Immunometabolism

Question 1

Define Immunometabolism

Answer 1

The metabolic state of immune cells and its dynamic changes during homeostasis and inflammation

Question 2

Two main themes of immunometabolism

Answer 2

1. Whole body metabolism

2. Cellular bioenergetics

Question 3

What is whole body metabolism?

Answer 3

• 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

Question 4

What is cellular bioenergetics?

Answer 4

• 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

Question 5

Macrophage accumulation in adipose tissue is the result of?

Answer 5

• 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

Question 6

True or False: Mounting of an immune response and functional programming within a cell is associated with innate metabolic change

Answer 6

True

Question 7

What is flow cytometry?

Answer 7

• 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

Question 8

What is a flow cytometer?

Answer 8

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

Question 9

Workflow of a flow cytometry experiment

Answer 9

1. Cell isolation
   - Separation
   - Tissue dissociation
   - Single cell suspension
2. Staining
   - Antibodies
   - Reactive dyes
3. Fixation
4. Acquisition
5. Data Analysis

Question 10

Can apoptosis be easily measured with flow cytometry?

Answer 10

Yes

Question 11

Example of files used in data analysis of flow cytometry?

Answer 11

FCS files

Question 12

What does light scatter indicate?

Answer 12

• Indicates size and granularity
• Also indicative of the type of immune cell
• Usually measured with Blue Laser
• Not based on fluorescence

Question 13

Fluorescence discrimination

Answer 13

• Light emission by fluorophores
• Multiple parameters simultaneously
• Intensity directly correlates with protein expression

Question 14

What is FACS?

Answer 14

Fluorescence activated cell sorting

• Slightly different machine to flow cytometer
• Preparatory technique
• Separation of cell populations
• Bulk or single cell sorting

Question 15

Downstream applications of FACS

Answer 15

• Cell culture
• Metabolic assay (Seahorse)
• Protein extraction
• DNA/RNA extraction

Question 16

Applications of flow cytometry

Answer 16

• Diagnostics
• Research
  • Immunology (immunophenotyping)
  • Cancer biology (useful for lymphoma)
  • Cancer therapy
  • Microbiology
  • Environmental science

Question 17

What is Agilent Seahorse?

Answer 17

A Metabolic Kinetics Assay

Question 18

What do Agilent Seahorse XF assays measure?

Answer 18

The rates of change of the two key energy metabolism pathways

Question 19

What can Seahorse be used for?

Answer 19

Can be used in obstetrics, neuronal work (characterising mitochondrial respiration), testing drugs that affect mitochondria respiration

Question 20

In the metabolic assay, what is first injected?

Answer 20

Oligomycin (Complex V in electron transport chain) is first injected. This inhibits ATP synthase
ETC has to work v hard since its membrane potential has been disrupted

Question 21

XF Glycolysis Stress Test

Answer 21

Measure basal glycolysis, then glucose, then introduce oligomycin (shuts down ATP via inhibiting ATP synthase)
Cells can only use glycolysis (not ETC) to produce ATP → stress

Question 22

Applications of Seahorse XF Technology

Answer 22

Immunology
Cancer
Toxicology
Obesity, diabetes and metabolic disorders

Question 23

What have a greater capacity for mitochondrial respiration than effector cells?

Answer 23

Memory cells

Question 24

Can you take a tissue biopsy and isolate cells for metabolic assay?

Answer 24

No