An orally active carbon monoxide-releasing molecule enhances beneficial gut microbial species to combat obesity in mice.

Carbon monoxide (CO), a gaseous signaling molecule, has shown promise in preventing body weight gain and metabolic dysfunction induced by high fat diet (HFD), but the mechanisms underlying these effects are largely unknown. An essential component in response to HFD is the gut microbiome, which is significantly altered during obesity and represents a target for developing new therapeutic interventions to fight metabolic diseases. Here, we show that CO delivered to the gut by oral administration with a CO-releasing molecule (CORM-401) accumulates in faeces and enriches a variety of microbial species that were perturbed by a HFD regimen. Notably, Akkermansia muciniphila, which exerts salutary metabolic effects in mice and humans, was strongly depleted by HFD but was the most abundant gut species detected after CORM-401 treatment. Analysis of bacterial transcripts revealed a restoration of microbial functional activity, with partial or full recovery of the Krebs cycle, ß-oxidation, respiratory chain and glycolysis. Mice treated with CORM-401 exhibited normalization of several plasma and fecal metabolites that were disrupted by HFD and are dependent on Akkermansia muciniphila's metabolic activity, including indoles and tryptophan derivatives. Finally, CORM-401 treatment led to an improvement in gut morphology as well as reduction of inflammatory markers in colon and cecum and restoration of metabolic profiles in these tissues. Our findings provide therapeutic insights on the efficacy of CO as a potential prebiotic to combat obesity, identifying the gut microbiota as a crucial target for CO-mediated pharmacological activities against metabolic disorders.

Uveitic glaucoma-like features in Yap conditional knockout mice.

Glaucoma is a multifactorial neurodegenerative disease characterized by the progressive and irreversible degeneration of the optic nerve and retinal ganglion cells. Despite medical advances aiming at slowing degeneration, around 40% of treated glaucomatous patients will undergo vision loss. It is thus of utmost importance to have a better understanding of the disease and to investigate more deeply its early causes. The transcriptional coactivator YAP, an important regulator of eye homeostasis, has recently drawn attention in the glaucoma research field. Here we show that Yap conditional knockout mice (Yap cKO), in which the deletion of Yap is induced in both Müller glia (i.e. the only retinal YAP-expressing cells) and the non-pigmented epithelial cells of the ciliary body, exhibit a breakdown of the aqueous-blood barrier, accompanied by a progressive collapse of the ciliary body. A similar phenotype is observed in human samples that we obtained from patients presenting with uveitis. In addition, aged Yap cKO mice harbor glaucoma-like features, including deregulation of key homeostatic Müller-derived proteins, retinal vascular defects, optic nerve degeneration and retinal ganglion cell death. Finally, transcriptomic analysis of Yap cKO retinas pointed to early-deregulated genes involved in extracellular matrix organization potentially underlying the onset and/or progression of the observed phenotype. Together, our findings reveal the essential role of YAP in preserving the integrity of the ciliary body and retinal ganglion cells, thereby preventing the onset of uveitic glaucoma-like features.

Metabolomics for personalized medicine: the input of analytical chemistry from biomarker discovery to point-of-care tests.

Metabolomics refers to the large-scale detection, quantification, and analysis of small molecules (metabolites) in biological media. Although metabolomics, alone or combined with other omics data, has already demonstrated its relevance for patient stratification in the frame of research projects and clinical studies, much remains to be done to move this approach to the clinical practice. This is especially true in the perspective of being applied to personalized/precision medicine, which aims at stratifying patients according to their risk of developing diseases, and tailoring medical treatments of patients according to individual characteristics in order to improve their efficacy and limit their toxicity. In this review article, we discuss the main challenges linked to analytical chemistry that need to be addressed to foster the implementation of metabolomics in the clinics and the use of the data produced by this approach in personalized medicine. First of all, there are already well-known issues related to untargeted metabolomics workflows at the levels of data production (lack of standardization), metabolite identification (small proportion of annotated features and identified metabolites), and data processing (from automatic detection of features to multi-omic data integration) that hamper the inter-operability and reusability of metabolomics data. Furthermore, the outputs of metabolomics workflows are complex molecular signatures of few tens of metabolites, often with small abundance variations, and obtained with expensive laboratory equipment. It is thus necessary to simplify these molecular signatures so that they can be produced and used in the field. This last point, which is still poorly addressed by the metabolomics community, may be crucial in a near future with the increased availability of molecular signatures of medical relevance and the increased societal demand for participatory medicine.

Immune components of early breastmilk: Association with maternal factors and with reported food allergy in childhood.

