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
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
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
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.
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
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.
Hypothesis for the function of IFI6 & IFI27 genes..
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
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
Empty GFP virus as well as IFI6 & IFI27 showed relatively similar titration data
Via flow cytometry, we selected for MonoMac1 cells that were transduced with both the eGFP vector/virus and iCherry vector/virus
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
