Stepwise Release of Biologically Active HMGB1 during HSV-2 Infection

BACKGROUND: High mobility group box 1 protein (HMGB1) is a major endogenous danger signal that triggers inflammation and immunity during septic and aseptic stresses. HMGB1 recently emerged as a key soluble factor in the pathogenesis of various infectious diseases, but nothing is known of its behaviour during herpesvirus infection. We therefore investigated the dynamics and biological effects of HMGB1 during HSV-2 infection of epithelial HEC-1 cells. METHODOLOGY/PRINCIPAL FINDINGS: Despite a transcriptional shutdown of HMGB1 gene expression during infection, the intracellular pool of HMGB1 protein remained unaffected, indicating its remarkable stability. However, the dynamics of HMGB1 was deeply modified in infected cells. Whereas viral multiplication was concomitant with apoptosis and HMGB1 retention on chromatin, a subsequent release of HMGB1 was observed in response to HSV-2 mediated necrosis. Importantly, extracellular HMGB1 was biologically active. Indeed, HMGB1-containing supernatants from HSV-2 infected cells induced the migration of fibroblasts from murine or human origin, and reactivated HIV-1 from latently infected T lymphocytes. These effects were specifically linked to HMGB1 since they were blocked by glycyrrhizin or by a neutralizing anti-HMGB1 antibody, and were mediated through TLR2 and the receptor for Advanced Glycation End-products (RAGE). Finally, we show that genital HSV-2 active infections also promote HMGB1 release in vivo, strengthening the clinical relevance of our experimental data. CONCLUSIONS: These observations target HMGB1 as an important actor during HSV-2 genital infection, notably in the setting of HSV-HIV co-infection

PTCH1+/− Dermal Fibroblasts Isolated from Healthy Skin of Gorlin Syndrome Patients Exhibit Features of Carcinoma Associated Fibroblasts

Gorlin's or nevoid basal cell carcinoma syndrome (NBCCS) causes predisposition to basal cell carcinoma (BCC), the commonest cancer in adult human. Mutations in the tumor suppressor gene PTCH1 are responsible for this autosomal dominant syndrome. In NBCCS patients, as in the general population, ultraviolet exposure is a major risk factor for BCC development. However these patients also develop BCCs in sun-protected areas of the skin, suggesting the existence of other mechanisms for BCC predisposition in NBCCS patients. As increasing evidence supports the idea that the stroma influences carcinoma development, we hypothesized that NBCCS fibroblasts could facilitate BCC occurence of the patients. WT (n = 3) and NBCCS fibroblasts bearing either nonsense (n = 3) or missense (n = 3) PTCH1 mutations were cultured in dermal equivalents made of a collagen matrix and their transcriptomes were compared by whole genome microarray analyses. Strikingly, NBCCS fibroblasts over-expressed mRNAs encoding pro-tumoral factors such as Matrix Metalloproteinases 1 and 3 and tenascin C. They also over-expressed mRNA of pro-proliferative diffusible factors such as fibroblast growth factor 7 and the stromal cell-derived factor 1 alpha, known for its expression in carcinoma associated fibroblasts. These data indicate that the PTCH1+/− genotype of healthy NBCCS fibroblasts results in phenotypic traits highly reminiscent of those of BCC associated fibroblasts, a clue to the yet mysterious proneness to non photo-exposed BCCs in NBCCS patients

Intrinsically High Capacity of Animal Cells From a Symbiotic Cnidarian to Deal With Pro-Oxidative Conditions

