Interferon-gamma and Interleukin 6 Modulate the Susceptibility of Macrophages to Human Immunodeficiency Virus Type 1 Inf

1. Interferon-gamma and Interleukin 6 Modulate the Susceptibility of Macrophages to Human Immunodeficiency Virus Type 1 Infection Zaitseva, M.; Lee, S.; Lapham, C.; Taffs, R.; King, L.; Romantseva, T.; Manischewitz, J.; and Golding, H. 2000. Blood: 96(9):3109-3117. Presented by: Alice Cioara

2. Cytokines • Important effector molecules • May exert multiple effects on viral entry and reverse transcription as well as reactivation • Proinflammatory cytokines • Interferon-gamma • Interleukin 6

3. Interferon  • Maps to chromosome 12q24.1 • Is a dimeric protein • Interferon  (IFN- ) and receptor complexes are rapidly internalized by endocytosis • Has antiviral and antiparastic activities • Inhibits the proliferation of a number of normal and transformed cells

4. Interferon  • A modulator of T-cell growth and functional differentiation • Regulates the expression of MHC class 2 genes • In macrophages – stimulates release of reactive oxygen species • Involved in processes of bone growth and inhibits bone resorption • Mediates host defense

5. Interferon  • Is a potent activator of mononuclear phagocytes • Produced by: T and B cells, natural killer cells, and lymphocytes • Activated by antigens • Has both suppressive and enhancing effects

6. Interleukin 6 • Maps to 7p21-p14 • Involved in T cell proliferation • Influences antigen-specific immune responses • Physiological stimuli for synthesis: • Bacterial endotoxins

7. Interleukin 6 • Produced by: • Monocytes, fibroblasts and endothelial cells • After stimluation – also produced by: macrophages T-cells and B-lymphocytes • Limited to leukocyte subpopulations • Promotes inflammatory events • Serves a protective role

8. Chemokines • Superfamily composed of 20 different leukocyte chemoattractants • Small proinflammatory proteins that recruit and activate leukocytes and inhibit the viral co-receptor function • Are chemotactic cytokines • 2 categories • CC (beta) • CXC (alpha)

9. Chemokines • CC (beta) • Non-syncytium-inducing • Marcophagetropic variants use R5 viruses • Slow to replicate • Do not infect CD4+ T-cell lines • CXC (alpha) • Syncytium-inducing • T-cell line adapted use CXC chemokine receptor 4 (CXCR4)

10. HIV • CC chemokine are fusion coreceptors for IV entry into macrophages • CCR5 – major determinant of entry • Macrophages are the main target and serve as a reservoir of HIV • Macrophages more efficiently infected by M-tropic HIV strains • Activation of Macrophages

11. HIV • “The cytokine action created during the onset of an opportunistic infection or as a result of local inflammation may affect HIV infection of macrophages by providing a more favorable environment for emerging T-tropic viruses”

12. IFN  AND Il-6 • To determine whether proinflammatory cytokines affect the ability of the macrophages to fuse with X4 envelope-expressing cells Procedure: • Macrophages were generated • Treated with cytokines (IFN , TNF, IL-6, and IL10) • Combined with cells expressing a T-tropic IIIB envelope

13. IFN  AND Il-6 • Only IFN  & IL-6 exhibited enhancement in the numbers of syncytia • Monocyte-derived macrophages (MDM) obtained • Mixed (ratio 1:1) with X4 envelope-expressing target cells. • Quantified # of syncytia and analyzed

14. IFN  AND Il-6

15. Infection of MDMs with X4 • To see if there is a correlation between enhancement of X4 HIV-1 viral entry and an increase in fusion of cytokine-treated MDMs • Analysis of viral DNA at 48 hours using PCR was performed • MDM incubated with INF  or IL6 • Washed to remove cytokines • Infected with HIV-1(NL4-3) • DNA extracted after infection

16. Infection of MDMs with X4

17. Viral DNA in cytokine-treated MDMs • To determine if an increase in the primary production of treated viral DNA would increase viral reproduction: • 6-day MDMs incubated with cytokines • Washed and then infected with HIV(LAI)

18. Viral DNA in cytokine-treated MDMs

19. Viral DNA in cytokine-treated MDMs • Conclusion of Data: • IL-6 & INF  increase efficiency of X4-tropic viral entry, thus resulting in an increased production of infection of MDMs with T-tropic HIV-1

