HMGB1 transcription and expression during HSV-2 infection.

<p>HEC-1 cells were infected at 0.1 or 1.0 pfu/cell. A time-course analysis of HMGB1 mRNA (A) and protein (B) was performed by quantitative RT-PCR and western blot, respectively, with 18S rRNA and GAPDH protein as controls. RNA quantification was performed according to the 2^{- ΔΔCT} method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016145#pone.0016145-Livak1" target="_blank">[50]</a>. (C) HMGB1 was detected more than 4 days in HEC-1 cells treated with actinomycin D. GADPH and P53 were used as controls. M  =  mock-infected cells. Results are representative of 3 independent experiments.</p

Correlations between HMGB1 concentrations and HSV-2 shedding in cervico-vaginal samples collected from HSV-2-infected women.

<p>Eighteen genital samples were collected from women seropositive for HSV-2. Viral culture was performed on Vero cells, and HSV-2 DNA was quantified with real-time PCR. HMGB1 was quantified with a commercial ELISA and a band-shift assay, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016145#pone.0016145-Chen1" target="_blank">[32]</a>.</p

Band-shift assay for HMGB1 detection in cervicovaginal secretions from HSV-2-infected women.

<p>Ten microliters of cervicovaginal specimens (#1–18) collected from 18 women seropositive for HSV-2 were mixed with radiolabeled hemicatenated DNA (hcDNA). HMGB1-hcDNA shifted complexes were analyzed by electrophoresis on nondenaturing polyacrylamide gel, using a band-shift assay, as described in (30). The amount of HMGB1 was calculated from the percentage of shifted hcDNA, quantified with ImageJ software (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016145#pone-0016145-t001" target="_blank">table 1</a>).</p

HEC-1 cell death during HSV-2 infection.

<p>HEC-1 cells, infected at 0.1 or 1.0 pfu/cell, were analyzed at various times post-infection for (A) HSV-2 productive infection, assessed by plaque assay, (B) cell viability, assessed by trypan blue exclusion, (C) apoptosis, assessed by western blot against PARP and pro-caspase 3, and (D) necrosis, assessed by extracellular LDH release over total LDH. (E) Necrosis was measured in mock and HSV-2-infected cells in the presence and absence of Z-VAD, an inhibitor of apoptosis. Results are either representative of at least 3 independent experiments (A, B, C) or are means and SD from 3 independent experiments (D, E).</p

HMGB1 released by HSV-2-infected cells activates fibroblast migration and stimulates HIV-1 expression.

<p>MRC5 fibroblasts were subjected to migration assays in the presence of (A) human recombinant HMGB1 or (B) supernatants from mock (M) or HSV-2-infected HEC-1 cells. HMGB1 concentrations reached 270 ng/ml in the supernatants collected from infected cells, versus 50 ng/ml in mock-infected cell media. Control experiments used glycyrrhizin or neutralizing antibodies against HMGB1 or its receptors RAGE, TLR-4 and TLR-2. Data are expressed as -fold increases in cell migration compared to non treated control cells. ACH-2 cells, that contain a latent HIV-1 provirus, were grown in the presence of (C) human recombinant HMGB1 or (D) supernatants from mock (M) or HSV-2-infected HEC-1 cells. P24 antigen was measured by ELISA after 36 h. Data are expressed as the -fold increase in p24 antigen compared with non treated control cells. Virion-free supernatants were obtained by ultracentrifugation. Results are either representative of several experiments (A, C, D) or expressed as the mean and SD of 3 independent experiments (B).</p

HMGB1 mobility is altered by HSV-2 infection.

<p>HEC-1 cells were treated with TNF-α (2 ng/ml) and CHX (10 µg/ml). At various times post-induction, western blot was performed on whole cell extracts to detect PARP and pro-caspase 3. A nuclear fraction obtained after NP-40-induced membrane permeabilization was also subjected to western blot to detect chromatin-bound HMGB1 (bottom). (A) Time-course analysis of HMGB1 in the cytoplasm and nucleus after HSV-2 infection of HEC-1 cells at 0.1 and 1.0 pfu/cell. M  =  mock-infected cells. (B) Time-course analysis of mobile (M) and chromatin-bound (CB) HMGB1 during HSV-2 infection, following membrane permeabilization by NP40. (C) Measurement of extracellular HMGB1 by ELISA after infection of HEC-1 cells by HSV-2. Results are representative of 3 independent experiments.</p