International audienceThe cnidarian-dinoflagellate symbiosis is a mutualistic intracellular association based on the photosynthetic activity of the endosymbiont. This relationship involves significant constraints and requires co-evolution processes, such as an extensive capacity of the holobiont to counteract pro-oxidative conditions induced by hyperoxia generated during photosynthesis. In this study, we analyzed the capacity of Anemonia viridis cells to deal with pro-oxidative conditions by in vivo and in vitro approaches. Whole specimens and animal primary cell cultures were submitted to 200 and 500 μM of H 2 O 2 during 7 days. Then, we monitored global health parameters (symbiotic state, viability, and cell growth) and stress biomarkers (global antioxidant capacity, oxidative protein damages, and protein ubiquitination). In animal primary cell cultures, the intracellular reactive oxygen species (ROS) levels were also evaluated under H 2 O 2 treatments. At the whole organism scale, both H 2 O 2 concentrations didn’t affect the survival and animal tissues exhibited a high resistance to H 2 O 2 treatments. Moreover, no bleaching has been observed, even at high H 2 O 2 concentration and after long exposure (7 days). Although, the community has suggested the role of ROS as the cause of bleaching, our results indicating the absence of bleaching under high H 2 O 2 concentration may exculpate this specific ROS from being involved in the molecular processes inducing bleaching. However, counterintuitively, the symbiont compartment appeared sensitive to an H 2 O 2 burst as it displayed oxidative protein damages, despite an enhancement of antioxidant capacity. The in vitro assays allowed highlighting an intrinsic high capacity of isolated animal cells to deal with pro-oxidative conditions, although we observed differences on tolerance between H 2 O 2 treatments. The 200 μM H 2 O 2 concentration appeared to correspond to the tolerance threshold of animal cells. Indeed, no disequilibrium on redox state was observed and only a cell growth decrease was measured. Contrarily, the 500 μM H 2 O 2 concentration induced a stress state, characterized by a cell viability decrease from 1 day and a drastic cell growth arrest after 7 days leading to an uncomplete recovery after treatment. In conclusion, this study highlights the overall high capacity of cnidarian cells to cope with H 2 O 2 and opens new perspective to investigate the molecular mechanisms involved in this peculiar resistance

Emergence of antibodies endowed with proteolytic activity against High-mobility group box 1 protein (HMGB1) in patients surviving septic shock

International audienceHigh-mobility group box 1 (HMGB1) concentration in serum or plasma has been proposed as an important biological marker in various inflammation-related pathologies. We previously showed that low titer autoantibodies against HMGB1 could emerge during the course of sepsis. Importantly their presence was positively related with patients' survival. In this study, we focused on plasma samples from 2 patients who survived sepsis and exhibited high titer antibodies to HMGB1. These antibodies were proved to be specific for HMGB1 since they did not bind to HMGB2 or to human serum albumin. Following IgG purification, it has shown that both patients secreted HMGB1-hydrolyzing autoantibodies in vitro. These findings suggested that proteolytic antibodies directed against HMGB1 can be produced in patients surviving septic shock

Resilience to ocean acidification: decreased carbonic anhydrase activity in sea anemones under high pCO2 conditions

Non-calcifying photosynthetic anthozoans have emerged as a group that may thrive under high carbon dioxide partial pressure ( pCO2) conditions via increased productivity. However, the physiological mechanisms underlying this potential success are unclear. Here we investigated the impact of high pCO2 on the dissolved inorganic carbon (DIC) use in the temperate sea anemone Anemonia viridis. We assessed the impacts of long-term exposure to high pCO2, i.e. sampling in situ natural CO2 vents (Vulcano, Italy), and short-term exposure, i.e. during a 3 wk controlled laboratory experiment. We focused on photo-physiological parameters (net photosynthesis rates, chlorophyll a content and Symbiodinium density) and on carbonic anhydrase (CA) activity, an enzyme involved in the energy-demanding process of DIC absorption. Long-term exposure to high pCO2 had no impact on Symbiodinium density and chlorophyll a content. In contrst, short-term exposure to high pCO2 induced a significant reduction in Symbiodinium density, which together with unchanged net photosynthesis resulted in the increase of Symbiodinium productivity per cell. Finally, in both in situ long-term and laboratory short-term exposure to high pCO2, we observed a significant decrease in the CA activity of sea anemones, suggesting a change in DIC use (i.e. from an HCO3- to a CO2 user). This change could enable a shift in the energy budget that may increase the ability of non-calcifying photosynthetic anthozoans to cope with ocean acidification

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

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

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