The impact of BCG dose and revaccination on trained immunity.

The innate immune system can display heterologous memory-like responses termed trained immunity after stimulation by certain vaccinations or infections. In this randomized, placebo-controlled trial, we investigated the modulation of Bacille Calmette-Guérin (BCG)-induced trained immunity by BCG revaccination or high-dose BCG administration, in comparison to a standard dose. We show that monocytes from all groups of BCG-vaccinated individuals exerted increased TNFα production after ex-vivo stimulation with various unrelated pathogens. Similarly, we observed increased amounts of T-cell-derived IFNγ after M. tuberculosis exposure, regardless of the BCG intervention. NK cell cytokine production, especially after heterologous stimulation with the fungal pathogen Candida albicans, was predominantly boosted after high dose BCG administration. Cytokine production capacity before vaccination was inversely correlated with trained immunity. While the induction of a trained immunity profile is largely dose- or frequency independent, baseline cytokine production capacity is associated with the magnitude of the innate immune memory response after BCG vaccination.

The impact of BNT162b2 mRNA vaccine on adaptive and innate immune responses.

The mRNA-based BNT162b2 protects against severe disease and mortality caused by SARS-CoV-2 via induction of specific antibody and T-cell responses. Much less is known about its broad effects on immune responses against other pathogens. Here, we investigated the adaptive immune responses induced by BNT162b2 vaccination against various SARS-CoV-2 variants and its effects on the responsiveness of immune cells upon stimulation with heterologous stimuli. BNT162b2 vaccination induced effective humoral and cellular immunity against SARS-CoV-2 that started to wane after six months. We also observed long-term transcriptional changes in immune cells after vaccination. Additionally, vaccination with BNT162b2 modulated innate immune responses as measured by inflammatory cytokine production after stimulation - higher IL-1/IL-6 release and decreased IFN-α production. Altogether, these data expand our knowledge regarding the overall immunological effects of this new class of vaccines and underline the need for additional studies to elucidate their effects on both innate and adaptive immune responses.

Induction of trained immunity by influenza vaccination - impact on COVID-19.

Non-specific protective effects of certain vaccines have been reported, and long-term boosting of innate immunity, termed trained immunity, has been proposed as one of the mechanisms mediating these effects. Several epidemiological studies suggested cross-protection between influenza vaccination and COVID-19. In a large academic Dutch hospital, we found that SARS-CoV-2 infection was less common among employees who had received a previous influenza vaccination: relative risk reductions of 37% and 49% were observed following influenza vaccination during the first and second COVID-19 waves, respectively. The quadrivalent inactivated influenza vaccine induced a trained immunity program that boosted innate immune responses against various viral stimuli and fine-tuned the anti-SARS-CoV-2 response, which may result in better protection against COVID-19. Influenza vaccination led to transcriptional reprogramming of monocytes and reduced systemic inflammation. These epidemiological and immunological data argue for potential benefits of influenza vaccination against COVID-19, and future randomized trials are warranted to test this possibility.

Intralesional Corticosteroid Administration in the Treatment of Keloids: A Scoping Review on Injection Methods.

BACKGROUND: Intralesional corticosteroid administration (ICA) is a first-line treatment for keloids. However, its clinical results are still highly variable and often suboptimal. Treatment results may strongly be influenced by various operator-dependent factors. The aim of this study was to map the details of ICA in keloids described in randomized controlled trials (RCTs), hence presenting the scientific practice of a first-line treatment for keloids in the best available evidence. SUMMARY: A systematic search was performed on PubMed, Ovid MEDLINE, Ovid EMBASE, and CENTRAL. Eligible studies were RCTs including patients with keloids treated with intralesional corticosteroids. Treatment and study design-related data were charted on a predefined form. Thirty-eight RCTs were included for data extraction. Triamcinolone acetonide was used in 37 (97.4%) studies. Dosing per cm2 could only be compared among ten (26%) studies and varied from 1 to 20 mg. The maximum dose per session varied from 20 to 80 mg. Local anesthetics were administered in seven (20%) RCTs. Treatment intervals varied from weekly to monthly, with 4 weeks most frequently (50%) used. Needle size was reported in eleven (29%) studies and varied from 26 to 30-gauge. Syringe size was specified in four (11%) studies, being 1 mL. The injection level was described in eleven (29%) studies. Blanching as endpoint was reported in ten (26%) studies. Outcome measures varied widely, from height, surface area, or volume, to Vancouver Scar Scale, Patient and Observer Scar Assessment Scale, pain and itch scores, patient satisfaction, and different efficacy rates. Only six studies had a follow-up of ≥6 months. Recurrence was identified in two studies with 18 weeks and 1 year of follow-up. Adverse events were reported in 23 (61%) studies.

