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.