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Acetyl and other acyl groups from different short-chain fatty acids (SCFA) competitively modify histones at various lysine sites. To fully understand the functional significance of such histone acylation, a key epigenetic mechanism, it is crucial to characterize the cellular sources of the corresponding acyl-CoA molecules required for the lysine modification. Like acetate, SCFAs such as propionate, butyrate and crotonate are thought to be the substrates used to generate the corresponding acyl-CoAs by enzymes known as acyl-CoA synthetases. The acetyl-CoA synthetase, ACSS2, which produces acetyl-CoA from acetate in the nucleocytoplasmic compartment, has been proposed to also mediate the synthesis of acyl-CoAs such as butyryl- and crotonyl-CoA from the corresponding SCFAs. This idea is now widely accepted and is sparking new research projects. However, based on our direct in vitro experiments with purified or recombinant enzymes and structural considerations, we demonstrate that ACSS2 is unable to mediate the generation of non-acetyl acyl-CoAs like butyryl- and crotonyl-CoA. It is therefore essential to re-examine published data and corresponding discussions in the light of this new finding.

The connection between imbalances in the human gut microbiota, known as dysbiosis, and various diseases has been well established. Current techniques for sampling the small intestine are both invasive for patients and costly for healthcare facilities. Most studies on human gut microbiome are conducted using faecal samples, which do not accurately represent the microbiome in the upper intestinal tract. A pilot clinical investigation, registered as NCT05477069 and sponsored by the Grenoble Alpes University Hospital, is currently underway to evaluate a novel ingestible medical device (MD) designed for collecting small intestinal liquids by Pelican Health. This study is interventional and monocentric, involving 15 healthy volunteers. The primary objective of the study is to establish the safety and the performance of the MD when used on healthy volunteers. Secondary objectives include assessing the device’s performance and demonstrating the difference between the retrieved sample from the MD and the corresponding faecal sample. Multi-omics analysis will be performed, including metagenomics, metabolomics, and culturomics. We anticipate that the MD will prove to be safe without any reported adverse effects, and we collected samples suitable for the proposed omics analyses in order to demonstrate the functionality of the MD and the clinical potential of the intestinal content.

The spread of antibiotic-resistant bacteria poses a substantial threat to morbidity and mortality worldwide. Carbapenem-resistant Pseudomonas aeruginosa (CRPA) are considered “critical-priority” bacteria by the World Health Organization (WHO) since 2017 taking into account criteria such as patient mortality, global burden disease, and worldwide trend of multi-drug resistance (MDR). Indeed P. aeruginosa can be particularly difficult to eliminate from patients due to its combinatory antibiotic resistance, multifactorial virulence, and ability to over-adapt in a dynamic way. Research is active, but the course to a validated efficacy of a new treatment is still long and uncertain. What is new in the anti–P. aeruginosa clinical development pipeline since the 2017 WHO alert? This review focuses on new solutions for P. aeruginosa infections that are in active clinical development, i.e., currently being tested in humans and may be approved for patients in the coming years. Among 18 drugs of interest in December 2021 anti–P. aeruginosa development pipeline described here, only one new combination of β-lactam/β-lactamase inhibitor is in phase III trial. Derivatives of existing antibiotics considered as “traditional agents” are over-represented. Diverse “non-traditional agents” including bacteriophages, iron mimetic/chelator, and anti-virulence factors are significantly represented but unfortunately still in early clinical stages. Despite decade of efforts, there is no vaccine currently in clinical development to prevent P. aeruginosa infections. Studying pipeline anti–P. aeruginosa since 2017 up to now shows how to provide a new treatment for patients can be a difficult task. Given the process duration, the clinical pipeline remains unsatisfactory leading best case to the approval of new antibacterial drugs that treat CRPA in several years. Beyond investment needed to build a robust pipeline, the Community needs to reinvent medicine with new strategies of development to avoid the disaster. Among “non-traditional agents”, anti-virulence strategy may have the potential through novel and non-killing modes of action to reduce the selective pressure responsible of MDR.

COVID-19 is caused by the human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in widespread morbidity and mortality. CD4+ T cells, CD8+ T cells and neutralizing antibodies all contribute to control SARS-CoV-2 infection. However, heterogeneity is a major factor in disease severity and in immune innate and adaptive responses to SARS-CoV-2. We performed a deep analysis by flow cytometry of lymphocyte populations of 125 hospitalized SARS-CoV-2 infected patients on the day of hospital admission. Five clusters of patients were identified using hierarchical classification on the basis of their immunophenotypic profile, with different mortality outcomes. Some characteristics were observed in all the clusters of patients, such as lymphopenia and an elevated level of effector CD8+CCR7- T cells. However, low levels of T cell activation are associated to a better disease outcome; on the other hand, profound CD8+ T-cell lymphopenia, a high level of CD4+ and CD8+ T-cell activation and a high level of CD8+ T-cell senescence are associated with a higher mortality outcome. Furthermore, a cluster of patient was characterized by high B-cell responses with an extremely high level of plasmablasts. Our study points out the prognostic value of lymphocyte parameters such as T-cell activation and senescence and strengthen the interest in treating the patients early in course of the disease with targeted immunomodulatory therapies based on the type of adaptive response of each patient.

