Induction of anti-tumor immunity and T-cell responses using nanodelivery systems engrafting TLR-5 ligand

Induction of activated T-cell responses is a prerequisite for the development of vaccine against intracellular infection and for the control of cancer. Particulate nanoscale delivery systems deliver antigens intracellularly and help in inducing T-cell responses. T-cell responses can be further augmented by targeting these particles to dendritic cells, which have the ability to induce both innate and adaptive immune responses. Flagellin, which acts as a TLR-5 ligand, has been extensively explored for its adjuvant activity. The paper under evaluation reports a novel vaccine delivery platform technology for induction of a T-cell response using a nanoscale liposome containing antigen and a small synthetic peptide representing TLR-5-binding motifs of flagellin. Vaccination using this nanodelivery system activated dendritic cells through TLR-5 activation and induced both innate and adaptive immune responses. Such novel delivery systems can improve modern vaccine formulation, particularly for the generation of activated T-cell responses and anti-tumor immunity.     

Evidence of placental translation inhibition and endoplasmic reticulum stress in the etiology of human intrauterine growth restriction

Unexplained intrauterine growth restriction of the fetus (IUGR) results from impaired placental development, frequently associated with maternal malperfusion. Some cases are complicated further by preeclampsia (PE+IUGR). Here, we provide the first evidence that placental protein synthesis inhibition and endoplasmic reticulum (ER) stress play key roles in IUGR pathophysiology. Increased phosphorylation of eukaryotic initiation factor 2alpha suggests suppression of translation initiation in IUGR placentas, with a further increase in PE+IUGR cases. Consequently, AKT levels were reduced at the protein, but not mRNA, level. Additionally, levels of other proteins in the AKT-mammalian target of rapamycin pathway were decreased, and there was associated dephosphorylation of 4E-binding protein 1 and activation of glycogen synthase kinase 3beta. Cyclin D1 and the eukaryotic initiation factor 2B epsilon subunit were also down-regulated, providing additional evidence for this placental phenotype. The central role of AKT signaling in placental growth regulation was confirmed in Akt1 null mice, which display IUGR. In addition, we demonstrated ultrastructural and molecular evidence of ER stress in human IUGR and PE+IUGR placentas, providing a potential mechanism for eukaryotic initiation factor 2alpha phosphorylation. In confirmation, induction of low-grade ER stress in trophoblast-like cell lines reduced cellular proliferation. PE+IUGR placentas showed elevated ER stress with the additional expression of the pro-apoptotic protein C/EBP-homologous protein/growth arrest and DNA damage 153. These findings may account for the increased microparticulate placental debris in the maternal circulation of these cases, leading to endothelial cell activation and impairing placental development.     

PKR-like endoplasmic reticulum kinase (PERK) activation following brain ischemia is independent of unfolded nascent proteins

Transient global brain ischemia results in an immediate inhibition of protein translation upon reperfusion. During early brain reperfusion protein synthesis is inhibited by alpha subunit of eukaryotic initiation factor 2 (eIF2α) phosphorylation by the PKR-like endoplasmic reticulum kinase (PERK). Normally, PERK is held in an inactive, monomeric state by the binding of the endoplasmic reticulum (ER) chaperone GRP78 to the lumenal end of PERK. The prevailing view is that ER stress leads to the accumulation of unfolded proteins in the ER lumen. GRP78 dissociates from PERK to bind these accumulated unfolded proteins, leading to PERK activation, phosphorylation of eIF2α, and inhibition of translation. To determine if an increase in unfolded nascent proteins following transient brain ischemia contributes to PERK activation, protein synthesis was blocked by intracerebral injection of anisomycin prior to induction of ischemia. Anisomycin inhibited protein synthesis by over 99% and reduced newly synthesized proteins in the ER to ∼20% of controls. With an ER nearly devoid of newly synthesized proteins, PERK was still activated and was able to phosphorylate eIF2α in CA1 neurons during reperfusion. These data strongly argue that PERK activation is independent of the large increase in unfolded nascent proteins within the ER following transient global brain ischemia.     