Overcoming immune dysfunction in the elderly: trained immunity as a novel approach.

People with advanced age have a higher susceptibility to infections and exhibit increased mortality and morbidity as the ability of the immune system to combat infections decreases with age. While innate immune cells display functional defects such as decreased phagocytosis, chemotaxis and cytokine production, adaptive immune cells exhibit reduced receptor diversity, defective antibody production and a sharp decline in naive cell populations. Successful responses to vaccination in the elderly are critical to prevent common infections such as influenza and pneumonia, but vaccine efficacy decreases in older individuals compared with young adults. Trained immunity is a newly emerging concept that showed that innate immune cells possess non-specific immunological memory established through epigenetic and metabolic reprogramming upon encountering certain pathogenic stimuli. Clinical studies suggest that trained immunity can be utilized to enhance immune responses against infections and improve the efficiency of vaccinations in adults; however, how trained immunity responses are shaped with advanced age is still an open question. In this review, we provide an overview of the age-related changes in the immune system with a focus on innate immunity, discuss current vaccination strategies for the elderly, present the concept of trained immunity and propose it as a novel approach to enhance responses against infections and vaccinations in the elderly population.

A suppressive oligodeoxynucleotide expressing TTAGGG motifs modulates cellular energetics through the mTOR signaling pathway.

Immune-mediated inflammation must be down-regulated to facilitate tissue remodeling during homeostatic restoration of an inflammatory response. Uncontrolled or over-exuberant immune activation can cause autoimmune diseases, as well as tissue destruction. A151, the archetypal example of a chemically synthesized suppressive oligodeoxynucleotide (ODN) based on repetitive telomere-derived TTAGGG sequences, was shown to successfully down-regulate a variety of immune responses. However, the degree, duration and breadth of A151-induced transcriptome alterations remain elusive. Here, we performed a comprehensive microarray analysis in combination with Ingenuity Pathway Analysis (IPA) using murine splenocytes to investigate the underlying mechanism of A151-dependent immune suppression. Our results revealed that A151 significantly down-regulates critical mammalian target of rapamycin (mTOR) activators (Pi3kcd, Pdpk1 and Rheb), elements downstream of mTOR signaling (Rps6ka1, Myc, Stat3 and Slc2a1), an important component of the mTORC2 protein complex (Rictor) and Mtor itself. The effects of A151 on mTOR signaling were dose- and time-dependent. Moreover, flow cytometry and immunoblotting analyses demonstrated that A151 is able to reverse mTOR phosphorylation comparably to the well-known mTOR inhibitor rapamycin. Furthermore, Seahorse metabolic assays showed an A151 ODN-induced decrease in both oxygen consumption and glycolysis implying that a metabolically inert state in macrophages could be triggered by A151 treatment. Overall, our findings suggested novel insights into the mechanism by which the immune system is metabolically modulated by A151 ODN.

Alendronate modulates cytokine responses in healthy young individuals after BCG vaccination.

