AMI - Lecture 5 - Immunometabolism

Describe the M1 state of macrophages

• M1 → host defense, pro inflammatory, kills tumor cells, chronic inflammation, short lived

• metabolism;

  • rely on glycolysis to produce ATP

  • TCA cycle broken at specific points (isocitrate dehydrogenase) → leading to intermediates used for inflammatory molecules.

  • Less fatty acids produced than M2.

  • some AA made

Explain the M2 stage of macrophages

• M2 → homeostasis, slow, anti inflammatory, tissue repair, wound healing, promote tumor growth, long lived

• metabolism;

  • cells rely on oxphos for sustained ATP,

  • glycolysis downregulated,

  • TCA cycle active → support oxphos

  • PPP active (NADH production)

  • fatty acid synthesis (membrane repair)

  • aa production

Explain the arginine metabolism in M1 and M2

• the balance between inducible nitric oxide synthase (iNOS) and arginase is how you determine if MQ are in M1 or M2 phase

• M1 → iNOS is dominant, iNOS converts L-arginine into nitric oxide (NO), NO plays microbial + tumor killing role

  • in M1 the aa metabolism supports proliferation + NO production

  • LPS promotes M1

• M2 → arginase dominance, arginine metabolizes L-arginine into → ornithine + urea

  • ornithine further used to produce → proline + polyamines (both needed for tissue repair + cell proliferation)

  • aa metabolism in M2 supports arginine pathway activity

  • IL4 is anti inflammatory, promotes M2

GAPDH importance in M1, M2

• M1 → increased GAPDH supports glycolysis, powers pro inflammatory responses

• M2 → decreased reliance on GAPDH, because M2 is dependent on OXPHOS + TCA

• GAPDH activated when there is stress / cellular damage

• GAPDH → key enzyme in glycolysis

  • in resting MQ low glycolysis → GAPDH present, can bind mRNA in T cell → mRNAs not translated into pro inflammatory cytokines

  • When t cell / MQ is activated → GAPDH will be post translationally modified → mRNA will be translated → pro inflammatory cytokines formed (TNFalpha, IL1beta)

explain the broken TCA cycle in M1 in more detail

• disruption TCA cycle leads to → accumulation succinate + citrate

  • citrate drives production NO + inflammatory mediators

  • succinate stabilizes HIF1alpha, this promotes expression of glycolytic enzymes + inflammatory genes (IL1beta)

• succinate is secreted by M1 and reuptaken through GPR91 receptor → GPR91 activation amplifies inflammatory response.

  • when GPR91 receptor (succinate) was knocked out → tissue inflammation reduced in arthritis.

• thus broken TCA cycle → fuel the glycolytic + pro-inflammatory metabolism of M1

What role does itaconate play in macrophage metabolism

• itaconate is derived from citrate in LPS activated MQ.

  • it exhibits anti inflammatory + antimicrobial properties

  • it’s produced when citrate accumulates in cytosol by enzyme IRG1

    • IRG1 KO MQ → itaconate production absent → inflammatory response

    • normal MQ itaconate produced → tempering of inflammatory response

Explain the 4 mechanisms through which itaconate work

• itaconate inhibits succinate dehydrogenase (under debate) leading to less succinate accumulation → no stabilization HIF1alpha → less inflammation

• itaconate activates NRF2 by freeing it from KEAP1, resulting in NRF2 moving to nucleus → initiating transcription of anti oxidant + anti inflammatory genes → protects MQ from damage ROS

• itaconate inhibits bacterial enzymes (isocitrate lyase) preventing bacterial survival

• itaconate activates MAPK pathway leads to expression of ATF3. this is a transcription factor known to suppress pro inflammatory genes (IL1)

  • NRF2 activation also promotes transcription of ATF3

Explain how the seahorse extracellular flux analysis is used for studying cellular metabolism

• can be used to differentiate between M1 and M2 state

• ECAR → reflects glycolysis rate by measuring protein production

  • increase ECAR, increase glycolysis (more CO2 + lactate)

  • M1 → increased ECAR

  • M2 → lower ECAR

• OCR (oxygen consumption rate) → reflects oxphos by measuring the rate at which O2 is consumed

  • M1 → low OCR, NO production inhibits mitochondrial respiration, thus M1 rely on glycolysis

  • M2 → high OCR, depend on OXPHOS

• adding glucose to seahorse → increase ECAR in M1 MQ

• limitations seahorse → AA metabolism not directly measured as well as PPP

explain if and how M1 can go to M2 state

• M1 → M2 difficult

  • induced by anti-inflammatory cytokines (IL4, IL13, IL10)

  • can only be done by iNOS inhibition (reduces level of NO).

  • this pathway is difficult because M1 have high NO production, this inhibits oxphos (crucial for M2).

    • need to shift from TCA + glycolysis →oxphos

explain if and how M2 can go to M1 state

• M2 → M1 happens more often. M2 relies on oxphos, can switch to aerobic glycolysis under inflammatory stimuli → LPS, IFNgamma

  • upregulation iNOS → production NO → increased expression pro-inflammatory cytokines