Paper 2: Lyn-dependent signalling, DC activation of NK cells Flashcards
what is signal transduction controlled by
- stimulatory and inhibitory pathways regulated by kinases and phosphatases
inactive Lyn (3)
- anchored to plasma membrane
- SH3, SH2, and two kinase groups linked together
- SH2 domain is bound to own tail to clamp Lyn shut
active Lyn (3)
- anchored to plasma membrane
- SH3, SH2 and two kinase groups present in more linear chain
- SH2 domain no longer bound to own tail
regulation of Lyn (2)
- SH2 domain used to regulate activity and for inhibition
- when tail is bound, Lyn is inactive; when tail is’t bound, Lyn is inactive
Lyn (3)
- protein type
- expression in cells
- activation
- Src-family tyrosine kinase (SFK)
- expressed in all leukocytes except T cells
- activated by ligand binding to adhesion molecules, cytokine receptors, immunoreceptors, and TLRs
does Lyn amplify or restrict signal transduction
- depending on cell microenvironment, developmental stage, and type of stimulus, Lyn can either restrict or amplify signal transduction
what activates NK cells to their full potential
- secretion of cytokines from CD11c^high DCs activated by TLRs is required to induce NK cell IFN production and cytotoxicity
when does negative regulation by Lyn occur (2)
- when Lyn phosphorylates tyrosine residues within ITIMs present in inhibitory proteins at the plasma membrane
- results in antagonization of signalling
when does positive regulation by Lyn occur (2)
- when Lyn phosphorylates ITAMs on membrane proteins
- results in amplifying signals
Lyn is important for establishing inhibitory signalling, what occurs in Lyn-/- mice (4)
- enhanced BCR signalling and failure to establish inhibitory signalling
- leads to autoimmunity and over-activity of BCR; BCRs with antibodies to self are allowed to develop
- develop severe fatal autoimmune lupus and nephritis
- spleen and blood are packed with leukocytes that are normally contained and controlled by infection
what is the general phenotype of loss of Lyn
- perturbation in signalling balance and severe pathology as myloid cells proliferate uncontrollably
what does injection of LPS into mice model
- mimics pathophysiological consequences of sepsis
what is the purpose of figure 1
- to investigate if Lyn regulates endotoxin sensitivity in vivo
what was observed in figure 1 (2)
- Lyn^up/up mice were ~80-fold more sensitive to LPS than Lyn+/+ or Lyn-/- mice shown by the amounts of LPS required to drop body temperature
- Lyn^up mice had increased levels of macrophages, neutrophils, lymphocytes, eosinophils and TNF-a compared to wt mice after LPS inhalation
what were the findings from figure 1
- Lyn^up/up mice exhibit enhanced endotoxin-induced inflammation and morbidity
what was the purpose of figure 2
- to investigate if Lyn regulated endotoxin-induced cytokine production
what methods were used for figure 2A, 2B, and 2C (3)
- ELISA
- western blot
- flow cytometry
what was observed in figure 2A
- Lyn^up/up serum contained significantly higher concentrations of pro-inflammatory cytokine IL-12, IFN-g, IL-1a, IL-1B, TNF-a, IFN-a, and IL-6 and lower concentrations of IL-10 compared to Lyn+/+ mice after LPS injection
what was observed in figure 2B
- LPS-induced expression of proinflammatory mediator iNOS was elevated in kidney, spleen, lung, and liver of Lyn^up/up mice after LPS injection compared to wt
what was observed in figure 2C
- myloid cells in Lyn^up/up BM had enhanced expression of MHC class 1 and Mac-1 after LPS injection
what are the findings from figure 2
- Lyn promotes production of pro-inflammatory cytokines in response to endotoxin
why was IFN-g important in this paper (2)
- IFN-g production in response to LPS is directly linked to magnitude of innate immune responses in vivo
- blocking of IFN-g with neutralizing Abs can prevent fatal LPS-induced sepsis in mice
identifying intracellular proteins (4)
- to capture steady state of the cell, cells must be fixed/preserved or have Golgi Apparatus preserved
- cells can then be stained be permeabilized
- stain with fluorescent antibodies
- analysis on flow cytometer
what is the purpose of figure 3
- to investigate how Lyn modulated IFN-g production in LPS-treated mice
according to the literature, what cells are known to produce IFN-g (2)
- NK cells
- T cells
what was observed in figure 3A (3)
- Lyn^up/up CD4+ and CD8+ T cells showed little LPS-induced IFN-g production
- 85% of of splenic NK cells in Lyn^up/up mice expressed IFN-g compared to 20% in Lyn+/+ mice after LPS
- Lyn^up/up mice expressed more IFN-g per cell, as indicated by MFI
what was observed in figure 3B and 3C
- Rag-/- Lyn^up/up mice, which have no B, T, or NKT cells, still experienced LPS-induced morbidity and LPS-induced NK cell IFN-g production
what were the findings from figure 3
- NK cells are the primary source of IFN-g in LPS-challenged Lyn^up/up mice
how do NK cells acquire effector functions such as IFN-g production
- must be stimulated by either DCs or macrophages
what was the purpose of figure 4A and 4B
- investigate whether macrophages or DC subsets were responsible for stimulating NK cells to produce IFN-g in LPS-stimulated Lyn^up/up mice
what was observed in figure 4A
- different DC subsets were effective at inducing NK cells to secrete IFN-g in response to LPS, but macrophages were not
what method was used for figure 4A and 4B
- ELISA
what was observed in figure 4B
- of the DC subsets, CD11c^high DCs were the most potent activators of NK cell IFN-g production after LPS treatment
is it possible to remove entire populations of cells from mice (2)
- yes, use cell-specific DTR model
- have mice cell of interest express DTR so that when DT is injected, the cells undergo apoptosis
what was the purpose of figure 4C and 4D
- to investigate if DCs are required for excessive NK cell IFN-g production in Lyn^up/up mice
what was observed in figure 4C and 4D (2)
- without DC ablation, Lyn^up/up contained significantly more IFN-g+ NK cells and elevated IFN-g in serum than Lyn+/+ mice after LPS treatment
- both Lyn^up/up and Lyn+/+ mice had reduction in IFN-g+ NK cells and IFN-g serum levels when DCs were ablated after LPS treatment
what were the findings in figure 4C and 4D
- DC lineage is required for enhanced NK cell IFN-g production in Lyn^up/up mice
what was the purpose of figure 4E and 4F
- investigate whether DCs and NK cells are also required for LPS-induced morbidity in Lyn^up/up mice
what was observed in figure 4E and 4F
- 75% of Lyn^up/up mice rescued from LPS-induced morbidity after DC depletion
-100% of of Lyn^up/up mice rescued from LPS-induced morbidity after NK cell depletion
what were the findings from figure 4E and 4F
- DC and NK cells are required for LPS-induced morbidity in Lyn^up/up mice
