that would combine innate and adaptive immune memory induction.

HIV virus can persist in a latent but activatable chronic state in resting CD4 +T-cells and macrophages that are more persistent, leading to the requirement of lifelong combination antiretroviral therapy (cART). The latency of HIV-1 in macrophage cells is associated with increased chromatin condensation induced by histone methylation at HIV-1 integration sites. Innate immune training of macrophages has been shown to relieve chromosome condensation through epigenetic rewiring. We hypothesized that training of macrophages could enhance the reactivation of HIV-1 by opening the chromatin to facilitate transcription in response to Latency Reversing Agents (LRAs). We used beta-glucan, Syk inhibitor and Rutaecarpine to train the THP89GFP cell line, which is an experimental model for latently infected HIV-1 monocytes. We found that these trained THP89GFP cells have higher transcription of HIV-1 specific genes in response to PMA stimulation. To check the epigenetic changes associated with training in the proviral HIV-1 DNA of THP89GFP, we performed a chromatin immunoprecipitation for the histone mark, H3K27Ac. Consistent with the higher induction of transcription of HIV-1 genes, we observed that multiple regions of the HIV-1 LTR had higher deposition of H3K27Ac in the trained macrophages. Our studies thus show that induction of trained innate immunity may be a viable approach to the reactivation of latently infected HIV-1. This finding supports the hypothesis that innate immune training stimuli could be developed as novel candidates for enhancing reactivation of latently infected HIV-1, which may facilitate elimination of macrophage reservoirs. This work was supported by the Intramural Research Program of NIAID, NIH This work was supported by the Intramural Research Program of NIAID, NIH and Office of AIDS research.

The Bacille Calmette-Guerin (BCG) vaccine is a well-established inducer of innate immune memory (also termed trained immunity), causing increased cytokine production upon heterologous secondary stimulation. Innate immune responses are known to be influenced by season, but whether seasons impact induction of trained immunity is not known. To explore the influence of season on innate immune memory induced by the BCG vaccine, we vaccinated healthy volunteers with BCG either during winter or spring. Three months later, we measured the ex vivo cytokine responses against heterologous stimuli, analyzed gene expressions and epigenetic signatures of the immune cells, and compared these with the baseline before vaccination. BCG vaccination during winter induced a stronger increase in the production of pro-inflammatory cytokines by peripheral blood mononuclear cells (PBMCs) upon stimulation with different bacterial and fungal stimuli, compared to BCG vaccination in spring. In contrast, winter BCG vaccination resulted in lower IFNγ release in PBMCs compared to spring BCG vaccination. Furthermore, NK cells of the winter-vaccinated people had a greater pro-inflammatory cytokine and IFNγ production capacity upon heterologous stimulation. BCG had only minor effects on the transcriptome of monocytes 3 months later. In contrast, we identified season-dependent epigenetic changes in monocytes and NK cells induced by vaccination, partly explaining the higher immune cell reactivity in the winter BCG vaccination group. These results suggest that BCG vaccination during winter is more prone to induce a robust trained immunity response by activating and reprogramming the immune cells, especially NK cells. (Dutch clinical trial registry no. NL58219.091.16)

Promotion of trained innate immunity by nanoparticles

Immunological memory was long considered a trait exclusive to cells of the adaptive immune system. However, recent studies have shown that after activation of the innate immune system, innate immune cells may undergo long-term functional reprogramming characterized by the ability to mount either a stronger or attenuated inflammatory response upon reactivation. This phenomenon, which has been termed trained immunity and is a de facto innate immune memory, is regulated by a network of integrated metabolic and epigenetic rewiring. The endogenous mediators that modulate trained immunity in the host are only partially understood, but increasing evidence supports the concept that the interleukin (IL)-1 family of cytokines plays an important role. In this review, we will highlight key findings from studies that provide insight into the multifaceted roles of members of the IL-1 family for trained immunity. Finally, we will discuss how the recent advances of our understanding on the role of IL-1 cytokines in this field may lead to new therapeutic strategies for treatment of common conditions, such as IL-1-driven autoinflammatory diseases.

