1) S100A8 and S100A9 are proteins expressed by neutrophils and macrophages that are secreted during inflammation and recruit lymphocytes. 2) The study evaluated the effect of S100A8 and S100A9 on HIV-1 replication in primary CD4+ T cells and monocyte-derived macrophages (MDMs). 3) The results showed that S100A8 and S100A9 reduced HIV production in MDMs by decreasing expression of the HIV co-receptor CCR5, but did not inhibit HIV replication in CD4+ T cells or peripheral blood mononuclear cells (PBMCs).

1. S100A8 and S100A9, Endogenous Toll-like Receptor 4 (TLR4) Ligands, Inhibit HIV-1 Replication in Macrophages but not in CD4+ T Cells Terrence Brann, M.S. SAIC Frederick, Inc., NCI-Frederick, Frederick, Maryland, USA,

2. Introduction to S100A8 & S100A9 Expressed by neutrophils and macrophages, secreted during inflammation Recruit lymphocytes to sites of inflammation In this study, we evaluated the effect of soluble S100A8 and S100A9 on HIV-1 replication in primary CD4+ T cells and monocyte-derived macrophages (MDMs)

3. Recombinant S100 proteins < 0.01 Endotoxin Units/ug protein by Limulus Amoebocyte Lysate Assay

4. Healthy donor PBMC CD14+ selection AB serum CD4+ selection PHA + IL2 CD4+ T cells MDM PHA + IL2 PBMC Primary Cell Culture

5. CD4+ T cells MDM PBMC Infect with R5-HIV-1 Incubate with S100A8 or S100A9 for 7 days (PBMC or CD4+Tcells) or 10 days (MDM) Measure p24 by ELISA HIV-1 inhibition assay Infect with X4-HIV-1 Infect with R5-HIV-1 or X4-HIV-1

6. PBMC + X4 HIV-1 MDM + R5 HIV-1 CD4+ T-cells + X4 HIV-1 PBMC + R5 HIV-1 S100A8 & S100A9 dose response in HIV-1-infected cells S100A9 0 ug/ml 0.01 ug/ml 0.1 ug/ml 1.0 ug/ml

7. 3-day pre-treatment of MDM with S100A8 & S100A9

8. S100A8 and S100A9 decrease MDM cell-surface expression of HIV co-receptor CCR5 CD4 CCR5 Control S100A8-treated S100A9-treated

9. S100A8 and S100A9 effect on gene expression of CCR5

10. Conclusions S100A8 and S100A9 reduce HIV production in MDM, but not in CD4+ T cells or PBMCs S100A8 and S100A9 decreased expression of CCR5

11. Acknowledgements SAIC Frederick, Inc Tom Imamichi Hiromi Imamichi Joseph Adelsberger Michael Baseler NCI-Frederick Munehisa Takahashi Teizo Yoshimura NIAID/NIH H. Clifford Lane Funded by NCI Contract N01-CO-12400