PKR is required for macrophage apoptosis after activation of TLR-4. By Christina, Ania and Xiaofu
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1 PKR is required for macrophage apoptosis after activation of TLR-4 By Christina, Ania and Xiaofu
2 Introduction
3 Macrophage PAMP Bacteria LPS Gram -ve LTA Gram +ve activation Clearance
4 Macrophage PAMP Bacteria Apoptosis
5
6 TLR2 TLR9 TLR3 Figure 1 Signaling though TLR4 while inhibiting p38 leads to apoptosis
7 HKBA or apoptosis HeN = TLR4 wt HeJ = TLR4 mut SB=p38 inhibitor HKBA=heat killed B. anthracis
8 apoptosis When p38 is inhibited (by SB) only signalling via TLR4 leads to increased apoptosis TLR4 TLR2 TLR9 TLR3
9 normal experiment TLR4 CD4 TIR Domain (Toll-IL-1 receptor) p38 SB apoptosis CAD
10 FAS-L Fas Fas Fas insult Cytochrome C ProCaspase 9 Caspase 9 Caspase 8 ProCaspase 8 Caspase 3 ProCaspase 3 icad CAD CAD nucleus
11 Again this LPS/p38-inhibitor dependent apoptotic response is specific to signaling through TLR4. (see last slide for notes on DAPI and TUNEL)
12 Figure 2 The role of MyD88 and TRAF6
13 MyD88 -/- more apoptosis
14 TRAF6 -/- more apoptosis
15 TRAF6 -/- less JNK phosphorylation same p38 phosphorylation IKK less degadation
16 Poly (IC) LTA TLR3?? TLR4 TLR3 TLR2 TLR9
17 Role of PKR in apoptosis induced by TLR signaling
18 Induction of IFN in response to TLR signaling IFNbeta IFNalpha
19 Figure 3 PKR is necessary for LPSinduced INF signaling in macrophages PKR activation by LPS
20 PKR acts downstream of TRIF Normal MAPK and IKK activation in PKR-/-
21 PKR is required for INFbeta induction but is dispensable for induction of anti apoptotic genes PKR is required for inos induction and STAT1 phosphorylation
22 Figure 4 PKR is required for LPS-induced macrophage apoptosis PKR deficient BMDMs are resistant to LPS-induced apoptosis
23 Bacilllus anthracis Exotoxin Edema Factor (EF) adenylate cyclase, inhibits phagocytosis Lethal Factor (LF) Zn dependent protease, cleaves MAPKs and lyses macrophages Protective Ag (PA) binds to cellular receptors J Biomol Struct Dyn Dec;22(3):
24 PKR is required only for LPS induced apoptosis INF beta sensitizes BMDM to LPS-induced apoptosis
25 Type 1 IFN signaling in not required for LPS-induced macrophage apoptosis PKR activation inhibits A1/Bfl1 expression
26 eif2alpha phosphorylation is required for induction of macrophage apoptosis IRF3 is required for induction of macrophage apoptosis LPS PKR IFN induction macrophage apoptosis BUT macrophage apoptosis does not require IFN induction
27 Figure 5 Effect of PKR on bacteria-induced apoptosis G-ve G-ve G+ve - Both G-ve and G+ve bacteria induce apoptosis - PKR -/- strain much less susceptible - In addition to TLR4 engagement, bacterium needs to make its own contribution
28 Effect of PKR on bacteria-induced apoptosis - Non-infected population shows comparable number of live cells and little death - Infected PKR +/+ population displays much more apoptosis than PKR -/- population
29 Dependence of Apoptosis on eif-2α Phosphorylation and IRF3 - S51A: cannot phosphorylate eif-2α, cannot shut down translation - IRF3 activated by dsrna much like PKR; logical downstream signaling partner
30 Dependence of Apoptosis on a Functional TLR4 - TLR4 receptor in HeJ mice is inactive - Inactive TLR4 receptor associated with greatly diminished apoptotic response to bacteria
31 Summary TLR4 signaling is an inducer of apoptosis in macrophages. MyD88, TRAF6, and IKK all contribute to the anti-apoptotic pathway. PKR, already known to induce TI IFN, is a crucial component of TLR4- induced apoptosis in macrophages. Acts in part through suppression of synthesis of anti-apoptotic products as well as through IRF3. IFNβ sensitizes the cell to apoptosis but is not required.
32 Apoptosis Through TLR4: It Takes Two to Tango 2 nd Hit Needed Homo: SB B. anthracis: LT Yersinia: Salmonella: YopJ SPI2
33 Downstream PKR Effectors: Is Anyone Out There? Double KO Immunity? Co-precipitation?? Microarray Analysis?
34 Epilogue: PKR Targeting as Anti-Bacterial Therapy PKR inhibition: reduction of apoptosis better control of pathogens suppresion of inos PKR needed for virus suppression Short term inhibition sirna? Blocking of 2 nd Hit instead?
35 For Your Reference TUNEL Tdt is used to add labeled nucleotides on to the ends of DNA. The more the DNA is broken (apoptosis) the more nucelotides can be added and the stronger the signal (Janeway 647-8). DAPI Binds to the minor groove of DNA and stains chromosomes.
36 Hold Me, Thrill Me, Kiss Me, Kill me
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