Characteristics of Activated Monocyte Phenotype Support R5-Tropic Human Immunodeficiency Virus

Abstract

BACKGROUND: Microbial translocation has been recognized as an important factor in monocyte activation and contributing to AIDS pathogenesis with elevated plasma lipopolysaccharide (LPS) levels, as a marker for microbial translocation, seen in advanced HIV disease. Therefore, the current study was undertaken to assess monocyte activation in vitro by LPS and to determine its impact on monocyte phenotype. METHODS: Monocytes from non-HIV-infected donors were analyzed for CD14, CD16, CD69, TNFα, and CCR5 by flow cytometry pre- and post-stimulation with LPS. In-vitro cultures were then set up to expose non-activated and activated monocytes to R5-, X4-, and dual (R5/X4)-tropic viruses; and the amount of HIV present on the cells was assayed. RESULTS: Non-HIV-infected monocytes, after LPS stimulation, were confirmed to have an activated phenotype with increase in CD16 and CD69 surface expressions (p<0.05). The activation phenotype was supported by increase in TNFα production, p<0.05. The activated monocytes had increased surface CCR5 (from 21% to 98%; p=0.05); and were found to have more R5-tropic virus than non-activated monocytes (p<0.05). CONCLUSIONS: Following activation by LPS, non-HIV-infected monocytes were found to have increase in surface CCR5. These activated monocytes, when exposed to R5-tropic virus, were found to have more virus compared to non-activated monocytes. The significance of the findings could lie in explaining how microbial translocation plays a role in HIV progression; and possibly promoting CCR5-directed strategies in treating HIV.

Figure 1

Representative Scatter Plots of M/MΦ Surface Expression Markers Following LPS Stimulation. Cells were stained for CD14, CD16, CD69, TNFα, and CCR5. The scatter plots show examples of the post-LPS stimulation experiments, n=3. Panel A: Monocytes gated on forward- and side-scatter plots; Panel B: Compared to pre-LPS stimulation, expression of CD14 on isolated monocytes remained stable before and after stimulation; 87% versus 92%, p>0.05; Panel C: CCR5 expression on CD14 monocytes increased from 21% to 99%, p=0.06; Panel D: CD69 expression after LPS stimulation on CD14 monocytes increased from to 18% to 91%, p<0.05; Panel E: CD16 expression increased from 9.5% to 89% after LPS stimulation, p<0.05; Panel F: TNFα production increased from 0% to 33% after LPS stimulation, p<0.05.

Figure 2

Relative Amounts of Virus Recovered from Activated versus Non-Activated Monocytes. A: Higher amount of BaL virus (R5-tropic) recovered from activated monocytes compared to non-activated monocytes, p<0.05, n=3; B & C: For both LAI (X4-tropic) and p89.6 (R5/X4-tropic), the amount of virus recovered from activated monocytes did not differ from non-activated monocytes, p>0.05, n=3 for both viruses.