Bacillus Calmette-Guérin (BCG) vaccination induces memory characteristics in innate immune cells and their progenitors, a process called trained immunity mediated by epigenetic and metabolic reprogramming. Cholesterol synthesis plays an amplifying role in trained immunity through mevalonate release. Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, can inhibit cholesterol synthesis. We explored their effects on trained immunity induced by BCG in a placebo-controlled clinical study (NL74082.091.20) in young, healthy individuals. Participants receiving single-dose oral alendronate on the day of BCG vaccination had more neutrophils and plasma cells one month after treatment. Alendronate led to reduced proinflammatory cytokine production by PBMCs stimulated with heterologous bacterial and viral stimuli one month later. Furthermore, the addition of alendronate transcriptionally suppressed multiple immune response pathways in PBMCs upon stimulation. Our findings indicate that N-BPs modulate the long-lasting effects of BCG vaccination on the cytokine production capacity of innate immune cells.

Seasonal variation in BCG-induced trained immunity.

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).

MMR vaccination induces trained immunity via functional and metabolic reprogramming of γδ T cells.

The measles, mumps, and rubella (MMR) vaccine protects against all-cause mortality in children, but the immunological mechanisms mediating these effects are poorly known. We systematically investigated whether MMR can induce long-term functional changes in innate immune cells, a process termed trained immunity, that could at least partially mediate this heterologous protection. In a randomized, placebo-controlled trial, 39 healthy adults received either the MMR vaccine or a placebo. Using single-cell RNA-Seq, we found that MMR caused transcriptomic changes in CD14+ monocytes and NK cells, but most profoundly in γδ T cells. Monocyte function was not altered by MMR vaccination. In contrast, the function of γδ T cells was markedly enhanced by MMR vaccination, with higher production of TNF and IFN-γ, as well as upregulation of cellular metabolic pathways. In conclusion, we describe a trained immunity program characterized by modulation of γδ T cell function induced by MMR vaccination.

Evaluation of the safety and immunological effects of Bacillus Calmette-Guérin in combination with checkpoint inhibitor therapy in a patient with neuroendocrine carcinoma: a case report.

BACKGROUND: Immune checkpoint inhibitors have revolutionized therapy of advanced and metastatic cancers. However, a significant proportion of patients do not respond to immune checkpoint inhibitors or develop resistance. Therefore, novel therapies or combinations of therapies that may act synergistically are needed. It has been suggested that induction of trained immunity may increase the response to immune checkpoint inhibitor therapy, through reprogramming myeloid cells toward an antitumor phenotype. On the other hand, activation of the immune system also carries the risk of potentially sustaining tumorgenicity and increasing immune- related toxicity. CASE PRESENTATION: We report the case of a 37-year-old Dutch male suffering from gastric neuroendocrine carcinoma with liver metastases and high risk for an unfavorable outcome, who was treated with a combination of programmed cell death protein 1 inhibitor nivolumab and the trained immunity-inducer Bacillus Calmette-Guérin vaccine as a salvage therapy. Three doses of BCG vaccine were administered at 3-month intervals, in conjunction with the immune checkpoint inhibitor regimen. At a certain point, radiation therapy was added to the treatment regimen. During the combination of these therapies, the patient developed immune-mediated colitis, which necessitated discontinuation of all treatments. Bacillus Calmette-Guérin vaccination induced a trained immune response with elevated monocyte-derived interleukin-6 and interleukin-1ß production capacity. From the first vaccination with Bacillus Calmette-Guérin until 3 months after the last vaccination with Bacillus Calmette-Guérin, the patient displayed only mild progression of the primary tumor and no progression of the metastases. CONCLUSION: In this study, we show the feasibility to combine checkpoint inhibitor therapy with inducers of trained immunity in a patient with an aggressive neuroendocrine tumor. Autoimmune side effects are common under programmed cell death protein 1 inhibitor therapy, which was considered the most likely cause of colitis, although an additive effect of Bacillus Calmette-Guérin vaccination or radiotherapy cannot be excluded. The patient displayed only mild progression during the combination therapy, but larger studies are warranted to fully explore the potential benefit of trained immunity inducers as an adjuvant to immune checkpoint inhibitor therapy.

The fungal-derived compound AM3 modulates pro-inflammatory cytokine production and skews the differentiation of human monocytes.

