Comparative characterization of the infant gut microbiome and their maternal lineage by a multi-omics approach.

The human gut microbiome establishes and matures during infancy, and dysregulation at this stage may lead to pathologies later in life. We conducted a multi-omics study comprising three generations of family members to investigate the early development of the gut microbiota. Fecal samples from 200 individuals, including infants (0-12 months old; 55% females, 45% males) and their respective mothers and grandmothers, were analyzed using two independent metabolomics platforms and metagenomics. For metabolomics, gas chromatography and capillary electrophoresis coupled to mass spectrometry were applied. For metagenomics, both 16S rRNA gene and shotgun sequencing were performed. Here we show that infants greatly vary from their elders in fecal microbiota populations, function, and metabolome. Infants have a less diverse microbiota than adults and present differences in several metabolite classes, such as short- and branched-chain fatty acids, which are associated with shifts in bacterial populations. These findings provide innovative biochemical insights into the shaping of the gut microbiome within the same generational line that could be beneficial in improving childhood health outcomes.

Further Insights into the Gut Microbiota of Cow's Milk Allergic Infants: Analysis of Microbial Functionality and Its Correlation with Three Fecal Biomarkers.

Cow's milk allergy (CMA) is one of the most prevalent food allergies in children. Several studies have demonstrated that gut microbiota influences the acquisition of oral tolerance to food antigens at initial stages of life. Changes in the gut microbiota composition and/or functionality (i.e., dysbiosis) have been linked to inadequate immune system regulation and the emergence of pathologies. Moreover, omic sciences have become an essential tool for the analysis of the gut microbiota. On the other hand, the use of fecal biomarkers for the diagnosis of CMA has recently been reviewed, with fecal calprotectin, α-1 antitrypsin, and lactoferrin being the most relevant. This study aimed at evaluating functional changes in the gut microbiota in the feces of cow's milk allergic infants (AI) compared to control infants (CI) by metagenomic shotgun sequencing and at correlating these findings with the levels of fecal biomarkers (α-1 antitrypsin, lactoferrin, and calprotectin) by an integrative approach. We have observed differences between AI and CI groups in terms of fecal protein levels and metagenomic analysis. Our findings suggest that AI have altered glycerophospholipid metabolism as well as higher levels of lactoferrin and calprotectin that could be explained by their allergic status.

Deciphering the role of platelets in severe allergy by an integrative omics approach.

BACKGROUND: Mechanisms causing the onset and perpetuation of inflammation in severe allergic patients remain unknown. Our previous studies suggested that severe allergic inflammation is linked to platelet dysfunction. METHODS: Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) samples were obtained by platelet-apheresis from severe (n = 7) and mild (n = 10) allergic patients and nonallergic subjects (n = 9) to perform platelet lipidomics by liquid chromatography coupled to mass spectrometry (LC-MS) and RNA-seq analysis. Significant metabolites and transcripts were used to identify compromised biological pathways in the severe phenotype. Platelet and inflammation-related proteins were quantified by Luminex. RESULTS: Platelets from severe allergic patients were characterized by high levels of ceramides, phosphoinositols, phosphocholines, and sphingomyelins. In contrast, they showed a decrease in eicosanoid precursor levels. Biological pathway analysis performed with the significant lipids revealed the alteration of phospholipases, calcium-dependent events, and linolenic metabolism. RNAseq confirmed mRNA overexpression of genes related to platelet activation and arachidonic acid metabolism in the severe phenotypes. Pathway analysis indicated the alteration of NOD, MAPK, TLR, TNF, and IL-17 pathways in the severe phenotype. P-Selectin and IL-17AF proteins were increased in the severe phenotype. CONCLUSIONS: This study demonstrates that platelet lipid, mRNA, and protein content is different according to allergy severity. These findings suggest that platelet load is a potential source of biomarkers and a new chance for therapeutic targets in severe inflammatory pathologies.

A method based on plateletpheresis to obtain functional platelet, CD3+ and CD14+ matched populations for research immunological studies.

BACKGROUND: In previous studies with peripheral blood cells, platelet factors were found to be associated with severe allergic phenotypes. A reliable method yielding highly concentrated and pure platelet samples is usually not available for immunological studies. Plateletpheresis is widely used in the clinics for donation purposes. In this study, we designed a protocol based on plateletpheresis to obtain Platelet-Rich Plasma (PRP), Platelet-Poor Plasma (PPP) as well as CD3+ and CD14+ cells matched samples from a waste plateletpheresis product for immunological studies. METHODS: Twenty-seven subjects were voluntarily subjected to plateletpheresis. PRP, PPP and blood cell concentrate contained in a leukocyte reduction system chamber (LRSC) were obtained in this process. CD3+ and CD14+ cells were isolated from the LRSC by density-gradient centrifugation and positive magnetic bead isolation. RNA was isolated from PRP, CD3+ and CD14+ cell samples and used for transcriptomic studies by Affymetrix. PRP and PPP samples were used for platelet protein quantification by multiplex assays. RESULTS: A reliable high yield method to obtain matched samples of PRP, PPP, CD3+ and CD14+ from a single donor for RNA and protein analyses has been designed. The RNA quality indicators (RQI) routinely used for other cell types were not suitable for platelet RNA characterization. Despite this, the platelet RNA was valid for transcriptomic studies by Affymetrix, as platelet transcripts obtained in our previous studies were confirmed in PRP samples. Platelet samples were enriched in platelet factors as determined in protein multiplex analysis. CONCLUSIONS: We have developed a method that yields not only high content and pure platelet samples from a single donor but also CD3+ and CD14+ matched samples that can be used for RNA and protein analyses in immunological studies.