Low-level dietary deoxynivalenol and acute exercise stress result in immunotoxicity in BALB/c mice

We hypothesized that acute exercise stress would exacerbate immunosuppressive effects of sub-acute exposure to dietary deoxynivalenol (DON). Male BALB/c mice were fed 0 or 2 mg DON/kg diet for 14 days, 12 animals per dose, and then exercised to fatigue on a treadmill. Mice were euthanized by decapitation, trunk blood and spleens were collected. Single-cell suspensions of splenocytes were used to quantify immune function by plaque hemolysis and conconavalin-A (ConA) stimulated lymphocyte proliferation assays. Serum corticosterone level was determined by enzyme immunoassay. Only the nonexercised DON-fed mice showed significant splenocyte proliferation suppression, 32.9 +/- 17.9% of nonexercised controls (p = 0.021). Exercised controls and DON-fed exercised animals showed splenocyte proliferation of 68-75% of nonexercised controls. Antibody response to a T-dependent antigen, sheep red blood cells, was significantly less for exercised DON-fed mice than in controls (p = 0.031). Serum corticosterone levels were significantly higher for both exercised groups compared to the unexercised groups (p < 0.001). IL-4 secretion from mitogen-stimulated splenocytes was elevated by DON alone (p < 0.05) while IL-2 was elevated by DON with exercise stress (p < 0.05). Our hypothesis was confirmed with respect to T-lymphocyte-dependent antibody production, but not for splenocyte proliferation. Exercise stress abrogated DON-mediated suppression of splenocyte proliferation, perhaps mediated by induction of elevated stress hormones counteracting cytokine expression alterations of DON.     

Activation of c-Jun N-terminal kinases by ribotoxic stresses

The c-Jun N-terminal kinases （JNKs） are classic stress-activated protein kinases. Many cellular stresses have been shown to stimulate JNK activation. In this review, we focus on ribotoxic stresses based on their multiple biological potencies including anti-HIV-1 activity. Some of the functions of ribotoxins and the signaling transduction pathway that mediated are mentioned. Different from other stimulators, ribotoxic stresses act on special motifs of 28S rRNA in translationally active mammal ribosomes. Binding and damaging on the motif leads to JNK activation and subsequently biological response to the signal initiator, which is named ribotoxic stress response. Cellular ＆ Molecular Immunology. 2005;2（6）：419-425.     

Dengue infection of monocytic cells activates ER stress pathways, but apoptosis is induced through both extrinsic and intrinsic pathways

Monocytic cells are believed to be an important mediator of the pathology of dengue disease in cases of secondary infection where pre-existing antibodies from a first infection facilitate virus entry to Fc receptor bearing cells. In this study we assessed the induction of the ER stress in response to infection of monocytic U937 cells with all four DENV serotypes as well as the induction of apoptosis. Clear evidence of ER stress and the production of pro-apoptotic signals were observed, together with activation of caspase 9. Surprisingly, caspase 8 was also activated, independently of caspase 9, and this was accompanied by an increase in the expression of TNF-α, suggesting the simultaneous but independent activation of both extrinsic and intrinsic apoptosis pathways. Both the induction of ER stress and apoptosis were shown to be serotype independent.     

In vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis C

In hepatocytes, the accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and the unfolded protein response (UPR), mediated by the ER‐resident stress sensors ATF‐6, IRE1, and PERK. UPR‐responsive genes are involved in the fate of ER‐stressed cells. Cells carrying hepatitis C virus (HCV) subgenomic replicons exhibit in vitro ER stress and suggest that HCV inhibits the UPR. Since in vivo ER homeostasis is unknown in livers with chronic HCV infection, we investigated ER stress and the UPR in liver samples from untreated patients with chronic hepatitis C (CHC), in comparison with normal livers. Electron microscopy, western blotting, and real‐time RT‐PCR were used in liver biopsy specimens. Electron microscopy identified features showing ER stress in hepatocyte samples from patients with CHC; however, ‘ER‐stressed’ hepatocytes were found in clusters (3‐5 cells) that were scattered in the liver parenchyma. Western blot analysis confirmed the existence of hepatic ER stress by showing activation of the three ER stress sensors ATF‐6, IRE1, and PERK in CHC. Real‐time RT‐PCR showed no significant induction of UPR‐responsive genes in CHC. In contrast, genes involved in the control of diffuse processes such as liver proliferation, inflammation, and apoptosis were significantly induced in CHC. In conclusion, livers from patients with untreated CHC exhibit in vivo hepatocyte ER stress and activation of the three UPR sensors without apparent induction of UPR‐responsive genes. This lack of gene induction may be explained by the inhibiting action of HCV per se (as suggested by in vitro studies) and/or by our finding of the localized nature of hepatocyte ER stress. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

