Immunometabolism Flashcards
Define Immunometabolism
The metabolic state of immune cells and its dynamic changes during homeostasis and inflammation
Two main themes of immunometabolism
- Whole body metabolism
2. Cellular bioenergetics
What is whole body metabolism?
- Identification of specific immune cell populations resident in tissues (e.g. significant increase in immune infiltration into adipose tissue in obesity)
- Novel metabolism-altering factors they secrete highlight the dynamic communication between the immune system and adipocytes (e.g. TNF + insulin signalling)
- Interruption by immune cells can influence endogenous signalling pathways
What is cellular bioenergetics?
- Intracellular metabolic pathways control the effector functions of immune cells
- Study of real-time substrate utilisation and metabolic flux in specific immune populations that result in metabolic reprogramming of immune cells
- Vital metabolic pathways with direct connection to immune reprogramming: glycolysis, TCA cycle, oxidative phosphorylation, pentose phosphate pathway (PPP), fatty acid oxidation & fatty acid synthesis
Macrophage accumulation in adipose tissue is the result of?
- Recruitment, retention, and proliferation
- Abundant presence of tissue-resident macrophages are polarized toward repair & maintenance functions in lean adipose tissue
- Suggests a physiological role for these cells in homeostasis
- These macrophages are reprogrammed into an inflammatory phenotype in the setting of metabolic stress (e.g. obesity) leading to functional deterioration
True or False: Mounting of an immune response and functional programming within a cell is associated with innate metabolic change
True
What is flow cytometry?
- A technique used to detect and measure physical and chemical characteristics of a population of cells or particles
- A fluorescence-based technology for the high-throughput analysis of characteristics of cells or particles
- Need a single cell suspension - blood most common & easiest
What is a flow cytometer?
A machine composed of the following parts: 1. Fluidics • Sample acquisition/focusing system 2. Optics • Light source (laser) • Mirrors, lens and filters 3. Electronics • Fluorescence detectors 4. Computer software
Workflow of a flow cytometry experiment
- Cell isolation
- Separation
- Tissue dissociation
- Single cell suspension - Staining
- Antibodies
- Reactive dyes - Fixation
- Acquisition
- Data Analysis
Can apoptosis be easily measured with flow cytometry?
Yes
Example of files used in data analysis of flow cytometry?
FCS files
What does light scatter indicate?
- Indicates size and granularity
- Also indicative of the type of immune cell
- Usually measured with Blue Laser
- Not based on fluorescence
Fluorescence discrimination
- Light emission by fluorophores
- Multiple parameters simultaneously
- Intensity directly correlates with protein expression
What is FACS?
Fluorescence activated cell sorting
- Slightly different machine to flow cytometer
- Preparatory technique
- Separation of cell populations
- Bulk or single cell sorting
Downstream applications of FACS
- Cell culture
- Metabolic assay (Seahorse)
- Protein extraction
- DNA/RNA extraction
