Periodic Reporting for period 1 - HUMANITY (Human Lung Microbiota And Innate Immunity)
Période du rapport: 2019-03-25 au 2021-03-24
Lung microbiota is next human niche that is gaining traction rapidly worldwide. Using culture- independent techniques it has been shown that healthy human lungs harbour characteristic microbial communities. Studies on the deep lung are rare because the best sample to work with is Bronchoalveolar Lavage Fluid (BALF), which is an invasive endoscopy procedure generally not applied to healthy individuals. Despite the implication of lung-associated bacteria in various diseases and impact on organ stability after transplantation, our current understanding of resident lung microbiota and specific interaction with human immune system is poor, which will be investigated in this project and hence, it is both highly relevant and well-timed. This MSCA funded project was aimed at (i) investigating the lung microbiota composition under different immune states in the transplanted lungs and cultivate resident lung bacteria, (ii) Identify key species within resident lung bacteria and elicited immune signatures, (iii) define underlying mechanisms and specific interactions between key lung bacteria and immune cells. By working on these objectives, we were able to show that the microbial ecosystem in the deep lung is dynamic and can harbour discrete "pneumotypes", which are associated with specific immune reaction and impacts lung health. We were also able to show differential immune activation by both commensal and pathogens isolated from the human lung.
I worked with the Department of Respiratory Medicine at the Lausanne University Hospital (CHUV). Here, they collected samples and performed 16S rRNA amplicon sequencing on 234 longitudinal bronchoalveolar lavage (BAL) samples from lung transplant recipients over a period of 48 months. I combine this amplicon sequencing data with my culture-based data, host gene expression, patient metadata and machine learning approaches to explore the microbiota of the deep lung. Thereby showing that specific microbial communities or "pneumotypes" are associated with infection, rejection and proper functioning of the transplanted organ. In addition, I cultivated samples on different media under various oxygen concentrations, genotyped individual bacteria. I have created a large biobank called the Lung Microbiota Culture Collection (LUMICOL) from human BAL samples and made in publicly available and have started getting requests to acquire the collection by multiple research groups globally. I combined amplicon sequencing data with bacterial culture results, bacterial and commensal virus numbers, host gene expression data, and patient metadata and used machine learning approaches to explore the microbiota of the deep lung. Next, we wanted to understand how these individual species from the human lower respiratory tract influence human innate immunity. For this, I established a screening approach to identify key lung bacteria and associated immune response in macrophages, the dominant immune cells in lung tissues. For this, I utilised a variant of commercially available cell line called THP-1 human monocytes expressing a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the transcriptional control of Nuclear factor kappa (NFk)-B, the major transcription factor in inflammatory responses. Our aim here was to push the system to be phenotypically similar to the human alveolar macrophages. Hence, I optimised the differentiation procedure and used flow cytometry and mass cytometry in collaboration with Flow Cytometry Core Facility, EPFL, Lausanne, to investigate surface protein markers on macrophages to assess its immunotype. Using these macrophages, we establish a fast, reliable, scalable screening strategy using to investigate the inflammatory potential of diverse bacteria from human lung. We have also made small artifical communities (SACs) by mixing individual bacteria from LUMICOL, which are essentially guided by the observations we made about the different pneumotypes and their taxonomic composition. These SACs were also exposed to macrophages for assessing its inflammatory activity. For the second part of this objective, we initially planned for using animal models. Ongoing work includes host gene expression studies by RNA-seq. Finally, we are now investigating mechanisms by which specific lung bacteria or SACs induce immune changes. For this we aim to know what factors are responsible in both bacteria and host that play important role in these interactions. We have sequenced genomes of major lung bacteria in LUMICOL to know the genes and pathways that exist in the lung microbial ecosystem. We determine the recognition mechanism of lung bacteria by human Pathogen Recognition Receptors (PRRs) and when differentially recognised by macrophages. We are now close to showing that these interactions may be governed by variations in bacterial surface antigens like Lipopolysaccharide (LPS) and Peptidoglycan.
Before the start of this project there were some open questions in this field and our findings that takes it beyond the state of art. Little was talked about bacterial prevalence in lung bronchoalveolar lavage samples and also about the temporal dynamics of lung microbiota. Using our large cohort of lung transplant recipients collected upto 48 months, we show the most prevalent bacteria amongst these samples and show that these comprised the balanced pneumotype that is associated with clinical stability. Using probabilistic model, we show that the lung favours the "balanced" pneumotype that provides the best ecosystem for immune homeostasis. We also always worked with real bacterial numbers instead of relative abundances and combined bacteria culture data to solidify our claims. Alveolar Macrophages (AM) are the major surveying cells in the human alveolar space. But, there was a lack of a standardized in vitro model for studying the largely unknown AM-lung bacteria interactions. Here we present a model system that fills this gap. I reveal for the first time that typically non-pathogenic lung bacteria trigger differential inflammatory responses. We also show that our AM-like cells react to lung bacterial pathogens besides the classical microbe-associated molecular patterns (MAMPs). SACs resembling the balanced pneumotype confered varied degree of protection against inflammation induced by pathogens: S. aureus and P. aeruginosa. Currently, we are investigating underlying and previously unresolved mechanisms using RNAseq, cellular pathway inhibition and cytokine profile analysis (manuscript in preparation). We show novel interactions between human lung bacteria and macrophages, highlighting the importance lung commensals in immune homeostasis. Respiratory diseases are a major public health concern. Bacterial pathogens cause pneumonia or inflammatory exacerbations in pre-existing conditions. We are seeing the COVID-19 pandemic, caused by a respiratory virus, SARS-CoV-2. It is plausible that this will not be the last pandemic targeting the respiratory tract. An important preventive or therapeutic counter to these respiratory illnesses may lie in the already existing lung microbiota. Finally, we have uncovered the potential of LUMICOL as a major resource for individual bacteria or tailor-made communities with therapeutic potential in lung disease.