Optimal LC-MS metabolomic profiling reveals emergent changes to monocyte metabolism in response to lipopolysaccharide.

Introduction: Immunometabolism examines the links between immune cell function and metabolism. Dysregulation of immune cell metabolism is now an established feature of innate immune cell activation. Advances in liquid chromatography mass spectrometry (LC-MS) technologies have allowed discovery of unique insights into cellular metabolomics. Here we have studied and compared different sample preparation techniques and data normalisation methods described in the literature when applied to metabolomic profiling of human monocytes. Methods: Primary monocytes stimulated with lipopolysaccharide (LPS) for four hours was used as a study model. Monocytes (n=24) were freshly isolated from whole blood and stimulated for four hours with lipopolysaccharide (LPS). A methanol-based extraction protocol was developed and metabolomic profiling carried out using a Hydrophilic Interaction Liquid Chromatography (HILIC) LC-MS method. Data analysis pipelines used both targeted and untargeted approaches, and over 40 different data normalisation techniques to account for technical and biological variation were examined. Cytokine levels in supernatants were measured by ELISA. Results: This method provided broad coverage of the monocyte metabolome. The most efficient and consistent normalisation method was measurement of residual protein in the metabolite fraction, which was further validated and optimised using a commercial kit. Alterations to the monocyte metabolome in response to LPS can be detected as early as four hours post stimulation. Broad and profound changes in monocyte metabolism were seen, in line with increased cytokine production. Elevated levels of amino acids and Krebs cycle metabolites were noted and decreases in aspartate and ß-alanine are also reported for the first time. In the untargeted analysis, 154 metabolite entities were significantly altered compared to unstimulated cells. Pathway analysis revealed the most prominent changes occurred to (phospho-) inositol metabolism, glycolysis, and the pentose phosphate pathway. Discussion: These data report the emergent changes to monocyte metabolism in response to LPS, in line with reports from later time points. A number of these metabolites are reported to alter inflammatory gene expression, which may facilitate the increases in cytokine production. Further validation is needed to confirm the link between metabolic activation and upregulation of inflammatory responses.

Study protocol for the St James's Hospital, Tallaght University Hospital, Trinity College Dublin Allied Researchers' (STTAR) Bioresource for COVID-19.

Background: The current coronavirus disease 2019 (COVID-19) pandemic began in Ireland with the first confirmed positive case in March 2020. In the early stages of the pandemic clinicians and researchers in two affiliated Dublin hospitals identified the need for a COVID-19 biobanking initiative to support and enhance research into the disease. Through large scale analysis of clinical, regional, and genetic characteristics of COVID-19 patients, biobanks have helped identify, and so protect, at risk patient groups The STTAR Bioresource has been created to collect and store data and linked biological samples from patients with SARS-CoV-2 infection and healthy and disease controls. Aim: The primary objective of this study is to build a biobank, to understand the clinical characteristics and natural history of COVID-19 infection with the long-term goal of research into improved disease understanding, diagnostic tests and treatments. Methods: This is a prospective dual-site cohort study across two tertiary acute university teaching hospitals. Patients are recruited from inpatient wards or outpatient clinics. Patients with confirmed COVID-19 infection as well as healthy and specific disease control groups are recruited.  Biological samples are collected and a case report form detailing demographic and medical background is entered into the bespoke secure online Dendrite database. Impact: The results of this study will be used to inform national and international strategy on health service provision and disease management related to COVID-19. In common with other biobanks, study end points  evolve over time as new research questions emerge. They currently include patient survival, occurrence of severe complications of the disease or its therapy, occurrence of persistent symptoms following recovery from the acute illness and vaccine responses.

Pro-inflammatory Stimulation of Monocytes by ANCA Is Linked to Changes in Cellular Metabolism.

