Identification of Nitrated Immunoglobulin Variable Regions in the HIV-Infected Human Brain: Implications in HIV Infection and Immune Response

HIV can infiltrate the brain and lead to HIV-associated neurocognitive disorders (HAND). The pathophysiology of HAND is poorly understood, and there are no diagnostic biomarkers for it. Previously, an increase in inducible nitric oxide synthase levels and protein tyrosine nitration in the brain were found to correlate with the severity of HAND., In this study, we analyzed human brains from individuals who had HIV infection without encephalitis and with encephalitis/HAND and compared them to the brains of healthy individuals. We identified the nitrated proteins and determined the sites of modification using affinity enrichment followed by high-resolution and high-mass-accuracy nanoLC–MS/MS. We found that nitrated proteins were predominantly present in the HIV-infected individuals with encephalitis, and, interestingly, the modifications were predominantly located on immunoglobulin variable regions. Our molecular model indicated potential interactions with HIV envelope proteins and changes on the heavy and light chain interface upon the nitration and nitrohydroxylation of these residues. Therefore, our findings suggest a role for these modifications in the immune response, which may have implications in disease pathogenesis

Robust Two-Dimensional Separation of Intact Proteins for Bottom-Up Tandem Mass Spectrometry of the Human CSF Proteome

Cerebrospinal fluid (CSF) is produced in the brain by cells in the choroid plexus at a rate of 500 mL/day. It is the only body fluid in direct contact with the brain. Thus, any changes in the CSF composition will reflect pathological processes and make CSF a potential source of biomarkers for different disease states. Proteomics offers a comprehensive view of the proteins found in CSF. In this study, we use a recently developed nongel based method of sample preparation of CSF followed by liquid chromatography–high accuracy mass spectrometry (LC-MS) for MS and MS/MS analyses, allowing unambiguous identification of peptides/proteins. <i>Gel-eluted liquid fraction entrapment electrophoresis</i> (Gelfree) is used to separate a CSF complex protein mixture in 12 user-selectable liquid-phase molecular weight fractions. Using this high throughput workflow, we have been able to separate CSF intact proteins over a broad mass range (3.5–100 kDa) with high resolution (between 15 and 100 kDa) in 2 h and 40 min. We have completely eliminated albumin and were able to interrogate the low abundance CSF proteins in a highly reproducible manner from different CSF samples at the same time. Using LC-MS as a downstream analysis, we identified 368 proteins using MidiTrap G-10 desalting columns and 166 proteins (including 57 unique proteins) using Zeba spin columns with a 5% false discovery rate (FDR). Prostaglandin D2 synthase, Chromogranin A, Apolipoprotein E, Chromogranin B, Secretogranin III, Cystatin C, VGF nerve growth factor, and Cadherin 2 are a few of the proteins that were characterized. Gelfree-LC-MS is a robust method for the analysis of the human proteome that we will use to develop biomarkers for several neurodegenerative diseases and to quantitate these markers using multiple reaction monitoring

Soluble Insulin Receptor Levels (MFI) and HIV-seropositive women stratified by HAND.

a<p>MFI  =  Median Fluorescence Intensity; median (interquartile range [25^th and 75^th percentile]),</p>b<p>significant p value<0.05^*.</p

Soluble insulin receptor full-length (sIRαβ) and HIV.

<p>Soluble insulin receptor (sIR) intact or full-length (αβ) was measured in plasma (A) and CSF (B) of HIV-seropositive women (HIV+) (n = 34) and controls (HIV−) (n = 10, 5 with plasma and different 5 for CSF). The sIR subunits were determined using an ELISA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037358#pone.0037358-The1" target="_blank">[47]</a> Significantly higher levels of full-length sIR was observed in HIV-seropositive women in plasma and CSF when compared with controls (p<0.001 and p = 0.003 respectively). (MFI = Median Fluorescence Intensity).</p

Membrane insulin receptor (mIR), insulin receptor substrate 1 (IRS-1), and IRS-1 tyrosine phosphorylation levels in the CSF white cell pellet (CSF WCP) stratified by HAND.

a<p>For the mIR (MFI) and IRS-1 we analyzed 11 women with normal cognition, 8 with asymptomatic impairment, and 15 with symptomatic impairment. For IRS-1 tyrosine phosphorylation 5 women per group were analyzed;</p>b<p>significant p value<0.05^*;</p>c<p>MFI = Median Fluorescence Intensity; median (interquartile range [25^th and 75^th percentile]).</p

Soluble insulin receptor full-length (sIRαβ) stratified by HAND.

