HIV-1 and methamphetamine alter galectins -1, -3, and -9 in human monocyte-derived macrophages.

Macrophages are key elements of the innate immune system. Their HIV-1 infection is a complex process that involves multiple interacting factors and various steps and is further altered by exposure of infected cells to methamphetamine (Meth), a common drug of abuse in people living with HIV. This is reflected by dynamic changes in the intracellular and secreted proteomes of these cells. Quantification of these changes poses a challenge for experimental design and associated analytics. In this study, we measured the effect of Meth on expression of intracellular and secreted galectins-1, -3, and -9 in HIV-1 infected human monocyte-derived macrophages (hMDM) using SWATH-MS, which was further followed by MRM targeted mass spectrometry validation. Cells were exposed to Meth either prior to or after infection. Our results are the first to perform comprehensive quantifications of galectins in primary hMDM cells during HIV-1 infection and Meth exposure a building foundation for future studies on the molecular mechanisms underlying cellular pathology of hMDM resulting from viral infection and a drug of abuse-Meth.

SWATH-MS and MRM: Quantification of Ras-related proteins in HIV-1 infected and methamphetamine-exposed human monocyte-derived macrophages (hMDM).

HIV-1 infection of macrophages is a multistep and multifactorial process that has been shown to be enhanced by exposure to methamphetamine (Meth). In this study, we sought to identify the underlying mechanisms of this effect by quantifying the effect of Meth on the proteome of HIV-1-infected macrophages using sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) approach. The analyses identified several members of the Rab family of proteins as being dysregulated by Meth treatment, which was confirmed by bioinformatic analyses that indicated substantial alteration of vesicular transport pathways. Validation of the SWATH-MS was performed using an MRM based approach, which confirmed that Meth exposure affects expression of the Rab proteins. However, the pattern of expression changes were highly dynamic, and displayed high donor-to-donor variability. Surprisingly a similar phenomenon was observed for Actin. Our results demonstrate that Meth affects vesicular transport pathways, suggesting a possible molecular mechanism underlying its effect on HIV infection hMDM and a potential broader effect of Meth on cellular homeostasis.

Secreted Metabolome of Human Macrophages Exposed to Methamphetamine.

Macrophages comprise a major component of the human innate immune system that is involved in maintaining homeostasis and responding to infections or other insults. Besides cytokines and chemokines, macrophages presumably influence the surrounding environment by secreting various types of metabolites. Characterization of secreted metabolites under normal and pathological conditions is critical for understanding the complex innate immune system. To investigate the secreted metabolome, we developed a novel workflow consisting of one Reverse Phase (RP) C18 column linked in tandem with a Cogent cholesterol-modified RP C18. This system was used to compare the secreted metabolomes of human monocyte-derived macrophages (hMDM) under normal conditions to those exposed to methamphetamine (Meth). This new experimental approach allowed us to measure 92 metabolites, identify 11 of them as differentially expressed, separate and identify three hydroxymethamphetamine (OHMA) isomers, and identify a new, yet unknown metabolite with a m/z of 192. This study is the first of its kind to address the secreted metabolomic response of hMDM to an insult by Meth. Besides the discovery of novel metabolites secreted by macrophages, we provide a novel methodology to investigate metabolomic profiling.