Chronic granulomatous disease (CGD) is a primary immunodeficiency resulting from loss of function mutations in the reactive oxygen species generating phagocyte NADPH oxidase (NOX2). CGD patients are prone to infection, but also have an increased susceptibility to autoimmune diseases. The aim of this study was to investigate the role of NOX2 in the regulation of specific immunity. In both CGD patients and NOX2-deficient mice, we observed an alteration in the basal proportions of IgG subtypes. Upon immunization with curdlan—a dectin 1 agonist—NOX2-deficient mice showed increased production of IgG2c compared to controls, and restimulation of lymph node-derived cells led to increased production of IFNγ, but not IL-5, indicative hallmark of an enhanced Th1 response. T cell activation was increased in NOX2-deficient mice and a similar trend was observed in vitro when T cells were co-cultured with NOX2-deficient bone marrow-derived cells. In contrast, no difference in T cell activation was observed when NOX2-deficient T cells were co-cultured with wild-type BMDC. Following stimulation of NOX2-deficient dendritic cells (DCs), no difference in costimulatory molecules was observed, while there was an increase in the release of Th1-driving cytokines. In summary, both CGD patients and CGD mice have an altered IgG subtype distribution, which is associated with an increased IFNγ production. Thus, NOX2 within DCs appears to be an important regulator at the interface of innate and specific immunity, especially after activation of the dectin 1 pathway, limiting immune activation and the development of autoimmunity.

Pseudomonas aeruginosa (P.a) is one of the most critical antibiotic resistant bacteria in the world and is the most prevalent pathogen in cystic fibrosis (CF), causing chronic lung infections that are considered one of the major causes of mortality in CF patients. Although several studies have contributed to understanding P.a within-host adaptive evolution at a genomic level, it is still difficult to establish direct relationships between the observed mutations, expression of clinically relevant phenotypes, and clinical outcomes. Here, we performed a comparative untargeted LC/HRMS-based metabolomics analysis of sequential isolates from chronically infected CF patients to obtain a functional view of P.a adaptation. Metabolic profiles were integrated with expression of bacterial phenotypes and clinical measurements following multiscale analysis methods. Our results highlighted significant associations between P.a “metabotypes”, expression of antibiotic resistance and virulence phenotypes, and frequency of clinical exacerbations, thus identifying promising biomarkers and therapeutic targets for difficult-to-treat P.a infections

Live-attenuated bacterial vectors for antigens delivery have aroused growing interest in the field of cancer immunotherapy. Their potency to stimulate innate immunity and to promote intracellular antigen delivery into antigen-presenting cells could be exploited to elicit a strong and specific cellular immune response against tumor cells. We previously described genetically-modified and attenuated Pseudomonas aeruginosa vectors able to deliver in vivo protein antigens into antigen-presenting cells, through Type 3 secretion system of the bacteria. Using this approach, we managed to protect immunized mice against aggressive B16 melanoma development in both a prophylactic and therapeutic setting. In this study, we further investigated the antigen-specific CD8+ T cell response, in terms of phenotypic and functional aspects, obtained after immunizations with a killed but metabolically active P. aeruginosa attenuated vector. We demonstrated that P. aeruginosa vaccine induces a highly functional pool of antigen-specific CD8+ T cell able to infiltrate the tumor. Furthermore, multiple immunizations allowed the development of a long-lasting immune response, represented by a pool of predominantly effector memory cells which protected mice against late tumor challenge. Overall, killed but metabolically active P. aeruginosa vector is a safe and promising approach for active and specific antitumor immunotherapy.