Trained innate immunity and resistance to Mycobacterium tuberculosis infection

Trained immunity is the ability of the innate immune system to mount a heightened response to an environmental stimulus after a previous encounter with a noxious trigger. This effect is mediated by metabolic rewiring and epigenetic reprogramming in innate immune cells. In the context of neuroinflammation, trained immunity may represent a major contributor to the pathogenesis of neurological diseases, exerting both detrimental and potentially beneficial effects. While the general mechanisms and systemic implications of trained immunity are widely discussed, evidence in central nervous system (CNS) diseases remains fragmented and largely confined to individual pathological conditions. As a result, a comprehensive framework integrating these findings and identifying shared mechanisms across neurological disorders is still lacking. In this review, we explore the concept of trained immunity with a focus on neuroinflammatory and neurodegenerative diseases, synthetizing evidence from multiple CNS pathologies, including multiple sclerosis, Alzheimer's disease, Parkinson's disease, and cerebrovascular disorders. We first critically examine preclinical and experimental studies addressing innate immune memory in the CNS and subsequently integrate these findings with emerging clinical evidence, aiming to identify convergent mechanisms and disease-relevant immune memory signatures. Finally, we discuss potential therapeutic targets identified in preclinical settings and outline key unresolved issues, including the nature of triggering stimuli, thresholds, and temporal dynamics shaping innate immune memory in the CNS. By highlighting current limitations and defining critical questions for future research, this review presents a unifying perspective on trained immunity in neurological diseases and underscores the translational potential to modulate neuroinflammation and to influence disease progression.

Bacillus Calmette-Guérin (BCG) is a licensed prophylactic vaccine against tuberculosis (TB). Current TB vaccine efforts focus on improving BCG effects through recombination or genetic attenuation and/or boost with different vaccines. Recent years, it was revealed that BCG could elicit non-specific heterogeneous protection against other pathogens such as viruses through a process termed trained immunity. Previously, we constructed a recombinant BCG (rBCG-DisA) with elevated c-di-AMP as endogenous adjuvant by overexpressing di-adenylate cyclase of Mycobacterium tuberculosis DisA, and found that rBCG-DisA induced enhanced immune responses by subcutaneous route in mice after M. tuberculosis infection. In this study, splenocytes from rBCG-DisA immunized mice by intravenous route (i.v) elicited greater proinflammatory cytokine responses to homologous and heterologous re-stimulations than BCG. After M. tuberculosis infection, rBCG-DisA immunized mice showed hallmark responses of trained immunity including potent proinflammatory cytokine responses, enhanced epigenetic changes, altered lncRNA expressions and metabolic rewiring in bone marrow cells and other tissues. Moreover, rBCG-DisA immunization induced higher levels of antibodies and T cells responses in the lung and spleen of mice after M. tuberculosis infection. It was found that rBCG-DisA resided longer than BCG in the lung of M. tuberculosis infected mice implying prolonged duration of vaccine efficacy. Then, we found that rBCG-DisA boosting could prolong survival of BCG-primed mice over 90 weeks against M. tuberculosis infection. Our findings provided in vivo experimental evidence that rBCG-DisA with c-di-AMP as endogenous adjuvant induced enhanced trained immunity and adaptive immunity. What’s more, rBCG-DisA showed promising potential in prime-boost strategy against M. tuberculosis infection in adults.