Understanding Systemic and Local Inflammation Induced by Nasal Polyposis: Role of the Allergic Phenotype.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by persistent symptoms associated to the development of nasal polyps. To this day, the molecular mechanisms involved are still not well defined. However, it has been suggested that a sustained inflammation as allergy is involved in its onset. In this exploratory study, the aim was to investigate the effect of the allergic status in the development of CRSwNP. To achieve this, we recruited 22 patients with CRSwNP and classified them in non-allergic and allergic using ImmunoCAP ISAC molecular diagnosis. Plasma samples were analyzed using liquid chromatography coupled to mass spectrometry (LC-MS). Subsequently, significant metabolites from plasma that were commercially available were then analyzed by targeted analysis in some nasal polyps. Additionally, nasal polyp and nasal mucosa samples were examined for eosinophils, neutrophils, CD3+ and CD11c+ cells, as well as collagen deposition and goblet cell hyperplasia. We found that 9 out of the 22 patients were sensitized to some aeroallergens (named as allergic CRSwNP). The other 13 patients had no sensitizations (non-allergic CRSwNP). Regarding metabolomics, bilirubin, cortisol, lysophosphatidylcholines (LPCs) 16:0, 18:0 and 20:4 and lysophosphatidylinositol (LPI) 20:4, which are usually related to a sustained allergic inflammation, were unexpectedly increased in plasma of non-allergic CRSwNP compared to allergic CRSwNP. LPC 16:0, LPC 18:0 and LPI 20:4 followed the same trend in nasal polyp as they did in plasma. Comparison of nasal polyps with nasal mucosa showed a significant increase in eosinophils (p < 0.001) and neutrophils (p < 0.01) in allergic CRSwNP. There were more eosinophils in polyps of non-allergic CRSwNP than in their nasal mucosa (p < 0.01). Polyps from non-allergic CRSwNP had less eosinophils than the polyps of allergic CRSwNP (p < 0.05) and reduced amounts of collagen compared to their nasal mucosa (p < 0.001). Our data suggests that there is a systemic inflammatory response associated to CRSwNP in the absence of allergy, which could be accountable for the nasal polyp development. Allergic CRSwNP presented a higher number of eosinophils in nasal polyps, suggesting that eosinophilia might be connected to the development of nasal polyps in this phenotype.

Exploring novel systemic biomarker approaches in grass-pollen sublingual immunotherapy using omics.

BACKGROUND: Sublingual allergen-specific immunotherapy (SLIT) intervention improves the control of grass pollen allergy by maintaining allergen tolerance after cessation. Despite its widespread use, little is known about systemic effects and kinetics associated to SLIT, as well as the influence of the patient sensitization phenotype (Mono- or Poly-sensitized). In this quest, omics sciences could help to gain new insights to understand SLIT effects. METHODS: 47 grass-pollen-allergic patients were enrolled in a double-blind, placebo-controlled, multicenter trial using GRAZAX® during 2 years. Immunological assays (sIgE, sIgG4, and ISAC) were carried out to 31 patients who finished the trial. Additionally, serum and PBMCs samples were analyzed by metabolomics and transcriptomics, respectively. Based on their sensitization level, 22 patients were allocated in Mono- or Poly-sensitized groups, excluding patients allergic to epithelia. Individuals were compared based on their treatment (Active/Placebo) and sensitization level (Mono/Poly). RESULTS: Kinetics of serological changes agreed with those previously described. At two years of SLIT, there are scarce systemic changes that could be associated to improvement in systemic inflammation. Poly-sensitized patients presented a higher inflammation at inclusion, while Mono-sensitized patients presented a reduced activity of mast cells and phagocytes as an effect of the treatment. CONCLUSIONS: The most relevant systemic change detected after two years of SLIT was the desensitization of effector cells, which was only detected in Mono-sensitized patients. This change may be related to the clinical improvement, as previously reported, and, together with the other results, may explain why clinical effect is lost if SLIT is discontinued at this point.

Multi-omics analysis points to altered platelet functions in severe food-associated respiratory allergy.

BACKGROUND: Prevalence and severity of allergic diseases have increased worldwide. To date, respiratory allergy phenotypes are not fully characterized and, along with inflammation progression, treatment is increasingly complex and expensive. Profilin sensitization constitutes a good model to study the progression of allergic inflammation. Our aim was to identify the underlying mechanisms and the associated biomarkers of this progression, focusing on severe phenotypes, using transcriptomics and metabolomics. METHODS: Twenty-five subjects were included in the study. Plasma samples were analyzed using gas and liquid chromatography coupled to mass spectrometry (GC-MS and LC-MS, respectively). Individuals were classified in four groups-"nonallergic," "mild," "moderate," and "severe"-based on their clinical history, their response to an oral challenge test with profilin, and after a refinement using a mathematical metabolomic model. PBMCs were used for microarray analysis. RESULTS: We found a set of transcripts and metabolites that were specific for the "severe" phenotype. By metabolomics, a decrease in carbohydrates and pyruvate and an increase in lactate were detected, suggesting aerobic glycolysis. Other metabolites were incremented in "severe" group: lysophospholipids, sphingosine-1-phosphate, sphinganine-1-phosphate, and lauric, myristic, palmitic, and oleic fatty acids. On the other hand, carnitines were decreased along severity. Significant transcripts in the "severe" group were found to be downregulated and were associated with platelet functions, protein synthesis, histone modification, and fatty acid metabolism. CONCLUSION: We have found evidence that points to the association of severe allergic inflammation with platelet functions alteration, together with reduced protein synthesis, and switch of immune cells to aerobic glycolysis.