<p>Soluble insulin receptor (sIR) intact or full-length (αβ) subunit was measured by ELISA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037358#pone.0037358-The1" target="_blank">[47]</a> in plasma (A) and CSF (B) of HIV-seropositive women (HIV+) (n = 34) stratified by HAND into normal cognition (n = 11), asymptomatic impairment (n = 8), and symptomatic impairment (n = 15); and 10 HIV-negative controls (HIV−) (5 plasma were different women from 5 CSF). In plasma (A), levels of full-length sIR were significantly increased from controls in all HIV-seropositive women and it correlated with the severity of HAND (normal cognition [p = 0.003], asymptomatic impairment [p<0.001], and symptomatic impairment [p<0.001]). Also, women with symptomatic impairment had significant higher levels of full-length sIR when compared to those with normal cognition (p = 0.009). A similar trend was observed in CSF samples (B), although the only significant increased was observed in the women with symptomatic impairment when compared to controls. (MFI = Median Fluorescence Intensity).</p

Insulin Receptor Substrate 1 (IRS-1) tyrosine phosphorylation stratified by HAND.

<p>Insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation was determined in CSF cell pellets of 23 HIV-seropositive women (HIV+) stratified by HAND (7 with normal cognition, 7 with asymptomatic impairment, and 9 with symptomatic impairment) using flow cytometry. A significant decrease in IRS-1 tyrosine phosphorylation was observed between HIV-seropositive women with normal cognition and symptomatic impairment (p = 0.02). (MFI = Median Fluorescence Intensity).</p

K+ channel blockers do not affect CD107a expression on activated CD8+ T cells.

<p>(A) Freshly isolated CD8+ T cells were stimulated with anti-CD3/CD28 or anti-CD3 for 24 hours. Cells were then stained with a CD107a-specific mAb, or an IgG1 isotype control (filled histogram) at the indicated times. (B). CD8+ T cells were pretreated with ShK (10 nM), MgTX (30 nM), ChTX (50 nM) and TRAM-34 (500 nM) for 3 h, followed by stimulation with anti-CD3/CD28 or anti-CD3 alone for 6 hours. Surface expression levels of CD107a were then analyzed by flow cytometry. FACS plots shown are representative data from three separate experiments.</p

Kv1.3 expression is upregulated in activated CD8+ T cells and co-localizes with CD8.

<p>(A) Purified CD8+ T cells were stimulated with anti-CD3/CD28 for 3 days. Naïve and stimulated cells were then immunostained for Kv1.3 in combination with CD8 and subsequently viewed by immunofluorescence microscopy. Cellular nuclei were counterstained with DNA dye DAPI (blue). Kv1.3 detected by AF 594 fluorescence is shown in red, while CD8 detected by AF 488 fluorescence is shown in green. Colocalization is indicated by a yellow and/or orange color in the overlay panels. (B) An isotype-matched antibody was used as a negative control. Original magnification, ×100. Image is representative of three different donors. (C) Summary of percentages of activated CD8+ T cells expressing Kv1.3. In brief, 4 view fields/microscopic section were evaluated for Kv1.3+ CD8 cells stimulated with anti-CD3/CD28 or anti-CD3 alone for 3 days. The percentages of Kv1.3+ cells are based on the number of CD8+ T cells counted. Data are mean ± SD from one representative of three independent and reproducible experiments. Values that are significantly different from that of non-stimulated control are indicated as **, <i>p</i><0.01.</p

Inhibitory effects of Kv1.3 blockade in the differentiation and homeostatic maintenance of T_EM CD8 cells.

<p>(A) Naïve, T_CM, T_EMRA and T_EM CD8 subpopulations were sorted from CD8+ gated cell population based on surface markers of CCR7 and CD45RA. Sorted individual subsets within the respective gates shown were transduced with a DN-Kv1.x and GFP control. After 7 days of transfection, the percentages of each subset in gated GFP+ CD8+ cells were analyzed by flow cytometry. Gate for expression of GFP was established using untransduced controls. (B) FACS profiles are representative of three separate experiments using cells from different donors. The percentage of cells in each quadrant is indicated. (C) The percentages of CD8 subsets displaying GFP fluorescence from each single transfected subpopulation are presented as mean ± SD of three experiments. Values that are significantly different from that of GFP control are indicated as follows: *, <i>p</i><0.05, **, <i>p</i><0.01; ***, <i>p</i><0.005. (D) Representative FACS profiles of phenotypical changes of transduced T_CM and T_EM subsets 21 days after transfection. (E). FACS-sorted CCR7- (T_EM/T_EMRA) were labeled with PKH26 day (2×10–6 M), followed by stimulation with anti-CD3/CD28 for 24 h and then transduced with a lentiviral vector encoding the dominant-negative Kv1.x and GFP control alone at an MOI of ∼8. PKH26 fluorescence was analyzed by flow cytometry at days 0, 5 and 11. FACS plots shown are representative data from two experiments.</p