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

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

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

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

Kv1.3 channel blocker attenuates GrB mediated neural cell toxicity.

<p>Freshly isolated human CD8+ T cells were simulated with anti-CD3 or anti-CD3/CD28 in the presence or absence of MgTx. Cultured supernatants were collected at 3 days after stimulation. (A). Human neural cells cultured on poly-D-lysine pre-coated 96 well plates were pretreated with supernatants from non-activated CD8 T cells (Unstim.), anti-CD3 or anti-CD3/CD28-activated CD8+ T cells without MgTx (none), and with MgTx (MgTx). After 24 hours of treatment, CellQuanti-blue dye was added in each well for 30 minutes. Fluorescence was then detected using a plate reader. Cell viability was quantified by fluorescence intensity. (B). MgTx (30 nM) was added to culture media and incubated for 3 days. Human NPCs were treated with supernatants without MgTx (Ctrl), MgTx contained sups with vehicle treatment, with GrB alone (GrB) or MgTx containing sups plus GrB treatment (GrB/MgTx sups). Neurotoxicity was evaluated by cell viability quantified by fluorescence intensity. The fluorescence intensity in each group is plotted as percent relative to that in non-activated cells (Unstim.) or control cells (Ctrl). Data are mean of triplicate ± SD of one representative of three independent experiments. Values that are significantly different from that of vehicle treated control are indicated as *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.005.</p