BACKGROUND: Breastmilk (BM) may participate in driving gut barrier function and immunity in the neonate. We analyzed immune and growth factor concentrations in early BM and their association with maternal/environmental characteristics and with food allergy (FA) in childhood. METHODS: One BM sample was collected in maternity from some mothers in the EDEN birth cohort (n = 2002 mother-child dyads). A random selection was performed among available samples (subcohort, n = 272), for which all deliveries were full-term, various maternal/environmental characteristics were recorded, and parents answered yearly the question "Has a medical doctor diagnosed a FA in your child?" (26 parent-reported FA cases). Only samples collected between day 2 and day 6 post-partum were considered for descriptive analysis (n = 263). Samples for all other FA cases available were added to the subcohort (46 additional cases; "casecohort" design). Fifty cytokines, antibodies, and growth factor concentrations were determined using multiplexed kits and analyzed using robust statistical procedures. RESULTS: BM components exhibited wide concentration ranges and global day-to-day variation. Different clusters of correlated factors appeared, with components from the main cluster related to maternal diet during pregnancy. Primiparity was positively associated with eleven other components, whereas other factors (eg, maternal atopy and smoking) were related to fewer components. Finally, the casecohort design highlighted a positive association between CXCL10, TNFß, and IL-2 concentrations and reported FA in childhood. CONCLUSION: Beyond the unique description of early BM composition, we show that immune information transmitted to the neonate is related to various maternal factors and identified components associated with FA diagnosis in childhood.

Deep analysis of immune response and metabolic signature in children with food protein induced enterocolitis to cow's milk.

BACKGROUND: Food Protein-Induced Enterocolitis Syndrome (FPIES) is considered to be a non-IgE mediated food allergy. However, its pathogenesis remains poorly understood and biomarkers are lacking. We aimed to perform in-depth characterization of humoral and cellular immune responses in children with cow's milk (CM)-FPIES and investigated whether there is a FPIES metabolomic signature. METHODS: Children with CM-FPIES and control subjects with an IgE-mediated CM allergy (IgE-CMA), both avoiding CM, were recruited on the day of an oral food challenge. Blood samples were collected before the challenge. Total and specific levels of IgE, IgG1-4, IgA, IgM and IgD to various whey and casein allergens and to their gastroduodenal digestion products were measured in plasma, using plasma from CM-tolerant peanut allergic patients (IgE-PA, not avoiding CM) as additional controls. Cytokine secretion and cellular proliferation were analyzed after stimulation of PBMC with different CM allergens. Metabolomic profiles were obtained for plasma samples using liquid chromatography coupled to high-resolution mass spectrometry. RESULTS: Nine children with CM-FPIES and 12 control subjects (6 IgE-CMA and 6 IgE-PA) were included. In children with CM-FPIES, total Ig concentrations were lower than in control subjects, specific Ig against CM components were weak to undetectable, and no specific IgE against CM digestion products were detected. Moreover, in CM-FPIES patients, we did not find any Th cell proliferation or associated cytokine secretion after allergen reactivation, whereas such responses were clearly found in children with IgE-CMA. Plasma metabolic profiles were different between CM allergic patients, with significantly lower concentrations of various fatty acids and higher concentrations of primary metabolites such as amino acids in CM-FPIES compared to IgE-CMA patients. CONCLUSIONS: In CM-FPIES, both humoral and cellular specific immune responses are weak or absent, and this is not related to CM avoidance. A metabolomic signature was identified in patients with CM-FPIES that may be useful for the diagnosis and management of this disease.

Bee Venom Phospholipase A2, a Good "Chauffeur" for Delivering Tumor Antigen to the MHC I and MHC II Peptide-Loading Compartments of the Dendritic Cells: The Case of NY-ESO-1.

Bee venom phospholipase A2 (bvPLA2) is a small, 15kDa enzyme which hydrolyses many phospholipids through interfacial binding. The mutated bvPLA2H34Q (bvPLA2m), in which histidine-34 is replaced by glutamine, is not catalytically active. This protein has been shown to be a suitable membrane anchor and has been suggested as a suitable tumor-antigen vector for the development of novel dendritic cell-based vaccines. To confirm this feature, in this study the fusion protein PNY, composed of NY-ESO-1(NY(s)) fused to the C-terminus of bvPLA2m, was engineered. bvPLA2m enhanced the binding of NY(s) to the membrane of human monocyte-derived dendritic cells (DCs) and, once taken up by the cells, the antigen fused to the vector was directed to both MHC I and MHC II peptide-loading compartments. bvPLA2m was shown to increase the cross-presentation of the NY(s)-derived, restricted HLA-A*02 peptide, NY-ESO-1157-165(NY157-165), at the T1 cell surface. DCs loaded with the fusion protein induced cross-priming of NY(s)-specific CD8 + T-cells with greater efficiency than DCs loaded with NY(s). Sixty-five percent of these NY(s)-specific CD8+ T-cell lines could also be activated with the DCs pulsed with the peptide, NY157-165. Of these CD8+ T-cell lines, two were able to recognize the human melanoma cell line, SK-MEL-37, in a context of HLA-A*02. Only a small number of bvPLA2m CD8+ T-cell lines were induced, indicating the low immunogenicity of the protein. It was concluded that bvPLA2m can be used as a membrane-binding vector to promote MHC class II peptide presentation and MHC class I peptide cross-presentation. Such a system can, therefore, be tested for the preparation of cell-based vaccines.