Chronic inflammation is a major driver of atherosclerotic cardiovascular disease, and therapeutics that target inflammation reduce clinical cardiac events beyond levels seen with conventional strategies targeting cholesterol alone. Recent findings suggest innate immune cells maintain ‘memory’ of prior exposure to inflammatory stimuli, a phenomenon known as ‘trained immunity’. In response to inflammatory stimuli, macrophages undergo metabolic and epigenetic rewiring that primes them to mount an augmented response upon a second exposure. Oxidized low-density lipoproteins (oxLDL) have recently been shown to be potent triggers of trained immunity. While trained immunity has been shown to promote inflammation, little is known about how immune training impacts efferocytosis. Therefore, we hypothesize that trained immunity in macrophages promotes inflammation by impairing efferocytosis. We treated murine bone marrow progenitors with oxLDL for 24 hours, then washed and differentiated them into macrophages (BMDMs). Upon assessing efferocytosis, trained BMDMs were able to ingest a first apoptotic cell (AC) better than untrained BMDMs yet had an impaired ability to take up additional ACs, reflecting a defect in continual efferocytosis. Using an in vivo approach, we transplanted donor bone marrow from Ldlr -/- mice fed a chow or Western diet into naïve C57BL/6 recipients. After recovery, we elicited peritoneal macrophages to assess efferocytosis and found that recipients receiving marrow from Western diet fed Ldlr -/- mice not only displayed impaired efferocytosis, but also significantly upregulated PGE 2 production, suggesting impaired resolution. To determine whether PGE 2 is a mediator of trained immunity, we primed bone marrow progenitors with PGE 2 and differentiated them into BMDMs. We found that macrophages primed with PGE 2 elaborated higher levels of inflammatory cytokines in response to LPS stimulation than controls. Overall, these findings demonstrate that oxLDL/Western diet-training impinges on the resolution program by impairing efferocytosis and are durable effects that demonstrate heritability. Future directions include determining the impact of these findings on the development of atherosclerosis.

Innate immune cells can develop exacerbated immunologic response and long-term inflammatory phenotype following brief exposure to endogenous or exogenous insults, which leads to an altered response towards a second challenge after the return to a nonactivated state. This phenomenon is known as trained immunity (TI). TI is not only important for host defense and vaccine response but also for chronic inflammations such as cardiovascular and metabolic diseases such as atherosclerosis. TI can occur in innate immune cells such as monocytes/macrophages, natural killer cells, endothelial cells (ECs), and nonimmune cells, such as fibroblast. In this brief review, we analyze the significance of TI in ECs, which are also considered as innate immune cells in addition to macrophages. TI can be induced by a variety of stimuli, including lipopolysaccharides, BCG (bacillus Calmette-Guerin), and oxLDL (oxidized low-density lipoprotein), which are defined as risk factors for cardiovascular and metabolic diseases. Furthermore, TI in ECs is functional for inflammation effectiveness and transition to chronic inflammation. Rewiring of cellular metabolism of the trained cells takes place during induction of TI, including increased glycolysis, glutaminolysis, increased accumulation of tricarboxylic acid cycle metabolites and acetyl-coenzyme A production, as well as increased mevalonate synthesis. Subsequently, this leads to epigenetic remodeling, resulting in important changes in chromatin architecture that enables increased gene transcription and enhanced proinflammatory immune response. However, TI pathways and inflammatory pathways are separated to ensure memory stays when inflammation undergoes resolution. Additionally, reactive oxygen species play context-dependent roles in TI. Therefore, TI plays significant roles in EC and macrophage pathology and chronic inflammation. However, further characterization of TI in ECs and macrophages would provide novel insights into cardiovascular disease pathogenesis and new therapeutic targets. Graphic Abstract: A graphic abstract is available for this article.

Trained immunity is reshaping our understanding of host defense by demonstrating that innate immune cells once thought to lack memory can be reprogrammed to mount heightened responses to subsequent challenges. Unlike tolerance, differentiation, or priming, trained immunity relies on epigenetic and metabolic rewiring of resident myeloid cells, particularly in mucosal barriers such as the skin, gut, and lungs, where these cells provide continuous protection against toxins and pathogens. Here, we review recent advances showing how an initial stimulus endows monocytes and macrophages with long-lasting functional changes that can be either protective or maladaptive upon re-exposure. We highlight therapeutic opportunities that harness trained immunity to boost vaccine efficacy and discuss strategies to modulate this program in cancer and hyper-inflammatory disorders. Finally, we propose new directions for enhancing or dampening trained immunity to promote human health.