ER stress and impaired autophagy flux in neuronal degeneration and brain injury

Autophagy is a highly controlled lysosome-mediated function in eukaryotic cells to eliminate damaged or aged long-lived proteins and organelles. It is required for restoring cellular homeostasis in cell survival under multiple stresses. Autophagy is known to be a double-edged sword because too much activation or inhibition of autophagy can disrupt homeostatic degradation of protein and organelles within the brain and play a role in neuronal cell death. Many factors affect autophagy flux function in the brain, including endoplasmic reticulum (ER) stress, oxidative stress, and aging. Newly emerged research indicates that altered autophagy flux functionality is involved in neurodegeneration of the aged brain, chronic neurological diseases, and after traumatic and ischemic brain injuries. In search to identify neuroprotective agents that may reduce oxidative stress and stimulate autophagy, one particular neuroprotective agent docosahexaenoic acid (DHA) presents unique functions in reducing ER and oxidative stress and modulating autophagy. This review will summarize the recent findings on changes of autophagy in aging, neurodegenerative diseases, and brain injury after trauma or ischemic strokes. Discussion of DHA functions is focused on modulating ER stress and autophagy in regard to its neuroprotection and anti-tumor functions.     

Alcohol-induced IL-17A production in Paneth cells amplifies endoplasmic reticulum stress,apoptosis, and inflammasome-IL-18 activation in the proximal small intestine in mice

Gut microbial translocation contributes to alcoholic hepatitis. Using a mouse model of alcoholic hepatitis, we investigated the effects of chronic alcohol plus binge and found increased abundance of Paneth cells and IL-17A in the proximal small intestine (PSI). Alcohol increased IL-17A production and pro-apoptotic signaling evidenced by Bax, Bim, caspase-3, and caspase-8 increases via endoplasmic reticulum (ER) stress indicated by C/EBP homologous protein (CHOP) upregulation; this was prevented by the ER stress inhibitor, 4-PBA, in isolated crypts in vitro and in vivo. Mechanistically, IL-17 augmented alcohol-induced ER stress in isolated crypts. In vivo IL-17A blocking antibody administration in alcohol-treated mice attenuated ER stress-mediated apoptosis and IL-18 induction and prevented alcohol-induced impairment of tight junctions in the PSI and LPS translocation to the liver. Acute-on-chronic alcohol resulted in inflammasome activation, caspase-1 cleavage, and IL-18 production in the PSI. In vivo treatment with antibiotics or 4-PBA prevented CHOP upregulation and inflammasome activation. Our data suggest that alcohol upregulates innate immune mechanisms by increasing Paneth cell numbers and IL-17A release contributing to apoptosis amplification, inflammasome activation, and gut leakiness in the PSI. Binge alcohol-induced Paneth cell expansion, ER stress, and inflammasome activation in the PSI are modulated by the gut microbiome.     

The Unfolded Protein Response in Homeostasis and Modulation of Mammalian Immune Cells

The endoplasmic reticulum (ER) plays important roles in eukaryotic protein folding and lipid biosynthesis. Several exogenous and endogenous cellular sources of stress can perturb ER homeostasis leading to the accumulation of unfolded proteins in the lumen. Unfolded protein accumulation triggers a signal-transduction cascade known as the unfolded protein response (UPR), an adaptive mechanism which aims to protect cells from protein aggregates and to restore ER functions. Further to this protective mechanism, in immune cells, UPR molecular effectors have been shown to participate in a wide range of biological processes such as cell differentiation, survival and immunoglobulin and cytokine production. Recent findings also highlight the involvement of the UPR machinery in the maturational program and antigen presentation capacities of dendritic cells. UPR is therefore a key element in immune system homeostasis with direct implications on both adaptive and innate immune responses. The present review summarizes the knowledge on the emerging roles of UPR signaling cascades in mammalian immune cells as well as the consequences of their dysregulation in relation to the pathogenesis of several diseases.     