Clinical and experimental data suggest that pathogenesis in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is driven by ANCA-mediated activation of neutrophils and monocytes. While the role of neutrophils has been extensively investigated, the function of monocytes remains relatively understudied. We have previously demonstrated that stimulation of monocytes with anti-myeloperoxidase (MPO), but not anti-proteinase-3 (PR3), antibodies results in production of the pro-inflammatory cytokine IL-1ß. Changes in cellular metabolism, particularly a switch to glycolysis, have recently been linked to activation of immune cells and production of IL-1ß. Therefore, we investigated the metabolic profile of monocytes following ANCA stimulation. We found a significant increase in glucose uptake in anti-MPO stimulated monocytes. Interestingly, both anti-MPO and anti-PR3 stimulation resulted in an immediate increase in glycolysis, measured by Seahorse extracellular flux analysis. However, this increase in glycolysis was sustained (for up to 4 h) in anti-MPO- but not anti-PR3-treated cells. In addition, only anti-MPO-treated cells exhibited increased oxidative phosphorylation, a metabolic response that correlated with IL-1ß production. These data indicate that monocyte metabolism is altered by ANCA, with divergent responses to anti-MPO and anti-PR3 antibodies. These metabolic changes may underlie pathologic immune activation in ANCA associated vasculitis, as well as potentially contributing to the differing clinical phenotype between PR3- and MPO-ANCA positive patients. These metabolic pathways may therefore be potential targets for therapeutic intervention.

Pathogenesis of ANCA-associated vasculitis: an emerging role for immunometabolism.

ANCA-associated vasculitis (AAV) is a severe systemic autoimmune disease. A key feature of AAV is the presence of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) directed against myeloperoxidase (MPO) or proteinase-3 (PR3). ANCA are key to the pathogenesis of AAV, where they activate innate immune cells to drive inflammation. Pre-activation or 'priming' of immune cells appears to be important for complete cellular activation in AAV. The burgeoning field of immunometabolism has illuminated the governance of immune cell function by distinct metabolic pathways. There is ample evidence that the priming events synonymous with AAV alter immune cell metabolism. In this review we discuss the pathogenesis of AAV and its intersection with recent insights into immune cell metabolism.

Alkylating histone deacetylase inhibitors may have therapeutic value in experimental myeloperoxidase-ANCA vasculitis.

Current therapies for treating antineutrophil cytoplasm autoantibody (ANCA)-associated vasculitis include cyclophosphamide and corticosteroids. Unfortunately, these agents are associated with severe adverse effects, despite inducing remission in most patients. Histone deacetylase inhibitors are effective in rodent models of inflammation and act synergistically with many pharmacological agents, including alkylating agents like cyclophosphamide. EDO-S101 is an alkylating fusion histone deacetylase inhibitor molecule combining the DNA alkylating effect of Bendamustine with a pan-histone deacetylase inhibitor, Vorinostat. Here we studied the effects of EDO-S101 in two established rodent models of ANCA-associated vasculitis: a passive mouse model of anti-myeloperoxidase IgG-induced glomerulonephritis and an active rat model of myeloperoxidase-ANCA microscopic polyangiitis. Although pretreatment with EDO-S101 reduced circulating leukocytes, it did not prevent the development of passive IgG-induced glomerulonephritis in mice. On the other hand, treatment in rats significantly reduced glomerulonephritis and lung hemorrhage. EDO-S101 also significantly depleted rat B and T cells, and induced DNA damage and apoptosis in proliferating human B cells, suggesting a selective effect on the adaptive immune response. Thus, EDO-S101 may have a role in treatment of ANCA-associated vasculitis, operating primarily through its effects on the adaptive immune response to the autoantigen myeloperoxidase.

The Role of Proteomics in Biomarker Development for Improved Patient Diagnosis and Clinical Decision Making in Prostate Cancer.

Prostate Cancer (PCa) is the second most commonly diagnosed cancer in men worldwide. Although increased expression of prostate-specific antigen (PSA) is an effective indicator for the recurrence of PCa, its intended use as a screening marker for PCa is of considerable controversy. Recent research efforts in the field of PCa biomarkers have focused on the identification of tissue and fluid-based biomarkers that would be better able to stratify those individuals diagnosed with PCa who (i) might best receive no treatment (active surveillance of the disease); (ii) would benefit from existing treatments; or (iii) those who are likely to succumb to disease recurrence and/or have aggressive disease. The growing demand for better prostate cancer biomarkers has coincided with the development of improved discovery and evaluation technologies for multiplexed measurement of proteins in bio-fluids and tissues. This review aims to (i) provide an overview of these technologies as well as describe some of the candidate PCa protein biomarkers that have been discovered using them; (ii) address some of the general limitations in the clinical evaluation and validation of protein biomarkers; and (iii) make recommendations for strategies that could be adopted to improve the successful development of protein biomarkers to deliver improvements in personalized PCa patient decision making.