Biology subjects

Bonatti, M.

Publications and source records attributed to Bonatti, M..

4 recordsLinked to original sources

bioRxiv2024

Characterization of HLA-DR immunopeptidome of bronchoalveolar lavage cells in patients with newly diagnosed rheumatoid arthritis and healthy current-smoker controls

Evidence suggests that self-tolerance is breached in the lung prior to the clinical onset of rheumatoid arthritis (RA) in the joints. The human leukocyte antigen DR (HLA-DR) shared epitope (SE) represents the strongest genetic risk factor for sero-positive RA. However, to our knowledge, the HLA-DR immunopeptidome of the RA lung and its link to HLA-DR genotype has not been investigated to date. The objective of this study was to optimize the methods for characterizing the HLA-DR immunopeptidome of lung immune cells and apply it to newly diagnosed RA patients versus current-smoker healthy controls, as well as to investigate the connection with the HLA-DR genotype. The HLA-DR immunopeptidome method was improved to facilitate characterization from as few as 6 million bronchoalveolar lavage (BAL) cells per subject, consisting primarily of alveolar macrophages. This method was applied to newly diagnosed RA patients naive to treatment (n=9, LURA cohort), as well as healthy current-smoker controls (n=10, COSMIC cohort). For five of the RA patients, a 6-month follow-up after initiation of the standard-of-care treatment regime was also included. After isolation and purification, peptide samples were separated by nano-flow liquid chromatography coupled to an Orbitrap mass spectrometer equipped with ion mobility device (FAIMS). Mass spectra acquired in data dependent acquisition mode were then searched against a human proteome database. Subsequently, the identified peptides were deconvoluted to their predicted binding HLA-DR allele using MHCMotifDecon based on the sequenced genotype of the individual. An optimized sample preparation and analytic method enabled the detection of over 23,000 peptides from over 3,000 source proteins with between 1,000 and 5,000 peptides identified per sample. Notably, the application of FAIMS with three compensation voltages allowed for efficient transfer of 2+, 3+, and 4+ peptide ions while removing singly charged background ions. Hierarchical clustering revealed that the immunopeptidome was more driven by the HLA-DR genotype than by RA disease or sex. However, since the HLA-DR genotype is a strong risk factor for RA, these results are convoluted. When deconvoluting the peptides to their predicted binding allele, the HLA-DRB1 alleles *01:01, *04:01, *04:04, *04:05, *04:07, and *10:01 were consistently assigned more peptides than other alleles. Except for *04:07 these alleles belong to the SE risk factor alleles, providing a potential explanation between HLA-SE and RA pathogenesis. Native peptides from known citrullinated and non-modified RA autoantigens (such as -enolase and calreticulin) were detected and validated as binders in prediction algorithms. No significant differences were found between base line and follow-up (post-treatment) samples from RA patients. Taken together, this data characterizes the HLA-DR immunopeptidome in the lung of early RA in an unprecedented manner, which together with future immunogenicity studies will help our understanding of the connection between the lung and the pathogenesis of RA. Finally, more peptides predicted to bind to SE alleles and *04:07 compared to other alleles demands further study on the relative expression of HLA-DR alleles and presentation mechanisms to understand the implications for RA.

immunology↗

bioRxiv2024

microRNA and non-targeted proteome analysis of liquid biopsies from the distal lung collected by Particles in Exhaled Air (PExA) reveals presence from extracellular vesicles

Background. Early detection and longitudinal follow-up are essential for timely diagnosis and treatment for lung diseases. Currently, invasive methods are often required to examine the distal parts of the lungs. The growing need to explore the bio-molecular mechanisms in various lung diseases highlights the importance of non-invasive methods. The use of particles in exhaled air (PExA), a non-invasive technique for sampling of epithelial lining fluid from distal airways, is gaining attention. MicroRNAs (miRNAs) are crucial in modulating protein expression both intracellularly and intercellularly, often transported via extracellular vesicles. Dysregulated miRNAs have been linked to many pulmonary diseases, and their relative stability, especially when encapsulated in EVs, makes them promising biomarkers. Here we report for the first time multi-modal analysis of miRNAs and proteins in PExA, offering an opportunity to study the role of miRNAs in the pathophysiology of respiratory diseases in a non-invasive manner. Methods. Exhaled particles were collected from healthy subjects using the PExA 2.0 instrument utilizing the optimized PExA breathing maneuver. PExA samples collected on different types of impaction membranes were analyzed using a non-targeted mass spectrometry-based proteomics workflow optimized for single-cell detection, and a miRNAseq workflow optimized for low input starting material. Technical validation of a subset of the detected miRNAs was performed using custom-designed miRCURY LNA miRNA PCR assays. Pathway enrichment analyses for the detected proteins were performed using STRING. Results. Proteomic analysis consistently identified over 50 proteins across multiple types of impaction membranes, sample dilution series, and individuals down to a single PEx sPOT (24ng starting material). We observed a significant enrichment of proteins associated with extracellular vesicles, including blood microparticles, and secretory granules. miRNA-seq revealed 39 mature miRNAs, the majority of which have been previously reported to be detected in the airways. Some were also reported to be secreted by primary human airway epithelial cells via extracellular vesicles. miRNA-125b and the members of the let-7 family were among the most abundant miRNAs detected. Fluorometric assays showed significant RNase activity in both PExA and other lung-related samples, such as bronchoalveolar lavage fluid, suggesting that this activity originates from the airways and is independent of the sampling techniques used. The workflow for extraction and processing of the PExA collection membrane, tested with abundant synthetic miRNAs and analyzed using the miRCURY LNA miRNA PCR assay, yielded results comparable to control samples, indicating that the membrane material does not interfere with the assay. Conclusions. Using PExA, we identified several miRNAs reported to be dysregulated in pulmonary disorders. The enrichment of extracellular secretory components in the core protein list, along with the elevated RNAse activity in the respiratory tract, suggest that the detected miRNAs may be encapsulated within extracellular vesicles. These miRNAs are of particular interest due to their potential role in intercellular communication. Our findings suggest that PExA holds a potential as a non-invasive tool for studying extracellular vesicle-mediated miRNA cargo in the small airways.

