Transcriptional analysis of the host response to HIV-1 infection in CD4+ T lymphocytes monocytes and macrophages

Abstract

The human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS), a condition characterized by depletion of CD4+ T cells and other immune system dysfunctions. Due to its widespread geographic distribution and ease of 4 transmission, HIV has become a serious global healthcare concern. HIV-1 subtype C (HIV-1-C) 5 is of particular interest since it is the most rapidly expanding and is especially prevalent in 6 southern Africa. In addition, HIV-1-C is poorly studied in comparison to other subtypes. Despite HIV being extensively researched, much is still unknown about the host cell response 8 to infection at a molecular level and how this might be exploited for therapeutic intervention. Therefore, the purpose of this study was to use microarray-based transcriptomic analysis to gain an unbiased perspective of the host cell response in CD4+ T lymphocytes and macrophages when exposed to primary HIV-1-C viruses of different tropisms (R5-tropic, X4- tropic, dual-tropic). In order to achieve this overall aim, we first needed to develop and optimize protocols for the culture of primary HIV-1-C strains. A p24 ELISA assay was used to confirm successful viral replication, followed by functional titration using the GHOST reporter cell line. We found that the efficiency of replication of primary viral isolates was highly stochastic. While many of the strains cultured suffered severe losses of infectivity, we managed to produce infective stocks of dual-tropic and X4-tropic isolates. We next aimed to optimize cell culture protocols for CD4+ T lymphocytes and macrophages that would ensure maximal susceptibility to HIV. Since activation of CD4+ T cells is reported to enhance viral replication, we assessed the efficacy of various activation protocols. We found that antibody- mediated activation was highly successful. Flow cytometric analysis revealed increased cell proliferation and CD25 expression. In addition, CD4+ T-cell activation dramatically increased CXCR4 surface expression while CCR5 expression was diminished. Infection experiments with our dual-tropic isolate confirmed that these cells were susceptible to infection. In order to optimize macrophage cell culture conditions, we compared the effects of differentiation under the influence of different growth factors (M-CSF or GM-CSF). Successful differentiation was determined by phenotypic analysis using flow cytometry coupled with a functional phagocytosis assay. Additional analysis of co-receptor expression revealed extremely low levels of CXCR4 and relatively high CCR5 expression in both macrophage populations. The differentiated macrophages were completely refractory to infection with our dual-tropic isolate, making them unsuitable for further experiments. The final component of the study was the gene expression analysis itself, which was performed using activated CD4+ T lymphocytes exposed to a dual-tropic isolate (CM9). Differential gene expression analysis revealed altered expression patterns in a relatively small but functionally diverse group of genes involved in apoptosis, deubiquitination, transcriptional regulation and immune system functions. Apoptosis and deubiquitination were identified as statistically overrepresented functional pathways in our subsequent GO-based analysis. Comparison of our findings with previous studies performed using HIV-1-B seems to indicate that while many of the genes observed in our study have not specifically been detected previously, they do tend to belong to similar pathways.