P2X7 Receptor Inhibition Improves CD34 T-Cell Differentiation in HIV-Infected Immunological Nonresponders on c-ART

<div><p>Peripheral CD4+ T-cell levels are not fully restored in a significant proportion of HIV+ individuals displaying long-term viral suppression on c-ART. These immunological nonresponders (INRs) have a higher risk of developing AIDS and non-AIDS events and a lower life expectancy than the general population, but the underlying mechanisms are not fully understood. We used an <i>in vitro</i> system to analyze the T- and B-cell potential of CD34+ hematopoietic progenitor cells. Comparisons of INRs with matched HIV+ patients with high CD4+ T-cell counts (immune responders (IRs)) revealed an impairment of the generation of T-cell progenitors, but not of B-cell progenitors, in INRs. This impairment resulted in the presence of smaller numbers of recent thymic emigrants (RTE) in the blood and lower peripheral CD4+ T-cell counts. We investigated the molecular pathways involved in lymphopoiesis, focusing particularly on T-cell fate specification (Notch pathway), survival (IL7R-IL7 axis) and death (<i>Fas</i>, <i>P2X7</i>, <i>CD39/CD73</i>). <i>P2X7</i> expression was abnormally strong and there was no <i>CD73</i> mRNA in the CD34+ cells of INRs, highlighting a role for the ATP pathway. This was confirmed by the demonstration that <i>in vitro</i> inhibition of the P2X7-mediated pathway restored the T-cell potential of CD34+ cells from INRs. Moreover, transcriptomic analysis revealed major differences in cell survival and death pathways between CD34+ cells from INRs and those from IRs. These findings pave the way for the use of complementary immunotherapies, such as P2X7 antagonists, to restore T-cell lymphopoiesis in INRs.</p></div

Transcriptomic analysis of CD34+ cells in HIV-infected IRs and INRs.

<p>(A) Genes differentially expressed in the CD34+ cells of IRs (<i>n</i> = 7) and INRs (<i>n</i> = 10) are shown. Each column represents an individual sample, and each row, an individual gene, with normalized expression level indicated on a color scale (red = upregulation, green = downregulation). (B) The top 5 biological functions in Ingenuity analysis, based on activation <i>z</i>-score, an algorithm predicting the degree of activation or inactivation of the genes of the group concerned. Rank in the top 5 is indicated by the number after the #. <i>P</i>-values are shown. Biological functions upregulated (positive <i>z</i>-score) in IR patients are shown in red, and biological functions downregulated in IR patients (negative <i>z</i>-score) are shown in green.</p

Limiting dilution assays (LDAs) to determine the T-cell and B-cell differentiation potential of CD34+ cells.

<p>(A) LDA design. Conditions for determining the potential of CD34+ cells to generate T cells ^a and B cells ^b are shown. For further details, see the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005571#sec011" target="_blank">materials and methods</a>. (B, C) Cell cultures on D21 positive for T-cell precursors (B) defined as CD45RA^highCD7+CD5+CD1a+ cells, and B cells (C) defined as CD79a^intra+ cells. (D) Analysis of the T-cell potential of CD34+ cells. Each point on the graph represents the mean value from three independent experiments. (E) The presence of T-cell precursors and B cells was assessed with the ELDA webtool, applying the maximum likelihood method to the Poisson model. Mean values (min-max) for three experiments are indicated for each group and each set of conditions. NS for <i>P</i>>0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Immune activation and inflammation in HIV-uninfected individuals and HIV-infected patients.

<p>(A) Percentage of CD38^high cells among CD8+ T lymphocytes (HIV-, <i>n</i> = 18; IRs, <i>n</i> = 16; INRs, <i>n</i> = 16). (B, C, D) Plasma concentrations of IL-6 (B), CRP (C) and sCD14 (D) in HIV-uninfected (HIV-, <i>n</i> = 3) and HIV-positive individuals (IRs, <i>n</i> = 15; INRs, <i>n</i> = 16). Bars indicate the mean and standard error. The Kruskal-Wallis test was used to assess differences between groups. NS for <i>P</i>>0.05, *<i>P</i><0.05, **<i>P</i><0.01.</p