Abstract Mistletoe (Viscum album L.) is used in German-speaking European countries in the field of integrative oncology linking conventional and complementary medicine therapies to improve quality of life. Various companies sell extracts, fermented or not, for injection by subcutaneous or intra-tumoral route with a regulatory status of anthroposophic medicinal products (European Medicinal Agency (EMA) assessment status). These companies as well as anthroposophical physicians argue that complex matrices composed of many molecules in mixture are necessary for activity and that the host tree of the mistletoe parasitic plant is the main determining factor for this matrix composition. The critical point is that parenteral devices of European mistletoe extracts do not have a standard chemical composition regulated by EMA quality guidelines, because they are not drugs, regulatory speaking. However, the mechanism of mistletoe’s anticancer activity and its effectiveness in treating and supporting cancer patients are not fully understood. Because of this lack of transparency and knowledge regarding the matrix chemical composition, we undertook an untargeted metabolomics study of several mistletoe extracts to explore and compare their fingerprints by LC-(HR)MS(/MS) and 1H-NMR. Unexpectedly, we showed that the composition was primarily driven by the manufacturer/preparation method rather than the different host trees. This differential composition may cause differences in immunostimulating and anti-cancer activities of the different commercially available mistletoe extracts as illustrated by structure–activity relationships based on LC–MS/MS and 1H-NMR identifications completed by docking experiments. In conclusion, in order to move towards an evidence-based medicine use of mistletoe, it is a priority to bring rigor and quality, chemically speaking.

Abstract Molecular networking connects mass spectra of molecules based on the similarity of their fragmentation patterns. However, during ionization, molecules commonly form multiple ion species with different fragmentation behavior. As a result, the fragmentation spectra of these ion species often remain unconnected in tandem mass spectrometry-based molecular networks, leading to redundant and disconnected sub-networks of the same compound classes. To overcome this bottleneck, we develop Ion Identity Molecular Networking (IIMN) that integrates chromatographic peak shape correlation analysis into molecular networks to connect and collapse different ion species of the same molecule. The new feature relationships improve network connectivity for structurally related molecules, can be used to reveal unknown ion-ligand complexes, enhance annotation within molecular networks, and facilitate the expansion of spectral reference libraries. IIMN is integrated into various open source feature finding tools and the GNPS environment. Moreover, IIMN-based spectral libraries with a broad coverage of ion species are publicly available.

Microbiota-derived metabolites have biological importance for their host. Spermidine, a metabolite described for its protective effect in age-related diseases, is now studied for its role in the resolution of inflammation and gut homeostasis. Strategies to modulate its production in the gastrointestinal tract are of interest to increase host spermidine intakes. Here, we show that metabolic engineering can be used to increase spermidine production by the probiotic Escherichia coli Nissle 1917 (EcN), used in humans. First, we found that increasing the expression of genes involved in polyamine biosynthesis, namely the S-adenosylmethionine synthase speD and the spermidine synthase speE, resulted in an increase in spermidine produced and excreted by our engineered bacteria. The major drawback of this first attempt was the production of acetylated forms of spermidine. Next, we propose to solve this problem by increasing the expression of the spermidine exporter system MdtI/MdtJ. This second strategy had a major impact on the spermidine profile found in the culture supernatant. Our results demonstrate, for the first time, the feasibility of rationally engineering bacterial probiotic strains to increase their ability to deliver the microbiota-derived metabolite, spermidine. This work illustrates how metabolomic and synthetic biology can be used to design and improve engineered Live Biotherapeutic Products that have the potential to be used in personalized medicine.

BackgroundThe SARS-CoV-2 infection triggers excessive immune response resulting in increased levels of pro-inflammatory cytokines, endothelial injury, and intravascular coagulopathy. The complement system (CS) activation participates to this hyperinflammatory response. However, it is still unclear which activation pathways (classical, alternative, or lectin pathway) pilots the effector mechanisms that contribute to critical illness. To better understand the immune correlates of disease severity, we performed an analysis of CS activation pathways and components in samples collected from COVID-19 patients hospitalized in Grenoble Alpes University Hospital between 1 and 30 April 2020 and of their relationship with the clinical outcomes.MethodsWe conducted a retrospective, single-center study cohort in 74 hospitalized patients with RT-PCR-proven COVID-19. The functional activities of classical, alternative, and mannose-binding lectin (MBL) pathways and the antigenic levels of the individual components C1q, C4, C3, C5, Factor B, and MBL were measured in patients’ samples during hospital admission. Hierarchical clustering with the Ward method was performed in order to identify clusters of patients with similar characteristics of complement markers. Age was included in the model. Then, the clusters were compared with the patient clinical features: rate of intensive care unit (ICU) admission, corticoid treatment, oxygen requirement, and mortality.ResultsFour clusters were identified according to complement parameters. Among them, two clusters revealed remarkable profiles: in one cluster (n = 15), patients exhibited activation of alternative and lectin pathways and low antigenic levels of MBL, C4, C3, Factor B, and C5 compared to all the other clusters; this cluster had the higher proportion of patients who died (27%) and required oxygen support (80%) or ICU care (53%). In contrast, the second cluster (n = 19) presented inflammatory profile with high classical pathway activity and antigenic levels of complement components; a low proportion of patients required ICU care (26%) and no patient died in this group.ConclusionThese findings argue in favor of prominent activation of the alternative and MBL complement pathways in severe COVID-19, but the spectrum of complement involvement seems to be heterogeneous requiring larger studies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third coronavirus leading to a global health outbreak. Despite the high mortality rates from SARS-CoV-1 and Middle-East respiratory syndrome (MERS)-CoV infections, which both sparked the interest of the scientific community, the underlying physiopathology of the SARS-CoV-2 infection, remains partially unclear. SARS-CoV-2 shares similar features with SARS-CoV-1, notably the use of the angiotensin conversion enzyme 2 (ACE2) as a receptor to enter the host cells. However, some features of the SARS-CoV-2 pandemic are unique. In this work, we focus on the association between obesity, metabolic syndrome, and type 2 diabetes on the one hand, and the severity of COVID-19 infection on the other, as it seems greater in these patients. We discuss how adipocyte dysfunction leads to a specific immune environment that predisposes obese patients to respiratory failure during COVID-19. We also hypothesize that an ACE2-cleaved protein, angiotensin 1-7, has a beneficial action on immune deregulation and that its low expression during the SARS-CoV-2 infection could explain the severity of infection. This introduces angiotensin 1-7 as a potential candidate of interest in therapeutic research on CoV infections.