20. Role of CXCR4 in IFN  and IL-6 • To determine the fusion of X4-tropic envelope with cytokine-treated MDMs was CXCR4 dependent:

21. Role of CXCR4 in IFN  and IL-6 IL-6- and IFN- -induced increase in the fusion of macrophages with T-tropic envelope-expressing cells is CXCR4 dependent Experiment Cytokine Inhibitor #of syncytia with IIIB envelope 1 None None 5+1 IFN RANTES 45+8 2 None None 17+1 IFN  RANTES 72+9 3 None None 6+1 IL-6 RANTES 87+4 4 None None 1+1 IL-6 None 57+12

22. Role of CXCR4 in IFN  and IL-6 • The previous data exhibits that CXCR4 does in fact participate in the fusion between X4-tropic envelopes with cytokine-treated macrophages

23. Enhanced MDMs, X4-tropic and CXCR4 • Flow cytometry was done on the MDMs which were used in the fusion experiments to determine if there was a correlation between X4-tropic envelopes with enhanced MDMs and CXCR4 surface expression • 12 different experiments were conducted

24. Enhanced MDMs, X4-tropic and CXCR4

25. Enhanced MDMs, X4-tropic and CXCR4 • The previous data shows that the fusion between X4-tropic envelopes with the cytokine-treated MDMs does depend on CXCR4. • This fusion, however, is not due to an increase in the levels of surface CXCR4 expression.

26. SDF-1-induced Ca++ flux • To determine if IFN  or IL-6 caused conformational changes in CXCR4 (in macrophages) which resulted in the improvement of ligand binding

27. SDF-1-induced Ca++ flux • The previous data suggests that the fusion of X4-tropic envelope with the cytokine-treated MDMs does depend on CXCR4. • However, it is not ascribed to an increase in the level of CXCR4 expression

28. Effect of Cx and H7 on IFN  and IL-6 • To determine if X4-tropic envelopes fusion with enhanced MDMs is linked with Stat1/3 activation: • MDMs pretreated with an inhibitor of Stat1/3 = H7 • These cells then mixed with IIIB envelope-expressing cells

29. Effect of Cx and H7 on IFN  and IL-6 • H7 and cycloheximide inhibit IL-6- and IFN- -induced fusion of macrophages with T-tropic envelope-expressing cells Cytokine* Inhibitor No. of syncytia None None 7 ± 4 IL-6 None 72 ± 9 IL-6 Cx 6 ± 2 IL-6 H7 9 ± 1 None Cx 8 ± 4 None H7 3 ± 0 None None 14 ± 1 IFN None 61 ± 13 IFN Cx 13 ± 1 IFN H7 12 ± 2 None Cx 11 ± 1 None H7 9 ± 1

30. IFN  and CCR5 • The reduction in the R5-tropic viral entry and reproduction detected in the presence of IFN  could be mediated by down-modulation of CD48 and/or the expression of CCR5 • Fusion assays • Surface staining • Performed 11 experiments+

31. IFN  and CCR5

32. IFN  and CCR5 • Treatment of MDM with IFN  • Slight reduction of surface CCR5 • Inhibition of MDMs with M-tropic envelopes = significant

33. Effect of IL-6 and Macrophages • To determine if cytokines have the ability to change the susceptibility of macrophages to primary viral isolates

34. Effect of IL-6 and Macrophages • The previous data shows that proinflammatory cytokines do have the ability to increase the susceptibility of macrophages to the infection with multiple primary X4 viruses

35. Cytokine-treated MDMs secrete β-chemokine • To determine the mechanism liable for the effects of the fusion of MDMs with X4 and R5 envelope-expressing cells along with the cytokine treatment

36. Cytokine-treated MDMs secrete β-Chemokines • IFN- and IL-6 induce chemokine production in macrophages Treatment* MIP-1 (pg/mL) MIP-1 (pg/mL) None 143 ± 35 66 ± 3 IFN- 909 ± 37 1425 ± 12 IL-6 647 ± 5 2176 ± 44

37. Discussion • Exhibited: • IFN  and IL-6 exert a positive effect on the susceptibility of MDMs to infection with X4 HIV-1 • Induced an increase in X4 HIV-1 viral entry • Infection of MDMs • Promoted fusion of X4 and MDMs

38. Discussion • Inhibitory effect • IFN  on R5 viral entry and the R5 reproduction of MDMs & fusion with target cells • β–chemokines were detected from IFN  & IL-6 treated MDMs.