![7AAD
Active Caspase 3
Singlets
Live 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](https://image.slidesharecdn.com/48edea55-6f41-4dbd-a0c8-9800aeda9c48-160202171115/85/BTTP-Presentation-8-320.jpg)
BTTP Presentation
- 1. Monocyte Apoptosis Sensitivity in HIV-1 Disease Amanda Versace Dr. Luis Montaner Lab August 7, 2012 Biomedical Technician Training Program
- 2. Pluripotent Stem Cell Lymphoid Progenitor Myeloid Progenitor Natural Killer T cell B cell Erythrocyte What are monocytes?
- 3. 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
- 4. 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?
- 5. 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?
- 6. 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 Hypothesis ViralLoad Early AdvancedChronic Monocyte Apoptosis Sensitivity
- 7. Objective 1) Apoptosis Background CdCl2 Titration 2) Titration/Transduction of individual virus 3) 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.
- 8. 7AAD Active Caspase 3 Singlets Live 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
- 11. IFI6 IFI27 rtTA3 rtTA3 rtTA3 Empty Control IFI6 Transgene IFI27 Transgene
- 12. 96.9 %
- 14. 2.5 μL 2.5 μL 2.5 μL 5.0 μL
- 15. 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
- 16. GFP Alone Cherry Alone Empty GFP + Cherry IFI6 GFP + Cherry IFI27 GFP + Cherry
- 17. Cells Sorted 7/23/2012 Cultured 7/27/2012 Cultured 7/30/2012 Cultured 8/2/12 MCS-GFP Alone 250,000 1.04 M 2.63 M 15.8 M rtTA3-cherry Alone 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
- 18. non 0 non 0 non 1000 non 1000 non 100 non 100 non 1 non 1 non 10 non 10 MCS 0 MCS 0 MCS 1000 MCS 1000 MCS 100 MCS 100 MCS 1 MCS 1 MCS 10 MCS 10 IFI6 0 IFI6 0 IFI6 1000 IFI6 1000 IFI6 100 IFI6 100 IFI6 1 IFI6 1 IFI6 10 IFI6 10 IFI27 0 IFI27 0 IFI27 1000 IFI27 1000 IFI27 100 IFI27 100 IFI27 1 IFI27 1 IFI27 10 IFI27 10 0 ng/mL 1 ng/mL 10 ng/mL 100 ng/mL 1000 ng/mL
- 19. 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 μM CdCl2 35 μM CdCl2 35 μM CdCl2 35 μM CdCl2 35 μM CdCl2 35 μM CdCl 35 μM CdCl2 35 μM CdCl2 35 μM CdCl2
- 20. Acknowledgements
- 21. 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.
Editor's Notes
- #3: Monocyte Function – give rise to MΦ and DC; respond to inflammation signals move to infected tissues and differentiate into MΦ and DC which elicit innate immune response Macrophage Function – phagocytose cellular debris and pathogens; also stimulate lymphocytes and other immune cells to respond to pathogens
- #4: Innate Immunity importance in vaccine development 1) Activated macrophages kill phagocytosed microbes 2) Activated mΦ Stimulate acute inflammation through cytokine secretion (mainly TNF, IL-1, and other chemokines and lipid mediators) 3) Remove dead tissues and facilitate repair after infection is under control Mo/MΦ play an important role in the innate immune response to HIV infection Like many other immune cells, Mono/Macs undergo apoptosis throughout HIV disease.
- #5: HIV infects immune cells that express a glycoprotein on their membrane called CD4 (cluster differentiation 4). CD4+ cells include: Helper T cells, Mono/Mac, and Dendritic Cells
- #6: So now we know that HIV targets CD4+ cells and that T cells and Mono/Mac express CD4 and are infected by the virus. T cell function in Adaptive Immunity, but what about Innate Immunity? Monocyte count remains consistent throughout the course of HIV disease. Apoptosis increases during late stage and turnover increases during late stage.
- #7: Hypothesis for the function of IFI6 & IFI27 genes..
- #9: Check to see if apoptosis could be induced on MonoMac1 cell line 7AAD – fluorescent chemical compound that intercalates in ds DNA. It does not readily pass through a cell membrane so it useful as a cell viability stain because positive 7AAD staining is indicative of a compromised cell membrane. Caspase-3 – Caspase proteins are sequentially activated when cell apoptosis is executed therefore their presence (which we probe for using Caspase-3 antibody) is indicative of apoptosis. We chose 35 μM CdCl2 dose for 24 hours to allow for decreased apoptosis sensitivity (IFI6) and increased apoptosis sensitivity (IFI27). Looking for a volume of Cadmium Chloride that will cause approx 10-20% of cells to die
- #11: To establish a cell line that will over express our genes of interest upon induction of transcription (with doxy or tetracycline), vectors with appropriate promoter regions were designed. EF1 alpha – promoter region which drives transcription of rtTA3. (elongation factor which enzymatically delivers tRNAs to the ribosome) rtTA3 – binds to the Tet Responsive Element (TRE) which drives transcription of genes downstream of it IRES – “Internal Ribosome Entry Site” - a nucleotide sequence that allows for translation initiation in the middle of a mRNA sequence. IRES are often used by viruses as a means to ensure that viral translation is active during periods of time when host translation is inhibited. The cell may also use IRES to increase translation of certain proteins during cell division and apoptosis
- #14: Empty GFP virus as well as IFI6 & IFI27 showed relatively similar titration data
- #16: Via flow cytometry, we selected for MonoMac1 cells that were transduced with both the eGFP vector/virus and iCherry vector/virus
- #17: Using a flow cytometer with sorting capabilities we selected for ONLY transduced cells The red squares represent transduced cell populations - Purified cell lines with transgene The sorted cells were then cultured for growth/division and aliquoted for liquid nitrogen storage and future experiments
- #18: Cell Counts