Reprogramming lipid signalling in neutrophils for precision treatment of sepsis

Background

Sepsis is one of the leading causes of mortality around the globe and is characterised by both overwhelming infection and immune dysfunction. Neutrophils protect us from sepsis by engulfing bacteria into membrane-bound vesicles called phagosomes. However, despite the key role of neutrophils in controlling infection, it is unknown how the lipids that form the phagosome membrane change in sepsis patients and what consequences lipidomic remodelling has on cell function (e.g. bacterial killing/NET release) and shaping the overall immune response to invading micro-organisms (e.g. processing of antigen for presentation). Indeed, our recent data suggests that changes in phagosome lipids trigger a subset of neutrophil phagosomes to remain connected to the extracellular environment, with implications for phagosome fate and the overall inflammatory response to invading microorganisms. 

We therefore hypothesise that sepsis induces specific alterations in phagosome lipid signalling and that these changes may impair phagosome maturation and microbial killing. Rebalancing these lipid pathways could restore immune function and improve patient outcomes. 

Aims

Aim 1

• Define how the lipidome of neutrophil phagosomes changes during sepsis.
  • This will be achieved by feeding bacteria to neutrophils isolated from ICU sepsis patients and healthy controls. Phagosomes will then be isolated and the lipidome quantified using Liquid Chromatography-Mass Spectrometry (LC-MS). Whole-cell lipidomics will also be performed to provide a snapshot of whole-cell changes in behaviour. Samples will be taken from patients following admission to ICU and microbiology will be performed on all samples to identify the causative agent.
  • Patients will be stratified into successfully treated, intermediate response to therapy and non-responsive to treatment; thus enabling evaluation of whether phagosomal lipid signatures predict patient outcomes, supporting precision medicine stratification.
  • Phagosome maturation will be measured in parallel using established pH/ROS sensitive dyes, bacterial killing assays, antigen processing and presentation assays and immunostaining for markers of phagosome maturation (e.g. Rab5, Rab7, LAMP1).
• Correlative Light Electron Microscopy (CLEM) will define how the subcellular structure of neutrophils differs between sepsis patients and healthy controls. We hypothesise that more ‘leaky phagosomes’ will be identified in sepsis patients vs healthy controls.

Aim 2

• Investigate if changing the lipidome of neutrophil phagosomes modulates phagosome maturation in vitro.
  • Candidate lipid pathways from Aim 1 (e.g., PI3P, PI(3,5)P2, sphingolipids) will be manipulated using pharmacological modulators and the effect of changing specific lipid pathways assessed using the phagosome maturation assays defined above. 

Aim 3

• In vivo validation and translational assessment
  • Candidate lipid pathways from Aim 1 and 2 will be manipulated in zebrafish using pharmacological modulators and CRISPR/Cas9 knockout/overexpression systems.
  • Live imaging of infected zebrafish will capture in real time how changes in lipid metabolism alter how a neutrophil responds to infection- thus modelling sepsis progression in vivo. 

Training outcomes

1. Bioinformatics to integrate lipidomic and clinical data into a predictive biomarker model.
2. Develop key skills in imaging, image analysis, molecular biology and lipid analysis.
3. Work with cross-disciplinary teams (including clinicians, immunologists, imaging specialists and cell biologists) at the UoE and the UoG.