HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response

Background

The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when protein folding capacity is exceeded. This stress induces a cyto-protective signaling cascades termed the unfolded protein response (UPR) aimed at restoring homeostasis. While acute ER stress is lethal, chronic sub-lethal ER stress causes cells to adapt by attenuation of UPR activation. Hepatitis C virus (HCV), a major human pathogen, was shown to cause ER stress, however it is unclear whether HCV induces chronic ER stress, and if so whether adaptation mechanisms are initiated. We wanted to characterize the kinetics of HCV-induced ER stress during infection and assess adaptation mechanisms and their significance.

Methods and Findings

The HuH7.5.1 cellular system and HCV-transgenic (HCV-Tg) mice were used to characterize HCV-induced ER stress/UPR pathway activation and adaptation. HCV induced a wave of acute ER stress peaking 2–5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including IRE1 and EIF2α phosphorylation, ATF6 cleavage and XBP-1 splicing. Downstream target genes including GADD34, ERdj4, p58ipk, ATF3 and ATF4 were upregulated. CHOP, a UPR regulated protein was activated and translocated to the nucleus. Remarkably, UPR activity did not return to baseline but remained elevated for up to 14 days post infection suggesting that chronic ER stress is induced. At this time, cells adapted to ER stress and were less responsive to further drug-induced ER stress. Similar results were obtained in HCV-Tg mice. Suppression of HCV by Interferon-α 2a treatment, restored UPR responsiveness to ER stress tolerant cells.

Conclusions

Our study shows, for the first time, that HCV induces adaptation to chronic ER stress which was reversed upon viral suppression. These finding represent a novel viral mechanism to manipulate cellular response pathways.     

A new kind of carbohydrate array,its use for profiling antiglycan antibodies,and the discovery of a novel human cellulose-binding antibody

In this study, we use a novel glycan array to analyze the glycan-binding antibody repertoire in a pool of affinity-purified IgG collected from a healthy human population. The glycan array used is based on mono- and oligosaccharides covalently linked to the surface via a long linker at their reducing ends. They are thus presented to the medium with a well-defined orientation and are accessible for specific binding by glycan-binding proteins, such as antibodies and lectins. A novel anticellulose antibody was detected that binds specifically to beta4-linked saccharides with a preference for glucopyranose over galactopyranose residues. We also found previously known antiglycan antibodies against mono- and oligosaccharides that are constituents of commonly occurring bacterial polysaccharides. We propose that this array can facilitate high-throughput screening of glycan-binding proteins and the search for biomarkers for personalized medicine.     

NADH Oxidase Functions as an Adhesin in Streptococcus pneumoniae and Elicits a Protective Immune Response in Mice

The initial event in disease caused by S. pneumoniae is adhesion of the bacterium to respiratory epithelial cells, mediated by surface expressed molecules including cell-wall proteins. NADH oxidase (NOX), which reduces free oxygen to water in the cytoplasm, was identified in a non-lectin enriched pneumococcal cell-wall fraction. Recombinant NOX (rNOX) was screened with sera obtained longitudinally from children and demonstrated age-dependent immunogenicity. NOX ablation in S. pneumoniae significantly reduced bacterial adhesion to A549 epithelial cells in vitro and their virulence in the intranasal or intraperitoneal challenge models in mice, compared to the parental strain. Supplementation of Δnox WU2 with the nox gene restored its virulence. Saturation of A549 target cells with rNOX or neutralization of cell-wall residing NOX using anti-rNOX antiserum decreased adhesion to A549 cells. rNOX-binding phages inhibited bacterial adhesion. Moreover, peptides derived from the human proteins contactin 4, chondroitin 4 sulfotraferase and laminin5, homologous to the insert peptides in the neutralizing phages, inhibited bacterial adhesion to the A549 cells. Furthermore, rNOX immunization of mice elicited a protective immune response to intranasal or intraperitoneal S. pneumoniae challenge, whereas pneumococcal virulence was neutralized by anti-rNOX antiserum prior to intraperitoneal challenge. Our results suggest that in addition to its enzymatic activity, NOX contributes to S. pneumoniae virulence as a putative adhesin and thus peptides derived from its target molecules may be considered for the treatment of pneumococcal infections. Finally, rNOX elicited a protective immune response in both aerobic and anaerobic environments, which renders NOX a candidate for future pneumococcal vaccine.     

