Lecture 3: Macrophages

Question 1

What do macrophages do?

Answer 1

express surface receptors involved in ligand recognition and uptake; function to clear microbes, harmful complexes, apoptotic cells, and promote wound healing

Question 2

What do macrophage receptors recognise?

Answer 2

antibody/complement coated particles, carbohydrates, chemokine, or cytokines

Question 3

Where are tissue macrophages derived from?

Answer 3

Blood monocytes made and released from bone marrow

Question 4

What are the benefits of using mice?

Answer 4

• share over 90% same genes as humans
• small and easy to handle in the lab
• short generation times
• short life spans

Question 5

What are the 3 stages of haematopoiesis?

Answer 5

Primitive, Pro-definitive, and Definitive

Question 6

What happens in primitive stage?

Answer 6

Occurs in the extra embryonic yolk sac and produces short lived red blood cells and long lived macrophages that seed the foetus

Question 7

What happens in pro-definitive stage?

Answer 7

Begins in the yolk sac and produces progenitors that make blood cells until birth, and progenitors that seed the foetus

Question 8

What happens in definitive stage?

Answer 8

Begins in the embryo and produces HSCs that initially seed the foetal liver and then permanently colonise the bone marrow

Question 9

How are bone marrow chimeras made?

Answer 9

• bone marrow containing HSCs is transplanted from a donor to a recipient mouse
• mouse is irradiated to remove host HSCs
• removal of host cells makes too for donor bone marrow to engraft and produce immune cells

Question 10

How are donor and host cells distinguished from one another?

Answer 10

Congenic markers such as CD45.1 and CD45.2

Question 11

How are parabiotic chimeras made?

Answer 11

By surgically joining the blood circulation of 2 animals

Question 12

How are reporter mice made?

Answer 12

Have a cell, pick the gene of interest, and attach fluorescent protein so when gene is expressed, there is expression of fluorescent tag

Question 13

What is Cre recombinase?

Answer 13

An enzyme that recognises LoxP sequence which is placed either side of a genetic stop sequence

Question 14

What happens when Cre recombinase recognises LoxP?

Answer 14

Mediates excision of the stop sequence and expression of the fluorescent protein (which is located downstream of the stop sequence)

Question 15

What experiment was used to demonstrate monocytes do not become macrophages in the brain?

Answer 15

• took newborn mice, irradiated them, then transplanted donor bone marrow
• 1 month after transplant 50% of circulating monocytes came from donor but none were in the microglia
• same results after 3 months show monocytes do not become macrophages in the brain

Question 16

Why was there entry of macrophages into the brain in parabiotic chimeras but none in bone marrow chimeras?

Answer 16

Bone marrow chimeras use irradiation, which damages blood vessels, while parabiosis does not = microglia do not receive input from monocytes unless the blood-brain barrier is damaged

Question 17

What is Runx1?

Answer 17

Runx1 is a transcription factor expressed only in the yolk sac and is important for creating HSCs

Question 18

How was the Runx1 fate mapping reporter mouse used?

Answer 18

• put Cre recombinase under Runx1 control
• inject pregnant mice with tamoxifen to activate Cre
• any cell that expressed Runx1 also turns on YFP expression

Question 19

What did use of Cx3cr1-gfp reporter mouse show?

Answer 19

• GFP activation showed macrophages in the brain before definitive haematopoiesis even begins
• Also presence of GFP in yolk sac
• Both sites have macrophages early in development so likely from same origin = yolk sac is the source of the brain macrophages

Question 20

How did they use fate mapping reporter Runx1 mice to prove microglial macrophages come from the yolk sac?

Answer 20

• inject pregnant mice while runx1 expression is restricted to yolk sac
• look where YFP expression is
• similar levels of YFP in the yolk sac and brain suggesting same primitive origin

Question 21

How did different time points effect results from fate mapping reporter Runx1 mice?

Answer 21

Early injection: labels microglia but not blood monocytes or other tissue macrophages
Late injection: labels blood monocytes and macrophages but decreases microglia labelling

Shows primitive progenitors contribute to adult microglia but not adult leukocytes