We have analyzed the prebiotic effect of softwood hemicelluloses on specific bacterial species in vitro and on microbiota diversity and metabolism in healthy mice in vivo. The ethanol-precipitated glycans from the softwood hemicellulose autohydrolysate were able to stimulate in vitro the growth of Bifidobacterium adolescentis, but to a much lesser extent than that of adherent-invasive E. coli. B. adolescentis was the best producer of SCFA. When mice were fed with the ethanol precipitate fraction, the relative abundance of Bacteroidetes increased while that of Proteobacteria diminished, suggesting change towards a less obesogenic microbiome. Following treatment, lipid analysis showed a decrease in cholesterol, bile acids and free fatty acids, indicating a potential cardioprotection role. Furthermore, analysis of fermentation metabolites revealed an increase in metabolites important for immune health and well-being. Our results highlight the value of hemicelluloses as a potential source of prebiotics supporting both a healthy digestive tract and immune system.

ObjectiveRegular physical activity may be associated with improved lung function via reduced systemic inflammation, although studies exploring this mechanism are rare. We evaluated the role of C-reactive protein in blood, which is a common marker of systemic inflammation, on the association of physical activity with forced expiratory volume in one second and forced vital capacity.MethodsCross-sectional data on spirometry, C-reactive protein levels and self-reported physical activity (yes/no; ≥2 times and ≥1hr per week of vigorous physical activity) were available in the European Community Respiratory Health Survey (N = 2347 adults, 49.3% male, 28-56 years-old). A subsample was also assessed 10 years later using the International Physical Activity Questionnaire, and tertiles of Metabolic Equivalent of Task-minutes per week spent in vigorous, moderate and walking activities were calculated (N = 671, 49.6% male, 40-67 years-old). Adjusted cross-sectional mixed linear regression models and the "mediate" package in "R" were used to assess the presence of mediation.ResultsDespite positive significant associations between nearly all physical activity metrics with forced expiratory volume in one second and forced vital capacity, there was no evidence that C-reactive protein levels played a role. An influence of C-reactive protein levels was only apparent in the smaller subsample when comparing the medium to low tertiles of moderate activity (mean difference [95% CIs]: 21.1ml [5.2, 41.9] for forced expiratory volume in one second and 17.3ml [2.6, 38.0] for forced vital capacity).ConclusionsIn a population of adults, we found no consistent evidence that the association of physical activity with forced expiratory volume in one second or forced vital capacity is influenced by the level of C-reactive protein in blood.

Trillions of microorganisms, termed the “microbiota”, reside in the mammalian gastrointestinal tract, and collectively participate in regulating the host phenotype. It is now clear that the gut microbiota, metabolites, and intestinal immune function are correlated, and that alterations of the complex and dynamic host-microbiota interactions can have deep consequences for host health. However, the mechanisms by which the immune system regulates the microbiota and by which the microbiota shapes host immunity are still not fully understood. This article discusses the contribution of metabolites in the crosstalk between gut microbiota and immune cells. The identification of key metabolites having a causal effect on immune responses and of the mechanisms involved can contribute to a deeper insight into host-microorganism relationships. This will allow a better understanding of the correlation between dysbiosis, microbial-based dysmetabolism, and pathogenesis, thus creating opportunities to develop microbiota-based therapeutics to improve human health. In particular, we systematically review the role of soluble and membrane-bound microbial metabolites in modulating host immunity in the gut, and of immune cells-derived metabolites affecting the microbiota, while discussing evidence of the bidirectional impact of this crosstalk. Furthermore, we discuss the potential strategies to hear the sound of such metabolite-mediated crosstalk.