Plasminogen activator system in tonsils of HAART-treated HIV-1+ individual.

<p>Tonsils from one HIV-1+individual under cART (MA35) tested positive for HIV p24Gag in the germinative center of tonsil (panel A, OM 10x), and expressed both uPAR and c-uPAR (panel B; seven tonsils from uninfected individuals (#36–46) were used as reference control). Sections used for Western blotting were from snap-frozen tissue blocks, whereas lysates from stably transfected HEK cells expressing uPAR and c-uPAR were used as positive controls; because of high levels of expression of these antigens in HEK cells actin was not detected at exposure time used for showing actin from tissue lysate. Chemotaxis of (panel C) and HIV expression from (panel D) U1 cells were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070606#pone-0070606-g003" target="_blank">Figure 3</a>. Gray squares in panel D represent the value of input virus in the conditioned supernatants at the end of the 4 days of culture. Cell-associated HIV proteins were measured in U1 cells cultivated for 4 days with the stimuli reported at the bottom of the panel (E) and viral proteins were identified based on their molecular weight (right side of the panel). PMA (10 nM) was used as positive control for inducing virus expression (n = 3).</p

UPA expression in tonsils and lymph nodes of uninfected and HIV-1⁺ individuals.

<p>Tonsils from either uninfected (panels A-C) or HIV-1⁺ individuals (panels D-F) showed uPA⁺ cells (in brown) predominantly in the GC. The phenotype of uPA⁺ cells was demonstrated by double immunostaining (panels G-L): uPA (red) was expressed by B lymphocytes (CD20⁺, brown), FDC (CD35⁺, brown) and a few macrophages (CD68⁺, brown), but not T cells (** CD3⁺, brown). Panels G-L: OM 10x. Panels M-O show OM at 100x. Brown staining in panels G-H is indicated by an arrow. Immunohistochemical score for the number of uPA+ cells was evaluate for lymphoid organs of uninfected and HIV-1⁺ individuals; all lymphoid organs (panel M), normal vs. hyperplastic lymphoid organs (panel N). Vertical and horizontal bars represent mean and SEM. Differences for the number of uPA⁺ cells in the different tissues and populations were tested by the two-tailed Mann-Whitney test (p values are indicated by asterisk * = 0.01–0.05; **0.001–0.01).</p

Soluble and cleaved form of uPAR differently modulates chemotactic activity and HIV expression by conditioned microenvironments.

<p>Chemotaxis of chronically infected monocytic cells was tested (n = 3) in response to: A. tonsil culture medium (TCM) and TCM added of recombinant stimuli or purified c-suPAR and suPAR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070606#pone.0070606-Masucci1" target="_blank">[76]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070606#pone.0070606-Fazioli1" target="_blank">[77]</a>; B. conditioned supernatant from uninfected tonsils (Nil-c), R5 infected tonsils (R5-c) and X4 infected tonsils (X4-c) immunodepleted with either control immunoglobulins (IP w Ig) or polyclonal anti-human uPAR Ab (IP w M2) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070606#pone.0070606-Resnati1" target="_blank">[47]</a> or M2-depleted supernatants reconstituted with 100 ng/ml of purified c-suPAR or suPAR. Migrated cells toward TCM was 25±9 (n = 3). HIV-1 expression from U1 cells was tested (n = 3) by cultivating them with the same stimuli and conditions used and described for chemotaxis assay (panels C-D). Gray squares in panel D represent the value of input virus in the conditioned supernatants at the end of the 4 days of culture. CM from <i>ex vivo</i> uninfected and infected tonsils were collected after 9 days of histoculture. Vertical and horizontal bars represent mean and SEM. Two-tailed paired T test was used for statistical analysis, and p values are indicated by asterisks (* = 0.01–0.05; ** = 0.001–0.01; *** = <0.001).</p

Clinical parameters of seronegative and HIV-1+individuals used for retrospective analysis.

<p>Tonsils with histological diagnosis of follicular hyperplasia from HIV infected and uninfected individuals were analyze.</p><p>Lymph nodes with follicular hyperplasia and normal histology from HIV infected and uninfected individuals were use. n; number of individuals, y; years, M; male, F; Female.</p><p>Age is report as median and range of distribution in brackets.</p

UPAR expression in tonsils and lymph nodes of uninfected and HIV-1⁺ individuals.

<p>Figures are representative of tonsils and lymph nodes from uninfected and HIV-1⁺ individuals with hyperplastic and normal histology. Sections used for IHC were obtained from formalin-fixed paraffin-embedded tissues, and the clinical parameters of donors are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070606#pone-0070606-t001" target="_blank">Table 1</a>. Tonsils from either uninfected (panels A-C) or HIV-1+ individuals (panels D-F) show uPAR⁺ cells present both in the GC (arrows, GC) and in the inter-follicular area (IA). UPAR was expressed by macrophages (panel G, double immunohistochemistry with CD68 shown in brown and uPAR in red), FDC (panel H, double immunohistochemistry with CD35 shown in brown and uPAR in red) and endothelial cells (panel I, uPAR shown in brown) in both groups of patients. Panels A-F: Optical Magnification (OM) 10x, panels G and H: OM 100x, panel I: OM 40x. Brown staining in panels G-H is indicated by an arrow. Immunohistochemical score for the number of uPAR⁺ cells was evaluate for lymphoid organs of uninfected and HIV-1⁺ individuals; all lymphoid organs (panel L), normal vs. hyperplastic lymphoid organs (panel M). Vertical and horizontal bars represent mean and SEM. Differences for the number of uPAR⁺ cells in the different tissues and populations were tested by the two-tailed Mann-Whitney test (p values are indicated by asterisk * = 0.01–0.05; **0.001–0.01).</p