physiology↗

bioRxiv2024

Transcriptomic analysis reveals lipid metabolism and macrophage involvement associated with nintedanib treatment in a rat bleomycin model

INTRODUCTIONIdiopathic pulmonary fibrosis (IPF) is a progressive and irreversible lung disease with a poor prognosis. While pirfenidone and nintedanib offer some benefits, they cannot cure IPF. Nintedanib inhibits various proliferative pathways and has antifibrotic effects, but its molecular mechanisms and impact on the lung transcriptome in vivo remain unclear. This study aims to evaluate nintedanibs transcriptomic profile in a rat model of bleomycin-induced lung fibrosis. METHODOLOGY/PRINCIPAL FINDINGSLung fibrosis was induced by two intratracheal administrations of bleomycin. Nintedanib protocol included three weeks of daily oral treatments beginning seven days after the first bleomycin dose. Left lungs were processed for histological evaluation using an automated fibrosis quantification system and the Ashcroft Score, while the right lungs were used for RNA sequencing to conduct differential expression and correlation network analysis (WGCNA). WGCNA modules were examined by cell and pathway enrichment analysis. Lipid peroxidation was assessed through the measurement of malondialdehyde in right lung lysates. Bleomycin induced significant fibrotic lesions, as confirmed by the histological evaluations. Nintedanib reduced fibrotic lesion size by about 15% and decreased severe Ashcroft scores. When compared to controls, the number of differentially expressed genes decreased from over 2000 to barely more than 400 after nintedanib treatment. WGCNA identified two gene clusters correlated to histological parameters, with nintedanib-treated animals showing gene expression levels similar to control animals. One cluster was associated with mesenchymal cells and extracellular matrix-related pathways, in line with the known anti-fibrotic effect of nintedanib. The second cluster, involving principally macrophages, was related to lipid metabolism, potentially uncovering a new mechanistic role of nintedanib in modulating lung fibrosis. CONCLUSIONS/SIGNIFICANCEThe mechanisms involving macrophages and lipid metabolism, influenced by nintedanib in this study, may open new research directions to better inquire the role of this cellular type in tissue repair and pathological lung fibrosis.

pharmacology and toxicology↗

bioRxiv2023

Global meta-analysis reveals overall benefits of silvopastoral systems for biodiversity

Domestic livestock grazing accounts for roughly one quarter of the worlds terrestrial surface and is a leading driver of biodiversity loss. Yet, it also provides a critical livelihood for nearly one billion smallholder farmers, creating a paradox that highlights the need for conservation strategies to balance human and ecological needs. Silvopastoral systems (SPS) integrate trees with livestock pastures, offering a promising solution to boost livestock productivity while safeguarding natural areas and biodiversity. However, evidence for the biodiversity benefits provided by SPS is limited to studies focusing on specific geographic regions or taxa. Through a global meta-analysis of 45 studies spanning 15 countries, four biogeographic regions, and seven taxa, we provide the first quantitative synthesis evaluating how SPS affect biodiversity and community stability relative to treeless pastures and natural forests. Overall, we show that SPS harbor higher levels of biodiversity (i.e., richness, abundance, and diversity) and stability than treeless pastures, and perform comparably to nearby forests. However, variations exist across regions and taxa, with the strongest positive responses in tropical dry regions and for low-mobility taxa like invertebrates and plants. Mammals, birds, and soil microorganisms, on the other hand, showed no significant biodiversity differences between treeless pastures and SPS. Thus, integrating SPS and protected areas as complementary components of a multifunctional landscape will be key to halting multi-taxa biodiversity loss and building sustainable livestock systems. Our findings support the conservation potential of SPS, while underscoring the need for strategic implementation to maximize benefits for biodiversity conservation.

ecology↗