A monoclonal antibody to human B lymphoblastoid cells activates human and murine T lymphocytes

A B lymphoblastoid cell line can provide a comitogenic, accessory signal for mitogen-treated T cells. In a study evaluating the antigenic determinant of such cells that mediate this effect, a monoclonal antibody (I57) was raised against the Daudi cell line. This antibody was found to interact with a 30-kDa protein on these cells and had agonistic properties. It enhanced the B lymphoblastoid accessory cell and interleukin 1 (IL-1)-dependent stimulation of PHA-treated murine thymocytes. The stimulatory effect of I57 on PHA-treated thymocytes was more pronounced at high, supraoptimal concentrations of the lectin. This was in contrast with the effect of IL-1 that failed to stimulate these cells treated with PHA at high concentrations. I57 also enhanced stimulation of thymocytes treated with IL-2 alone or with both PHA and IL-2. I57 exhibited by itself mitogenic activity for human T cells. These cells, treated with IL-2, were further stimulated by I57. I57 seems to be different from other agonistic antibodies that have been described so far.     

LifeMap Discovery?: The Embryonic Development,Stem Cells,and Regenerative Medicine Research Portal

LifeMap Discovery™ provides investigators with an integrated database of embryonic development, stem cell biology and regenerative medicine. The hand-curated reconstruction of cell ontology with stem cell biology; including molecular, cellular, anatomical and disease-related information, provides efficient and easy-to-use, searchable research tools. The database collates in vivo and in vitro gene expression and guides translation from in vitro data to the clinical utility, and thus can be utilized as a powerful tool for research and discovery in stem cell biology, developmental biology, disease mechanisms and therapeutic discovery. LifeMap Discovery is freely available to academic nonprofit institutions at http://discovery.lifemapsc.com     

Migration cues induce chromatin alterations

Directed cell migration is a property central to multiple basic biological processes. Here, we show that directed cell migration is associated with global changes in the chromatin fiber. Polarized posttranslational changes in histone H1 along with a transient decrease in H1 mobility were detected in cells facing the scratch in a wound healing assay. In parallel to the changes in H1, the levels of the heterochromatin marker histone H3 lysine 9 tri-methylation were elevated. Interestingly, reduction of the chromatin-binding affinity of H1 altered the cell migration rates. Moreover, migration-associated changes in histone H1 were observed during nuclear motility in the simple multicellular organism Neurospora crassa . Our studies suggest that dynamic reorganization of the chromatin fiber is an early event in the cellular response to migration cues.     

Defects in CD8+ regulatory T cells in the lamina propria of patients with inflammatory bowel disease

Mucosal tolerance is believed to be partly mediated by regulatory T cells. Intestinal epithelial cells (IECs) may play an important role in the generation of such regulatory cells, because they are able to process and present Ag to T cells. Furthermore, we have previously demonstrated that IECs are able to generate regulatory CD8(+) T cells in vitro. In the present study, we have analyzed lamina propria (LP) lymphocytes for the presence of such regulatory CD8(+) T cells in normal individuals as well as in patients with inflammatory bowel disease (IBD). The results of the present study show that LP CD8(+) T cells derived from normal controls possess regulatory activity, whereas both unfractionated LP lymphocytes and purified LP CD4(+) T cells do not. The LP CD8(+) T cells suppress Ig production by pokeweed mitogen-stimulated PBMCs by 31-80%, in a cell contact-dependent manner. No significant difference in suppression between CD28(+) and CD28(-)CD8(+) LP T cells was observed. In contrast to CD8(+) T cells from normal LP, CD8(+) T cells isolated from LP of IBD patients, did not suppress Ig production by pokeweed mitogen-stimulated PBMC (five of six ulcerative colitis specimens; six of six Crohn's disease specimens). Furthermore, we demonstrate that the frequency of TCR Vbeta5.1-positive CD8(+) T cells, which we previously have demonstrated to be regulatory and to be expanded by IECs in vitro, is decreased in IBD LP compared with normal LP. In conclusion, this study demonstrates that CD8(+) T cells with regulatory activity are present in the LP of normal healthy individuals, but not in patients with IBD, suggesting that these cells might play an active role in mucosal tolerance.