The proper functioning of the immune system depends on an appropriate balance between pro-inflammation and anti-inflammation. When the balance is disrupted and the system is excessively biased towards inflammation, immune responses cannot return within the normal range, which favors the onset of diseases of autoimmune or inflammatory nature. In this scenario, it is fundamental to find new compounds that can help restore this balance and contribute to the normal functioning of the immune system in humans. Here, we show the properties of a fungal compound with a strong safety profile in humans, AM3, as an immunomodulatory molecule to decrease excessive cytokine production in human cells. Our results present that AM3 treatment of human peripheral blood mononuclear cells and monocytes decreased their pro-inflammatory cytokine secretion following the challenge with bacterial lipopolysaccharide. Additionally, AM3 skewed the differentiation profile of human monocytes to macrophages towards a non-inflammatory phenotype without inducing tolerance, meaning these cells kept their capacity to respond to different stimuli. These effects were similar in young and elderly individuals. Thus, the fungal compound, AM3 may help reduce excessive immune activation in inflammatory conditions and keep the immune responses within a normal homeostatic range, regardless of the age of the individual.

Differences in Immune Responses in Individuals of Indian and European Origin: Relevance for the COVID-19 Pandemic.

During the coronavirus disease 2019 (COVID-19) pandemic, large differences in susceptibility and mortality due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have been reported between populations in Europe and South Asia. While both host and environmental factors (including Mycobacterium bovis BCG vaccination) have been proposed to explain this, the potential biological substrate of these differences is unknown. We purified peripheral blood mononuclear cells from individuals living in India and the Netherlands at baseline and 10 to 12 weeks after BCG vaccination. We compared chromatin accessibility between the two populations at baseline, as well as gene transcription profiles and cytokine production capacities upon stimulation. The chromatin accessibility of genes important for adaptive immunity was higher in the Indians than in the Europeans, while the latter had more accessible chromatin regions in genes of the innate immune system. At the transcriptional level, we observed that the Indian volunteers displayed a more tolerant immune response to stimulation, in contrast to a more exaggerated response in the Europeans. BCG vaccination strengthened the tolerance program in the Indians but not in the Europeans. These differences may partly explain the different impact of COVID-19 on the two populations. IMPORTANCE In this study, we assessed the differences in immune responses in individuals from India and Europe. This aspect is of great relevance, because of the described differences in morbidity and mortality between India and Europe during the pandemic. We found a significant difference in chromatin accessibility in immune cells from the two populations, followed by a more balanced and effective response in individuals from India. These exciting findings represent a very important piece of the puzzle for understanding the COVID-19 pandemic at a global level.

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.

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.

Immune Memory in Aging: a Wide Perspective Covering Microbiota, Brain, Metabolism, and Epigenetics.

Non-specific innate and antigen-specific adaptive immunological memories are vital evolutionary adaptations that confer long-lasting protection against a wide range of pathogens. Adaptive memory is established by memory T and B lymphocytes following the recognition of an antigen. On the other hand, innate immune memory, also called trained immunity, is imprinted in innate cells such as macrophages and natural killer cells through epigenetic and metabolic reprogramming. However, these mechanisms of memory generation and maintenance are compromised as organisms age. Almost all immune cell types, both mature cells and their progenitors, go through age-related changes concerning numbers and functions. The aging immune system renders the elderly highly susceptible to infections and incapable of mounting a proper immune response upon vaccinations. Besides the increased infectious burden, older individuals also have heightened risks of metabolic and neurodegenerative diseases, which have an immunological component. This review discusses how immune function, particularly the establishment and maintenance of innate and adaptive immunological memory, regulates and is regulated by epigenetics, metabolic processes, gut microbiota, and the central nervous system throughout life, with a focus on old age. We explain in-depth how epigenetics and cellular metabolism impact immune cell function and contribute or resist the aging process. Microbiota is intimately linked with the immune system of the human host, and therefore, plays an important role in immunological memory during both homeostasis and aging. The brain, which is not an immune-isolated organ despite former opinion, interacts with the peripheral immune cells, and the aging of both systems influences the health of each other. With all these in mind, we aimed to present a comprehensive view of the aging immune system and its consequences, especially in terms of immunological memory. The review also details the mechanisms of promising anti-aging interventions and highlights a few, namely, caloric restriction, physical exercise, metformin, and resveratrol, that impact multiple facets of the aging process, including the regulation of innate and adaptive immune memory. We propose that understanding aging as a complex phenomenon, with the immune system at the center role interacting with all the other tissues and systems, would allow for more effective anti-aging strategies.

