Proteomic analyses of methamphetamine (METH)-induced differential protein expression by immature dendritic cells (IDC)

Abstract

In the US, the increase in methamphetamine (METH) use has been associated with increased human immunodeficiency virus (HIV-1) infection. Dendritic cells (DC) are the first line of defense against HIV-1. DC play a critical role in harboring HIV-1 and facilitate the infection of neighboring T cells. However, the role of METH on HIV-1 infectivity and the expression of the proteome of immature dendritic cells (IDC) has not been elucidated. We hypothesize that METH modulates the expression of a number of proteins by IDC that foster the immunopathogenesis of HIV-1 infection. We utilized LTR amplification, p24 antigen assay and the proteomic method of difference gel electrophoresis (DIGE) combined with protein identification through high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to analyze the effects of METH on HIV-1 infectivity (HIV-1 IIIB; CXCR4-tropic, X4 strain) and the proteomic profile of IDC. Our results demonstrate that METH potentiates HIV-1 replication in IDC. Furthermore, METH significantly differentially regulates the expression of several proteins including CXCR3, protein disulfide isomerase, procathepsin B, peroxiredoxin and galectin-1. Identification of unique, METH-induced proteins may help to develop novel markers for diagnostic, preventive and therapeutic targeting in METH using subjects.

FIGURE 1

Constitutive expression of biomarkers on mature (MDC) and immature (IDC) dendritic cells as measured by FACS analysis. Statistical significance was determined by Student’s t-test (n = 3 independent experiments).

FIGURE 2

Effect of METH on HIV-1 replication in IDC. (A) IDC were treated with or without METH, infected with HIV-1 IIIB overnight, washed and cultured for 48 hr. RNA was extracted and reverse transcribed followed by quantitative, real time PCR using primers specific for the LTR-R/U5 region of the HIV-1 genome. Differences in threshold cycle number are used to quantitate the relative amount of PCR target contained within each tube. Relative mRNA species expression was quantitated and expressed as transcript accumulation index (TAI), calculated using the comparative C_T method All data were controlled for quantity of RNA input by performing measurements on an endogenous reference gene, β-actin. (B) IDC were treated with or without METH, infected with HIV-1 IIIB overnight, washed and then cultured for 15 days. The culture supernatants were quantitated for p24 antigen using an ELISA method with a minimum detection range of 1 pg of p24 antigen/ml. All data are presented as the mean ± SD of 3 independent experiments. Statistical significance was calculated by Student’s t-test

FIGURE 2

Effect of METH on HIV-1 replication in IDC. (A) IDC were treated with or without METH, infected with HIV-1 IIIB overnight, washed and cultured for 48 hr. RNA was extracted and reverse transcribed followed by quantitative, real time PCR using primers specific for the LTR-R/U5 region of the HIV-1 genome. Differences in threshold cycle number are used to quantitate the relative amount of PCR target contained within each tube. Relative mRNA species expression was quantitated and expressed as transcript accumulation index (TAI), calculated using the comparative C_T method All data were controlled for quantity of RNA input by performing measurements on an endogenous reference gene, β-actin. (B) IDC were treated with or without METH, infected with HIV-1 IIIB overnight, washed and then cultured for 15 days. The culture supernatants were quantitated for p24 antigen using an ELISA method with a minimum detection range of 1 pg of p24 antigen/ml. All data are presented as the mean ± SD of 3 independent experiments. Statistical significance was calculated by Student’s t-test

FIGURE 3

Representative 2-D gel image of IDC cell lysate. IDC were treated with 100 μM METH for 24 hr. Total protein was isolated and subjected to DIGE analysis as described in methods. Representative 2-D gel image of proteins stained with SYPRO Ruby. Arbitrary numbers point to (arrows) the outline of statistically significant, differentially expressed proteins between control and METH treated IDC. Identity of each protein is shown in Table 2. Three separate experiments gave similar results.

FIGURE 4

Western blot analysis of the effect of METH on the differential expression of proteins by IDC. IDC were treated with 100 μM METH for 24 hr. Total protein was isolated and subjected to western blots. Densitometry analyses were done using a Syngene Image Analyzer with Gene Tools Analysis Software. Data were normalized to protein expression levels of β-actin (data not shown). The graphs show the % change in OD as measured by densitometry of bands from western blots. Statistical significance was determined by Student’s t-test (n = 3 independent experiments.