Innate immune defense during pneumonia in the elderly: implications of cell-specific changes in the metabolome and lipidome

BACKGROUND Pneumonia is the fourth most common cause of death globally, and the world’s leading infectious killer, responsible for an estimated 3 million deaths annually. Elderly persons are vulnerable to community-acquired pneumonia (CAP); almost half of all hospitalized patients with CAP are > 65 years of age. Many age-associated diseases are linked to deterioration of the immune system, a process termed immunosenescence. Immunosenescence amongst other has a major impact on innate responses against bacterial pathogens. Neutrophils (polymorphonuclear cells, PMNs) and monocytes are important cellular components of the innate immune response. The term immunometabolism relates to changes in intracellular metabolic pathways in immune cells that change their function. Current knowledge indicates a complex interplay between cellular metabolic reprogramming, and immunity, ageing and epigenetics (including DNA methylation). LINK BETWEEN PREVIOUSLY AWARDED AND CURRENT APPLICATION Both applications focus on innate host defense during CAP. The previously awarded ZonMW grant investigates the impact of altered DNA methylation in monocytes and PMNs, such as occurs during ageing, on innate host defense against respiratory pathogens and during pneumonia. To this end a prospective observational study in patients with CAP was initiated in which monocytes and PMNs were collected for detailed immunological analyses. The current application will investigate changes in the metabolome and lipidome of monocytes and PMNs in patients with CAP as compared with gender- and age-matched controls, and their association with immune responses. For these new analyses samples will be used that already have been collected as part of the original project. OVERALL OBJECTIVE To obtain insight in the impact of pneumonia on the metabolome and lipidome in monocytes and PMNs and to assess the association of cell-specific metabolomes and lipidomes with the responsiveness of these cells to bacterial agonists, as well as with the transcriptome and DNA methylome within the same cells. STUDY QUESTIONS (1) How does pneumonia impact on the metabolome and lipidome of circulating monocytes and PMNs, and how do the changes detected relate to the function of key metabolic pathways within these cells? (2) What is the association between the metabolome and lipidome in monocytes and PMNs, and their responses to CAP pathogens in vitro? (3) What is the association between the metabolome and lipidome in monocytes and PMNs, and the transcriptome and DNA methylome in the same cells? APPROACH Ad Study Question 1: We will analyze the metabolome and lipidome in purified monocytes and PMNs (stored as lysates specifically prepared for these analyses) from 54 patients admitted to the hospital with CAP, as well as from 34 gender- and age-matched controls. Monocyte and PMN metabolomes and lipidomes will be measured using ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry. Data will be analyzed through the bioinformatics pipeline available in Amsterdam UMC and metabolites and lipids will be grouped in metabolic pathways using pathway analysis tools. Ad Study Question 2: As part of the previously awarded grant monocytes and PMN responsiveness was tested by stimulations with bacterial agonists and measurements of cytokines and degranulation products in cell supernatants; RNA expression profiles were determined by RNA sequencing. The association between metabolome/lipidome and cellular responsiveness to bacterial agonists will be analyzed. Functional links will be assessed between metabolic/lipidomic changes in monocytes and immunological function using a human monocytic cell line and gene deletion by Crispr-Cas9 technology. Gene targets will be selected based on the analyses described above relating to metabolic/lipidomic and RNA expression profiles, and genetically modified cells will be analyzed for responsiveness toward bacterial agonists using a variety of readouts. Ad Study Question 3: As part of the previously awarded grant the transcriptome and DNA methylome of monocytes and PMNs are determined by RNA sequencing and Reduced Representation Bisulfite Sequencing. The association between the cell-specific metabolome/lipidome and the cellular transcriptome and DNA methylome will be evaluated using advanced bioinformatics and biostatistics. RELEVANCE AND INNOVATION CAP represents a major health burden in the elderly population. This project aims to explore novel research areas, linking the emerging field of age-associated cell-specific (monocytes and PMNs) changes in DNA methylation, the metabolome and lipidome with (impaired) host defense against pneumonia in the elderly. This project falls within several priorities of this call, i.e., it focuses on infections of the airways and the elderly, it makes use of interdisciplinary collaborations, and it builds on the previously awarded grant.