Prenatal diagnosis of a case with tetrasomy 9p confirmed by cytogenetics, FISH, microarray analysis and review.

OBJECTIVE: Tetrasomy 9p is a rare fetal condition. Cases are usually mosaic. Here, we present a non-mosaic tetrasomy 9p case with cytogenetic analysis, fluorescence in situ hybridization, microarray data, ultrasound findings, and phenotypic presentation. CASE REPORT: A pregnancy was referred to cytogenetic analysis because of increased nuchal translucency in prenatal ultrasound at 13 weeks of gestation. Prenatal laboratory analysis revealed an extra marker chromosome with a non-mosaic pattern. Ultrasonographic findings were unilateral cleft lip and palate, micrognathia, and atrioventricular septal defect at the 17th week; additionally, ventriculomegaly, left axis deviation of the fetal heart, and a single umbilical artery were determined at the 23rd week. CONCLUSION: Phenotypic severity in non-mosaic tetrasomy 9p widely differs depending on the chromosomal content. We recommend performing appropriate genetic tests in those pregnancies with the suspicion of tetrasomy 9p, evaluating the mosaic state, and following those cases with detailed ultrasonographic examinations.

LSM1 is the new candidate gene for neurodevelopmental disorder.

Neurodevelopmental disorders are a heterogeneous group of diseases. Clinical presentation often overlaps with neurodevelopmental disorders, and explaining the molecular origin often requires reverse phenotyping. Next-Generation Sequencing (NGS) allows fast and cost-effective high-throughput sequencing. Given this fact, NGS is a useful tool for reverse phenotyping, especially for rare diseases. We hereby present two similarly affected siblings with neurodevelopmental delay. Duo-whole exome sequencing was performed. The homozygous LSM1 variant was found as the most likely cause for the condition. Our report contributes to the literature on the phenotype the biallelic LSM1 mutations. Moreover, we highlight the importance of reverse phenotyping and reanalysis of the genetic data.

Single-cell RNA sequencing reveals induction of distinct trained-immunity programs in human monocytes.

Trained immunity refers to the long-lasting memory traits of innate immunity. Recent studies have shown that trained immunity is orchestrated by sustained changes in epigenetic marks and metabolic pathways, leading to an altered transcriptional response to a second challenge. However, the potential heterogeneity of trained-immunity induction in innate immune cells has not been explored. In this study, we demonstrate cellular transcriptional programs in response to 4 different inducers of trained immunity in monocyte populations at single-cell resolution. Specifically, we identified 3 monocyte subpopulations upon the induction of trained immunity, and replicated these findings in an in vivo study. In addition, we found gene signatures consistent with these functional programs in patients with ulcerative colitis, sepsis, and COVID-19, suggesting the impact of trained-immunity programs in immune-mediated diseases.

Neonatal BCG vaccination is associated with a long-term DNA methylation signature in circulating monocytes.

Trained immunity describes the capacity of innate immune cells to develop heterologous memory in response to certain exogenous exposures. This phenomenon mediates, at least in part, the beneficial off-target effects of the BCG vaccine. Using an in vitro model of trained immunity, we show that BCG exposure induces a persistent change in active histone modifications, DNA methylation, transcription, and adenosine-to-inosine RNA modification in human monocytes. By profiling DNA methylation of circulating monocytes from infants in the MIS BAIR clinical trial, we identify a BCG-associated DNA methylation signature that persisted more than 12 months after neonatal BCG vaccination. Genes associated with this epigenetic signature are involved in viral response pathways, consistent with the reported off-target protection against viral infections in neonates, adults, and the elderly. Our findings indicate that the off-target effects of BCG in infants are accompanied by epigenetic remodeling of circulating monocytes that lasts more than 1 year.