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Monocyte Apoptosis Sensitivity in HIV-1 Disease
Amanda VersaceDr. Luis Montaner Lab
August 7, 2012
Biomedical Technician Training Program
Pluripotent Stem Cell
Lymphoid Progenitor
Myeloid Progenitor
Natural Killer
T cell
B cell
Erythrocyte
What are monocytes?
Monocytes/Macrophages (mo/mΦ) mediate inflammation
• Kill phagocytosed microbes• Stimulate acute inflammation • Remove dead tissues and facilitate repair
Monocyte role in innate immunity
Further research may show that mo/mΦ apoptosis during HIV infection contributes to the pathogenesis of HIV disease
HIV infects white blood cells that express CD4.
Commonly known CD4+ cells are:• helper T-cells (CD4+ T Lymphocytes) • Monocytes & Macrophages
What role do monocytes play in HIV infection?
Due to the dramatic loss of CD4+ T cells during chronic and late stage HIV infection, T cells have been the primary focus of HIV cell pathology research.
So why focus on Monocytes & Macrophages?
Interferon stimulated gene (ISG) expression changes between chronic and advanced stage HIV disease. Some of these genes may contribute to monocyte apoptotic behavior.
Some ISGs may cause mo/mΦ to resist apoptosis during chronic stage while others cause apoptosis sensitivity in advanced stage disease.
IFI6 – Anti Apoptotic
gene
IFI27 – Pro Apoptotic
gene
To further study the apoptotic role of IFI6 & IFI27, we created stable, tet-inducible, monocytic cell lines with these transgenes
HypothesisVi
ral L
oad
Early AdvancedChronic
Mon
ocyt
eAp
opto
sisSe
nsiti
vity
Objective
1) Apoptosis Background CdCl2 Titration
2) Titration/Transduction of individual virus3) Co-Transduction 4) Sorting Grow up 5) Dose Response (Drug titrations/ RT PCR)6) Western Blot (to make sure RNA is translated) 7) Transduction using primary cells
Experimental Outline
By engineering stable, tet-inducible MonoMac1 cell lines with transgenes - IFI6 (anti-apoptotic), IFI27 (pro-apoptotic) – we will create a relevant system that will reproducibly show the impact of these genes on apoptosis sensitivity.
7AAD
Active Caspase 3
SingletsLive 0 μM
20 μM 30 μM 35 μM
40 μM 50 μM
Treated non-transduced cells with CdCl2 and titrated for [CdCl2] that induces approx. 10-20% apoptosis
16% avg apoptosis
MCS
Gene Inserts:•IFI6•IFI27
IFI6
IFI2
7
rtTA3
rtTA3
rtTA3
Empty Control
IFI6 Transgene
IFI27 Transgene
96.9 %
SS
C-A
(gra
nula
rity)
GFP (fluorescence)
2.5 μL
2.5 μL
2.5 μL
5.0 μL
Co-transduced 2 Million MonoMac1 cells with GFP vector (containing gene insert) and cherry vector (helper genes)
(+,+)(-,+)
(-,-) (+,-)
38.8 %
MCS-GFP + rtTA3-cherry
IFI27-GFP + rtTA3-cherry
IFI6-GFP + rtTA3-cherry
44.5 % 42.4 %
GFP
GFP
GFP
cherry cherry cherry
GFP Alone Cherry Alone
Empty GFP + Cherry IFI6 GFP + Cherry IFI27 GFP + Cherry
Cells Sorted7/23/2012
Cultured7/27/2012
Cultured7/30/2012
Cultured 8/2/12
MCS-GFP Alone 250,000 1.04 M 2.63 M 15.8 M
rtTA3-cherryAlone 79,000 400,000 3.68 M 22 M
MCS-GFP + cherry 140,000 1.2 M 2.31 M 8.8 M
IFI6-GFP + cherry 177,000 480,000 3.57 M 13.6 M
IFI27-GFP + cherry 102,000 960,000 3.89 M 19.9 M
non0
non1000
non100
non1
non10
MCS0
MCS1000
MCS100
MCS1
MCS10
IFI60
IFI61000
IFI6100
IFI61
IFI610
IFI270
IFI271000
IFI27100
IFI271
IFI2710
0 ng/mL 1 ng/mL 10 ng/mL 100 ng/mL 1000 ng/mL
MCS (control)
IFI 6(Anti Apoptotic)
IFI 27(Pro Apoptotic)
16 % Apoptosis ~ 16 % Apoptosis
~ 16 % Apoptosis
16 % Apoptosis ~ 12 % Apoptosis
~ 8 % Apoptosis
16 % Apoptosis
~ 20 % Apoptosis
~ 40 % Apoptosis
0 ng/mL Doxy
10 ng/mL Doxy
100 ng/mL Doxy
0 ng/mL Doxy
10 ng/mL Doxy
100 ng/mL Doxy
0 ng/mL Doxy
10 ng/mL Doxy
100 ng/mL Doxy
35 μMCdCl235 μMCdCl235 μMCdCl2
35 μMCdCl235 μMCdCl235 μMCdCl2
35 μMCdCl2
35 μMCdCl2
35 μMCdCl2
Acknowledgements
HIV contains a viral envelope made up of 2 glycoproteins (gp120 & gp41) which mediate its entry into a host cell.
How HIV Infects CD4+ Cells
Gp120 binds to CD4 protein.
• This binding initiates conformational change in gp120 which allows the virus to bind to co-receptors expressed on the host cell.
• CCR5 or CXCR4.
Gp41 then undergoes a structural change allowing HIV to fuse a peptide into the host cell. The viral and cell membranes are then fused together.
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