Macrophages Flashcards
What was Ralph Van Furth’s proposed mononuclear phagocyte system?
He proposed a model to explain why macrophages are not found circulating in blood
His MPS stated that:
– all cells arise from HSCs in the bone marrow
– monocytes in the blood enter tissue and differentiate into macrophages and dendritic cells
How have views on the mononuclear phagocyte system since changed?
Tissue-resident macrophages (e.g., Kupffer cells in the liver, alveolar macrophages in the lungs, microglia in the brain) are now recognized to arise from yolk sac or foetal liver precursors during embryonic development rather than monocyte differentiation
The MPS is now viewed as a dynamic system that includes both embryonically derived and monocyte-derived macrophages
Why are mice used to understand human immunology?
– share >90% of the same genes
– ability to control genetics
– easy to handle and easily maintained in a lab setting
– large numbers of animals quickly
– short lifespan, entire lifecycle can be studied in ~2y
many discoveries occur in mice, which then translate into humans eg HLA identified in mice, then 20y later in humans
What are the stages of haematopoiesis in mice?
each wave produces distinct hematopoietic progenitors
- primitive: from E7, occurs in extraembryonic yolk sac and produces short-lived RBCs and long lived macrophages that seed foetus
- pro-definitive: begins in the yolk sac and produces progenitors that make blood cells til birth, and progenitors seed foetus
– not as well defined as other two stages, still studied - definitive: from E10.5, begins in embryo and produces HSCs that first seed foetal liver and then permanently colonise the bone marrow
What are mouse chimeras?
BM chimeras made by transplanting BM containing HSCs from a donor to a recipient
mice are irradiated to remove host HSCs - the DNA damage leads to cell death, particularly in rapid dividing cells such as HSCs
donor and recipient distinguished by expression of congenic markers such as CD45.1 and CD45.2
parabiotic chimeras made by surgically joining blood circulation of 2 animals (not legal in UK - GOOD)
What is a reporter mouse?
used to genetically mark and track cells of interest
– constitutive or drug inducible eg tamoxifen
can use Cre recombinase to drive expression of fluorescent protein in a specific lineage of cells by positioning it under control of a cell specific promoter gene
when cre enzyme recognises loxP sequences it mediates excision of stop sequence and expression of fluorescent protein, located downstream of stop sequence
Explain how a knockout mouse is generated?
same logic as reporter mice, expect loxP sites flank the gene of interest
Cre is under control of promoter of gene of interest
How did microglia (brain resident macrophages) put van Furth’s model into question?
experiments showed that macrophages from donated monocytes do not develop into brain macrophages in newborn transplants
slight increase as mice ages
– explained through blood vessel damage via irradiation
– resolved via parabiotic mice, where there is no monocyte entry into the brain
conclusion
– postnatal microglia are maintained independently of circulating monocytes throughout life
How did fate mapping analysis show that adult microglia are derived from primitive macrophages?
Cz3cr1-gfp reporter mouse
– CXC3R1 is expressed by monocytes and microglia
- Runx1 is expressed only in yolk sac until E8
- inject pregnant mice with 4’OHT to activate Cre and turn on irreversible YFP expression
microglia were detectable in the brain at E9.5 before definitive hematopoiesis begins
similar levels of YFP expression seen in yolk sac and brain suggests that they have same primitive origin
– yolk sac
early injection up to E7.5 labels microglia but not other monocytes or other tissue macrophages
late injection from E7.5 labels blood monocytes and macrophages in other tissues
– labelling on microglia decreases
this shows that primitive progenitors contribute to adult microglia but not adult leukocytes
How did further experiments show other tissue resident macrophages are independent of blood monocyte renewal?
Myb knockout mice, a transcription factor required for development of HSCs
loss of Myb results only affects F4/80 low resident tissue macrophages
– demonstrates that F4/80 bright resident tissue macrophages do not develop from HSCs and must have different origin. yolk sac derived?
F4/80 bright macs and yolk sac-derived microglia cluster together
- non biased hierarchal clustering - cells thats share a similar gene expression profile cluster together
skin resident macrophages still develop when isolated from HSCs.
embryonic lim buds were transplanted from Cx3cr1-gfp mice at E10.5 into chicken embryos
transplanted limb buds cannot receive input from HSCs which develop after E10.5
12 days lter GFP+ tissue resident macrophages could be seen in skin of the limb
Fate mapping reporter mice show that tissue resident macrophages in most organs are from yolk-sac derive progenitors.
Csf1r is expressed by yolk-sac derived macrophages early in development
use Csf1r-Cre mice to label and track yolk sac-derived macrophages into adulthood by inducing expression of YFP
YFP is detected in F4/80 bright tissue macrohages but nor blood immune cells or F4/80 low tissue macrophages
F4/80 bright have different origin to low, which is primitive not definitive
non-irradiated chimeric mice show that tissue resident macrophages are from yolk-sac derived progenitors.
deletion of myb in adult mice makes chimerase without irradiation
deletion of HSCs, depending on organ, different amount of contribution from donor bone marrow in F4/80 bright
different with F4/80 low, all basically come from donor, these are dependent on Myb and HSCs
What is the only tissue resident macrophage that is dependent on renewal via blood monocytes?
gut macrophages
similar experiment used
was found that embryo permanent label of YFP faded over time, indicating continuous replacement from a new population
further experiment using Flt3 Cre reporter, a gene expressed by all HSCs
similar % of blood monocytes and gut macrophages labelled by FLt3 reporter mouse
Ccr2 expression is necessary for monocytes to enter gut
– knockout monocyes do not contribute to tissue macropahges in gut
– this experiment done through use of parabiotic chimeras
Long-live tissue-function specific macrophages reside in most tissues. Is this the case in intestinal macrophages?
was found that Tim-4 and CD4 expression divide macrophages into subsets
- neither
- only CD4
- both
Tim-4+CD4+ macrophages are infrequently replenished from blood monocytes
– these macrophages are dominant at birth and are maintained into adulthood
functions of each yet to be determined