Hierarchy of CD4 T cell epitopes of the ANRS Lipo5 synthetic vaccine relies on the frequencies of pre-existing peptide-specific T cells in healthy donors.

The Agence National de Recherche sur le SIDA et les hepatitis Lipo5 vaccine is composed by five long fragments of HIV proteins and was recently shown to induce in seronegative volunteers a CD4 T cell response largely dominated by the G2 fragment. To understand this response profile, we submitted the five HIV fragments to HLA-DR-binding assays and evaluated the frequency of naive Lipo5-specific CD4 T lymphocytes in the blood of 22 healthy individuals. We enumerated the Lipo5-specific T cell lines induced in vitro by weekly rounds of specific stimulation. Four peptides and hence not only G2 exhibited a broad specificity for HLA-DR molecules. In contrast, most of the T cell lines specific for Lipo5 reacted with G2, revealing a G2-specific T cell repertoire superior to 2 cells per million, whereas it is close to 0.4 for the other peptides. We also found good cross-reactivity of all the peptides with clade B and C variants and that G2 and P1 are able to recruit T cells that recognize HIV-infected cells. We therefore mainly observed very good concordance between the frequency to individual Lipo5 peptides among vaccinees in a large-scale vaccine trial and the distribution of peptide specificity of the in vitro induced T cell lines. These findings underline the role of the size of the epitope-specific naive repertoire in shaping the CD4 T cell response after vaccination and highlight the value of evaluating the naive repertoire to predict vaccine immunogenicity.

The signal peptide of the tumor-shared antigen midkine hosts CD4+ T cell epitopes.

BACKGROUND: The CD4 T cell response to the tumor antigen Midkine was unknown. RESULTS: Most of the T cell response to Midkine relies on T cell epitopes contained in its signal peptide. CONCLUSION: The signal peptide of Midkine is accessible to HLA class II pathway for CD4 T cell presentation. SIGNIFICANCE: It is a new function for signal peptides to contribute to tumor-specific CD4 T cell response. Because of the key role of CD4 T cell response in immunity to tumors, we investigated the CD4(+) T cell response to the recently identified tumor antigen Midkine (MDK). By weekly stimulations of T lymphocytes harvested from seven HLA-DR-typed healthy donors, we derived CD4(+) T cell lines specific for eight MDK peptides. Most of the T cell lines reacted with the peptides 9-23 and 14-28, located in and overlapping the MDK signal peptide, respectively. Accordingly, the MDK signal peptide appeared to be rich in good binders to common HLA-DR molecules. The peptide 9-23-specific T cell lines were specifically stimulated by autologous dendritic cells loaded with lysates of MDK-transfected cells or with lysates of tumor cells naturally expressing the MDK protein. One T cell line was stimulated by HLA-compatible MDK-transfected tumor cells. By contrast, the peptide 14-28-specific T cell lines were not stimulated in any of these conditions. Our data demonstrate that CD4(+) T cell epitopes present in the signal peptide can be accessible to recognition by CD4(+) T cells and may therefore contribute to tumor immunity, whereas a peptide overlapping the junction between the signal peptide and the mature protein is not.

Human CD4 T cell epitopes selective for Vaccinia versus Variola virus.

Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections.

In vitro human CD4+ T cell response to the vaccinia protective antigens B5R and A33R.

Subunit vaccine candidates against poxvirus infection induced protective humoral and cellular response in animal models but their immunogenicity in human remains unknown. We have therefore evaluated in vitro the CD4 T cell response of the major antigens B5R and A33R and characterized their CD4 T cell epitopes. Twelve peptides selected on the basis of their binding capacity to HLA-DR molecules, induced CD4 T lymphocytes harvested in healthy donors. In the A33R proteins two peptides are T cell stimulating for at least half of the donors and are restricted to multiple HLA-DR molecules in agreement with their broad specificity for HLA-DR molecules. In B5R, two peptides exhibited a good immunoprevalence but only one is a good binder to multiple HLA-DR molecules. One peptide was a moderate binder for multiple HLA-DR molecules, although it was efficiently presented to peptide-specific T cell lines. Altogether, our data demonstrated the capacity of B5R and A33R peptides to elicit a T cell response in multiple healthy donors and showed that promiscuity and immunoprevalence of CD4 T cell epitopes are not necessarily associated.