Gene therapy in macrophages

Question 1

Why is genetic background in macrophages important in inflammatory disease?

Answer 1

common genetic variation has an effect on the gene expression levels in macrophages

Question 2

Why are macrophages important in inflammatory diseases?

Answer 2

they are plastic cells where external stimuli affects the transcriptome and activation phenotype

Question 3

Give an example of where different external stimuli targeting the same receptor can affect the transcriptome and activation phenotype?

Answer 3

TLR4 stimulation at the cell membrane results in AP-1 and NFkB expression- inflammatory cytokiens whereas activation at the endosome results in activation of IRF3 and type 1 cytokines

Question 4

What do inflammatory cytokines and type 1 IFNs induce in macrophages?

Answer 4

antimicrobial functions; chemotaxis; phagocytosis; tissue reapir factors; metabolic regulatory

Question 5

What are the questions surroudn ing genetic control of inflammatory disease with regards to macrophages?

Answer 5

how does germline sequence variation control macrophage function; how does germline sequence variation regulating macrophages cause inflammatory disease?

Question 6

Why is crescentic GN in the rat used as a model in looking at macrophages in inflammatory disease?

Answer 6

crescentic GN is macrophage dependet

Question 7

What different rat strains used to look at macrophages role in crescentic GN?

Answer 7

LEW mouse strain is resistant to GN whereas WKY rat strain is susceptible when stimualted with nephrotoxic serum- what are the genetic determinants of this difference?

Question 8

How can the WKY strain and LEW strain be used to look at genetic control of inflammatory disease?

Answer 8

if cross the LEW resistnat strain and the WKY susceptbile strain, you create a heterogenous population with a range of phenotypes- look at the genes influencing this

Question 9

What is a QTL?

Answer 9

quantitative trait locus is a locus which correlates with variation of a quantitative trait in the phenotype of a population of organisms

Question 10

How are expression QTLs used to look at genetic risk?

Answer 10

germline variation results in a difference in mRNA levels due to SNPs resulting in quantitative trait transcript —eQTL is a genomic loci that explains the variation in expression levels of mRNA which alters the phenotype of the cell– changes inflammatory disease

Question 11

What is the difference between cis-eQTLs and trans-eQTLs?

Answer 11

cis- SNP affecting the QTL is close to the gene whereas trans- SNP is distal to the gene and both result in changes in the expression levels of the gene

Question 12

What is seen in the macrophages of WKY rats vs LEW rats?

Answer 12

WKY rats who increased fusion of macrophages to form multinucleating giant cells whereas LEW macorphages don’t

Question 13

When are fused macrophages seen ?

Answer 13

ostecloasts and granulomas

Question 14

Why is a model of multinucleated giant cell formation useful in GN?

Answer 14

in the nephritic glomerulus of WKY rats, multinucleated giant cells are seen

Question 15

What is the experimental design for looking at the genetics determining multinucleated giant cell formation?

Answer 15

cross the WKY and LEW rat strains to mix their DNA and create a broad rang of MGC formation and from there look at hte eQTLs

Question 16

What was found to be a genetic determinant of macrophage gene expression in WKY x LEW population?

Answer 16

SNP in chromosome 9 was a master regulator of 190 transcripts

Question 17

What gene family did the SNP in chromosome 9 correspond to?

Answer 17

Trem family especially Trem2

Question 18

How could the effect of Trem2 in macrophage gene expression be confirmed?

Answer 18

use SiRNA and look at the corresponding expression of different genes after the knock down

Question 19

Who was responsible for creating SiRNA?

Answer 19

Fire and Mello (2006 Nobel Prize)

Question 20

Why is SiRNA revolutionary in the field of biomedical science?

Answer 20

no longer have to genetically create KO mice strains, can be done in cell lines and can image in real time to see the effects of SiRNA

Question 21

What are siRNAs derived from?

Answer 21

double stranded RNAs which bind to an enzyme which cuts it up, on strand is selected and remains bound to a protein- which is directed to specific areas of the mRNA which then calatyses the breakdown of that mRNA

Question 22

What type of network does Trem2 regulate?

Answer 22

a trans network

Question 23

What is the function of Trem2 physiologically?

Answer 23

bone homeostasis by controlling hte rate of osteoclastogenesis

Question 24

What is the strongest trans-regatuled gene by Trem2?

Answer 24

Kcnn4

Question 25

What happened when Kcnn4 is silenced?

Answer 25

there is no multineacleated giant cell formation

Question 26

How was the finding that Kcnn4 is implicated in MGC formation further applied after siRNA

Answer 26

pharmacological blockade of Kcnn4- TRAM34 gave the same reduction in MGC formation in both human and rat macropahges

Question 27

What indicates that Kcnn4 is implicated in osteoclast formation?

Answer 27

when it is knocked out there are far fewer osteoclast numbers per well and this decreases with inceasing pharmacological inhibition with TRAM34

Question 28

Kcnn4 has been implicated in a trans-regulated network involvewd in osteoclast activity and macrophage multinucleation regulated by Trem2, but how does this relate to GN?

Answer 28

pharmacological blockade of Kcnn4 reduced susceptibility to glomerulonephritis after nephrotoxic serum injection in WKY rats - 50% reduction in proteinuria

Question 29

How did genetic deletion of Kcnn4 change susceptibility to arthritis?

Answer 29

reduced inflammation in collagen induced arthritis; reduced pannus, cartilage damage and bone damage

Question 30

How does Kcnn4 module bone homeostasis?

Answer 30

through formation and activation of osteclasts- with increased bone density in KO Kcnn4 mice

Question 31

What is the group that detemrined the role of Kcnn4 in glomerulonephritis; arthritis and bone mass?

Answer 31

Kang et al

Question 32

How does CRISPR/Cas9 work?

Answer 32

Cas9 and guide RNA target a DNA strand resulting a double strand break whichresults in nonhomologous repair resulting in cleaved DNA is just joined knocking it out or different DNA is added putting mutations at the targeted site resulting in a kock out straight away

Question 33

How was CRISPR/Cas9 used to look at regulators of TNF?

Answer 33

Cas9 mouse was transfected with guiding RNA to lots of different genes, knocking them out- the effect on TNFa production was looked at and the guiding RNA sequenced to determine the novel regulatorys

Question 34

How can CRISPR/Cas9 be used to looks at single cell transcriptomes?

Answer 34

barcoded beads are given to each cell as well as a specific gRNA, the single cells are then grouped by their guiding RNA and the cells transcriptome is then analysed

Question 35

What is the overall function of CRISPS technology?

Answer 35

enables precise and efficient genome editiing in living eukaryotic cells

Question 36

What is the CRISPR system based on?

Answer 36

an adaptive immune system used by microbes to defend themselves against invading viruses by recording and targeting their DNA sequences e.g CRISPR locus in E.coli strain matched the sequence of a phage that infected many E.coli strains

Question 37

What is the result of macrophage fusion?

Answer 37

leads to the formation of osteoclasts in bone and multinucleate giant cells in chronic inflammatory diseases

Question 38

What have genetic studies in regulation of macrophage multinucleation shown?

Answer 38

that germline variation can regulate the multinucleation and influence disease pathogenesis- murine model of TB- genetic looci were associated with production of inflam mediators by macrophages- presence of a heritable genetic component affecting MGC function and susceptibility to granulomatous diseases ; GWAS of MGC-associated diseases TB and GPA showed common variations assocaited with them

Question 39

What is the assocation of loss of function mutations in TREM2 in human bone disease?

Answer 39

can cause Nasu-Hakola disease where defective multinucleation in osteoclasts results in impaired bone resorption