Redox events in HTLV-1 Tax-induced apoptotic T-cell death

A number of studies implicate reactive oxygen intermediates in the induction of DNA damage and apoptosis. Recent studies suggest that the human T-cell leukemia virus type 1 (HTLV-1) Tax protein induces oxidative stress and apoptotic T-cell death. Activation of the T-cell receptor/CD3 pathway enhances the Tax-mediated oxidative and apoptotic effects. Tax-mediated apoptosis and oxidative stress as well as activation of nuclear factor-kappaB can be potently suppressed by antioxidants. This review focuses on Tax-dependent changes in the intracellular redox status and their role in Tax-mediated DNA damage and apoptosis. The relevance of these observations to HTLV-1 virus-mediated T-cell transformation and leukemogenesis are discussed.     

Immunological mechanisms contributing to the double burden of diabetes and intracellular bacterial infections

Diabetes has been recognized as an important risk factor for a variety of intracellular bacterial infections, but research into the dysregulated immune mechanisms contributing to the impaired host–pathogen interactions is in its infancy. Diabetes is characterized by a chronic state of low-grade inflammation due to activation of pro-inflammatory mediators and increased formation of advanced glycation end products. Increased oxidative stress also exacerbates the chronic inflammatory processes observed in diabetes. The reduced phagocytic and antibacterial activity of neutrophils and macrophages provides an intracellular niche for the pathogen to replicate. Phagocytic and antibacterial dysfunction may be mediated directly through altered glucose metabolism and oxidative stress. Furthermore, impaired activation of natural killer cells contributes to decreased levels of interferon-γ, required for promoting macrophage antibacterial mechanisms. Together with impaired dendritic cell function, this impedes timely activation of adaptive immune responses. Increased intracellular oxidation of antigen-presenting cells in individuals with diabetes alters the cytokine profile generated and the subsequent balance of T-cell immunity. The establishment of acute intracellular bacterial infections in the diabetic host is associated with impaired T-cell-mediated immune responses. Concomitant to the greater intracellular bacterial burden and potential cumulative effect of chronic inflammatory processes, late hyper-inflammatory cytokine responses are often observed in individuals with diabetes, contributing to systemic pathology. The convergence of intracellular bacterial infections and diabetes poses new challenges for immunologists, providing the impetus for multidisciplinary research.     

Photodynamic antibacterial effect of graphene quantum dots

Synthesis of new antibacterial agents is becoming increasingly important in light of the emerging antibiotic resistance. In the present study we report that electrochemically produced graphene quantum dots (GQD), a new class of carbon nanoparticles, generate reactive oxygen species when photoexcited (470 nm, 1 W), and kill two strains of pathogenic bacteria, methicillin-resistant Staphylococcus aureus and Escherichia coli. Bacterial killing was demonstrated by the reduction in number of bacterial colonies in a standard plate count method, the increase in propidium iodide uptake confirming the cell membrane damage, as well as by morphological defects visualized by atomic force microscopy. The induction of oxidative stress in bacteria exposed to photoexcited GQD was confirmed by staining with a redox-sensitive fluorochrome dihydrorhodamine 123. Neither GQD nor light exposure alone were able to cause oxidative stress and reduce the viability of bacteria. Importantly, mouse spleen cells were markedly less sensitive in the same experimental conditions, thus indicating a fairly selective antibacterial photodynamic action of GQD.     

Inhibition of JNK3 Promotes Apoptosis Induced by BH3 Mimetic S1 in Chemoresistant Human Ovarian Cancer Cells

Previous studies have suggested that the novel BH3 mimetic S1 could induce apoptosis in diverse tumor cell lines through endoplasmic reticulum (ER) stress or mitochondrial cell death pathways. The activation of c‐Jun N‐terminal kinase (JNK) through inositol requiring enzyme‐1 (IRE1) is closely connected to ER stress‐induced apoptosis. However, the role of JNK is complex, as there are different JNK subtypes and the function of each subtype is still not entirely clear. Here we found that the mRNA expression of JNK3 was continuously high in S1‐treated human ovarian cancer SKOV3/DDP cells using a human unfolded protein response (UPR) pathway PCR array. Pharmacological inhibition of JNK3 increased cell sensitivity to apoptosis induced by S1. Furthermore, inhibition of JNK3 induced accumulation of both acidic compartment and p62, and upregulated ROS production. Our results suggest that JNK3 plays a pro‐survival role during ER stress through preventing the block of autophagic flux and reducing oxidative stress in SKOV3/DDP cells. Inhibition of JNK3 may be a potential method to enhance the killing effect of the Bcl‐2 inhibitor S1. Anat Rec, 298:386–395, 2015. © 2014 Wiley Periodicals, Inc.