Gene therapy in macrophages Flashcards
Why is genetic background in macrophages important in inflammatory disease?
common genetic variation has an effect on the gene expression levels in macrophages
Why are macrophages important in inflammatory diseases?
they are plastic cells where external stimuli affects the transcriptome and activation phenotype
Give an example of where different external stimuli targeting the same receptor can affect the transcriptome and activation phenotype?
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
What do inflammatory cytokines and type 1 IFNs induce in macrophages?
antimicrobial functions; chemotaxis; phagocytosis; tissue reapir factors; metabolic regulatory
What are the questions surroudn ing genetic control of inflammatory disease with regards to macrophages?
how does germline sequence variation control macrophage function; how does germline sequence variation regulating macrophages cause inflammatory disease?
Why is crescentic GN in the rat used as a model in looking at macrophages in inflammatory disease?
crescentic GN is macrophage dependet
What different rat strains used to look at macrophages role in crescentic GN?
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?
How can the WKY strain and LEW strain be used to look at genetic control of inflammatory disease?
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
What is a QTL?
quantitative trait locus is a locus which correlates with variation of a quantitative trait in the phenotype of a population of organisms
How are expression QTLs used to look at genetic risk?
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
What is the difference between cis-eQTLs and trans-eQTLs?
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
What is seen in the macrophages of WKY rats vs LEW rats?
WKY rats who increased fusion of macrophages to form multinucleating giant cells whereas LEW macorphages don’t
When are fused macrophages seen ?
ostecloasts and granulomas
Why is a model of multinucleated giant cell formation useful in GN?
in the nephritic glomerulus of WKY rats, multinucleated giant cells are seen
What is the experimental design for looking at the genetics determining multinucleated giant cell formation?
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
What was found to be a genetic determinant of macrophage gene expression in WKY x LEW population?
SNP in chromosome 9 was a master regulator of 190 transcripts
What gene family did the SNP in chromosome 9 correspond to?
Trem family especially Trem2
How could the effect of Trem2 in macrophage gene expression be confirmed?
use SiRNA and look at the corresponding expression of different genes after the knock down
Who was responsible for creating SiRNA?
Fire and Mello (2006 Nobel Prize)
Why is SiRNA revolutionary in the field of biomedical science?
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
What are siRNAs derived from?
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
What type of network does Trem2 regulate?
a trans network
What is the function of Trem2 physiologically?
bone homeostasis by controlling hte rate of osteoclastogenesis
What is the strongest trans-regatuled gene by Trem2?
Kcnn4
What happened when Kcnn4 is silenced?
there is no multineacleated giant cell formation
How was the finding that Kcnn4 is implicated in MGC formation further applied after siRNA
pharmacological blockade of Kcnn4- TRAM34 gave the same reduction in MGC formation in both human and rat macropahges
What indicates that Kcnn4 is implicated in osteoclast formation?
when it is knocked out there are far fewer osteoclast numbers per well and this decreases with inceasing pharmacological inhibition with TRAM34
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?
pharmacological blockade of Kcnn4 reduced susceptibility to glomerulonephritis after nephrotoxic serum injection in WKY rats - 50% reduction in proteinuria
How did genetic deletion of Kcnn4 change susceptibility to arthritis?
reduced inflammation in collagen induced arthritis; reduced pannus, cartilage damage and bone damage
How does Kcnn4 module bone homeostasis?
through formation and activation of osteclasts- with increased bone density in KO Kcnn4 mice
What is the group that detemrined the role of Kcnn4 in glomerulonephritis; arthritis and bone mass?
Kang et al
How does CRISPR/Cas9 work?
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
How was CRISPR/Cas9 used to look at regulators of TNF?
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
How can CRISPR/Cas9 be used to looks at single cell transcriptomes?
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
What is the overall function of CRISPS technology?
enables precise and efficient genome editiing in living eukaryotic cells
What is the CRISPR system based on?
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
What is the result of macrophage fusion?
leads to the formation of osteoclasts in bone and multinucleate giant cells in chronic inflammatory diseases
What have genetic studies in regulation of macrophage multinucleation shown?
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
What is the assocation of loss of function mutations in TREM2 in human bone disease?
can cause Nasu-Hakola disease where defective multinucleation in osteoclasts results in impaired bone resorption