HIV-1 Infected Lymphoid Organs Upregulate Expression and Release of the Cleaved Form of uPAR That Modulates Chemotaxis and Virus Expression

<div><p>Cell-associated receptor for urokinase plasminogen activator (uPAR) is released as both full-length soluble uPAR (suPAR) and cleaved (c-suPAR) form that maintain ability to bind to integrins and other receptors, thus triggering and modulating cell signaling responses. Concerning HIV-1 infection, plasma levels of suPAR have been correlated with the severity of disease, levels of immune activation and ineffective immune recovery also in individuals receiving combination anti-retroviral therapy (cART). However, it is unknown whether and which suPAR forms might contribute to HIV-1 induced pathogenesis and to the related state of immune activation. In this regard, lymphoid organs represent an import site of chronic immune activation and virus persistence even in individuals receiving cART. Lymphoid organs of HIV-1⁺ individuals showed an enhanced number of follicular dendritic cells, macrophages and endothelial cells expressing the cell-associated uPAR in comparison to those of uninfected individuals. In order to investigate the potential role of suPAR forms in HIV-1 infection of secondary lymphoid organs, tonsil histocultures were established from HIV-1 seronegative individuals and infected <i>ex vivo</i> with CCR5- and CXCR4-dependent HIV-1 strains. The levels of suPAR and c-suPAR were significantly increased in HIV-infected tonsil histocultures supernatants in comparison to autologous uninfected histocultures. Supernatants from infected and uninfected cultures before and after immunodepletion of suPAR forms were incubated with the chronically infected promonocytic U1 cell line characterized by a state of proviral latency in unstimulated conditions. In the contest of HIV-conditioned supernatants we established that c-suPAR, but not suPAR, inhibited chemotaxis and induced virus expression in U1 cells. In conclusion, lymphoid organs are an important site of production and release of both suPAR and c-suPAR, this latter form being endowed with the capacity of inhibiting chemotaxis and inducing HIV-1 expression.</p></div

<i>Ex-vivo</i> HIV-1 infection of tonsil histocultures increases the soluble levels of CCL2MCP-1, suPAR, c-suPAR and the number of uPAR+ cells.

<p>Tonsil blocks were left either uninfected (Nil) or were infected with R5 or X4 HIV-1 strain. Every 3 days the culture conditioned supernatants were tested for the levels of viral replication (RT activity), lactate dehydrogenase (LDH), CCL2/MCP-1, suPAR, uPA and PAI-1. The results are reported as fold vs. Nil (absolute values are shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070606#pone.0070606.s001" target="_blank">Figure S1</a>). Panels A (n = 31), B (n = 31) and C (n = 28) show fold of HIV-1 replication, LDH and CCL2/MCP-1 levels vs. Nil, respectively, as measured in the culture supernatants The panels below show the IHC detection of HIV p24_Gag measured 6 days post-infection and hematoxylin-eosin staining at day 9 after infection. Panel D (n = 28) shows fold of suPAR levels vs. Nil measured in the culture supernatants. Panel E (n = 7) shows the IHC score in terms of number of uPAR⁺ cells in Nil and <i>ex-vivo</i> infected tonsil histocultures at the indicated time points after infection (Day 0 is the time of surgical removal); the right panels show the IHC of uPAR+ cells (brown) 9 days post-infection. Panel F shows western blot analysis evaluating for the presence of both uPAR and c-uPAR and of HIV-1 p24 _Gag release in the culture supernatants of tonsils left uninfected (Nil) or infected <i>in vitro</i> for the indicated time points after infection. Panels G (n = 21) and panel H (n = 21) show the fold of uPA and PAI-1 levels vs. Nil measured in the culture supernatants. The vertical and horizontal bars represent the mean and SEM. Black dots and triangles in panels A-E and G-H represent data from tonsils infected <i>ex-vivo</i> with R5 and X4 strain, respectively; white circles in panel E represent data from uninfected ex-vivo tonsils. Statistical significance is indicated by asterisks (*** = <0.001); 2-tailed paired t test (panel A), 2-tailed Wilcoxon signed-rank test (panels C and D). Pictures are at 40×magnification.</p

Gene Onthology Analysis of the differentially expressed genes following <i>MYBBP1A</i>-down-regulation in HeLa cells.

<p>N = total number of differentially expressed genes within the GO cathegory.</p><p>%: percent of the total number of differentially expressed genes.</p><p><i>p-value</i>: calculated on the basis of the enrichment of the category (ie number of genes affected/number of genes in the category).</p

<i>MYBBP1A</i> down-regulation induces apoptosis.

<p>(<b>A</b>) HeLa cells were transiently transfected with siRNA1, 2 or 3, or with control High-GC or Medium-GC oligonucleotides, or with Lipofectamine only, for 48 h. The figure shows the determination of early apoptotic cells by flow cytometry (i.e. Annexin V positive and 7AAD-negative) (left panel, circled gate) 48 h post transfection. The histogram on the right shows the quantification in the various samples at the different times after transfection. In untreated cells only 1% of the cells were in apoptosis. (<b>B</b>) Western-blot analysis of HeLa cells transiently transfected for 48 h with CTL (untreated cells), LIPO (treated only with lipofectamine), HIGH GC (transfected with High-GC control), siRNA1 (transfected with MYBBP1A-specific siRNA1). The immunoblot was performed against MYBBP1A, active Caspase 3 and Caspase 9; tubulin is shown as loading control.</p

Genes differentially expressed in <i>MYBBP1A</i>-down-regulated HeLa cells^*.

*<p>Data obtained with a human Affymetrix ST 1.0 chip. Analysis of gene expression profiling on HeLa cells transfected with siRNA3, harvested 48 h after transfection.</p>§<p>Genes whose expression level varied by more than 50% (p<0.0001).</p