Apolipoprotein H drives hepatitis B surface antigen retention and endoplasmic reticulum stress during hepatitis B virus infection.

BACKGROUND: Apolipoprotein H (APOH), also known as beta2-glycoprotein I (beta2-GPI), is an acute phase protein in hepatitis B virus (HBV) infection and binds to hepatitis B surface antigen (HBsAg) with high-affinity. APOH expression is upregulated by HBV and the large surface protein (LHBs), but also elevated in HBV-related hepatoma cells. Previous studies show that intracellular retention of HBsAg induces endoplasmic reticulum (ER) stress, a key driver of hepatocyte damage during chronic liver injury, but the mechanisms are unclear. We hypothesize that APOH mediates HBV-induced ER stress through increased retention of HBsAg. METHODS: VR-APOH-myc and VR-LHBs-flag plasmids were constructed by PCR using pcDNA3.1(-)-APOH or an HBV expression vector, respectively. APOH and ER stress markers were examined at protein and mRNA levels by Western Blot or RT-qPCR. HBsAg titer was assayed by ELISA. RNA-seq was performed to elucidate the transcriptional impact of APOH manipulation in HBV-producing cells (HepG2.2.15 cells). RESULTS: We found that HBV upregulates APOH expression in 293 T cells, and APOH overexpression subsequently inhibits secretion of HBsAg. Next, we show that LHBs overexpression in conjunction with APOH leads to ER stress in 293 T cells, as evidenced by production of the binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), as well as increased splicing of X-box binding protein 1 (XBP1). We further observed that loss of beta2-GPI reduced CHOP expression in HepG2.2.15 cells, while beta2-GPI overexpression enhanced CHOP production. CONCLUSION: The interaction of beta2-GPI and HBV initiates ER stress through driving intracellular retention of HBsAg and activates the UPR.

ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression.

Understanding the intrinsic mediators that render CD8+ T cells dysfunctional in the tumor microenvironment is a requirement to develop more effective cancer immunotherapies. Here, we report that C/EBP homologous protein (Chop), a downstream sensor of severe endoplasmic reticulum (ER) stress, is a major negative regulator of the effector function of tumor-reactive CD8+ T cells. Chop expression is increased in tumor-infiltrating CD8+ T cells, which correlates with poor clinical outcome in ovarian cancer patients. Deletion of Chop in T cells improves spontaneous antitumor CD8+ T cell immunity and boosts the efficacy of T cell-based immunotherapy. Mechanistically, Chop in CD8+ T cells is elevated primarily through the ER stress-associated kinase Perk and a subsequent induction of Atf4; and directly represses the expression of T-bet, a master regulator of effector T cell function. These findings demonstrate the primary role of Chop in tumor-induced CD8+ T cell dysfunction and the therapeutic potential of blocking Chop or ER stress to unleash T cell-mediated antitumor immunity.

Tissue factor pathway inhibitor attenuates ER stress-induced inflammation in human M2-polarized macrophages.

Endoplasmic reticulum (ER) stress has been shown to play a key role during the initiation and clinical progression of the cardiovascular diseases, such as atherosclerosis. We have recently shown that expression of tissue factor pathway inhibitor (TFPI) in human monocyte-derived macrophages (MDMs) was induced by cholesterol crystals (CC). In the present study we aimed to determine the role of TFPI under ER stress conditions using human MDMs. qRT-PCR and immunohistochemistry analysis were performed to determine the presence of the ER stress marker CCAAT/enhancer binding protein homologous protein (CHOP) and TFPI in human carotid plaque material and also in human MDMs polarized into pro-inflammatory M1 or anti-inflammatory M2 populations. CHOP mRNA levels were upregulated in the plaques compared to healthy vessels, and CHOP protein was localized in the same area as TFPI in the plaques. Both CHOP and TFPI mRNA levels were upregulated after CC treatment, especially in the M2 phenotype, and the ER stress inhibitor 4-phenylbutyric acid (PBA) reversed this effect. Furthermore, CC treatment increased the levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-8, which for TNF-α and IL-8 was inhibited by PBA, and reduced the levels of the anti-inflammatory cytokine IL-10 in M2-polarized macrophages. Knockdown of TFPI prior to CC treatment exacerbated TNF-α and IL-6 levels, but reduced IL-8 and IL-10 levels. Our results show that CC induce TFPI and cytokine expression in M2-polarized macrophages through activation of the ER stress pathway and that TFPI has a protective effect against TNF-α and IL-6 mediated inflammation. These mechanisms may have implications for the pathogenesis of atherosclerosis.

ERAP2 functional knockout in humans does not alter surface heavy chains or HLA-B27, inflammatory cytokines or endoplasmic reticulum stress markers.

INTRODUCTION: Single nucleotide polymorphisms in ERAP2 are strongly associated with ankylosing spondylitis (AS). One AS-associated single nucleotide polymorphism, rs2248374, causes a truncated ERAP2 protein that is degraded by nonsense-mediated decay. Approximately 25% of the populations of European ancestry are therefore natural ERAP2 knockouts. We investigated the effect of this associated variant on HLA class I allele presentation, surface heavy chains, endoplasmic reticulum (ER) stress markers and cytokine gene transcription in AS. METHODS: Patients with AS and healthy controls with either AA or GG homozygous status for rs2248374 were studied. Antibodies to CD14, CD19-ECD, HLA-A-B-C, Valpha7.2, CD161, anti-HC10 and anti-HLA-B27 were used to analyse peripheral blood mononuclear cells. Expression levels of ER stress markers (GRP78 and CHOP) and proinflammatory genes (tumour necrosis factor (TNF), IL6, IL17 and IL22) were assessed by qPCR. RESULTS: There was no significant difference in HLA-class I allele presentation or major histocompatibility class I heavy chains or ER stress markers GRP78 and CHOP or proinflammatory gene expression between genotypes for rs2248374 either between cases, between cases and controls, and between controls. DISCUSSION: Large differences were not seen in HLA-B27 expression or cytokine levels between subjects with and without ERAP2 in AS cases and controls. This suggests that ERAP2 is more likely to influence AS risk through other mechanisms.

The immunosuppressive activity of MDSCs requires expression of CHOP.

The stress sensor CHOP enhances the accumulation and inhibitory activity of MDSCs in tumors.

Differential effects of Escherichia coli subtilase cytotoxin and Shiga toxin 2 on chemokine and proinflammatory cytokine expression in human macrophage, colonic epithelial, and brain microvascular endothelial cell lines.

Subtilase cytotoxin (SubAB) is the prototype of a recently emerged family of AB5 cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). Its mechanism of action involves highly specific A-subunit-mediated proteolytic cleavage of the essential endoplasmic reticulum (ER) chaperone BiP. Our previous in vivo studies showed that intraperitoneal injection of purified SubAB causes a major redistribution of leukocytes and elevated leukocyte apoptosis in mice, as well as profound splenic atrophy. In the current study, we investigated selected chemokine and proinflammatory cytokine responses to treatment with SubAB, a nontoxic derivative (SubAA272B), or Shiga toxin 2 (Stx2) in human macrophage (U937), brain microvascular endothelial (HBMEC), and colonic epithelial (HCT-8) cell lines, at the levels of secreted protein, cell-associated protein, and gene expression. Stx2 treatment upregulated expression of chemokines and cytokines at both the protein and mRNA levels. In contrast, SubAB induced significant decreases in secreted interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in all three tested cell lines and a significant decrease in secreted IL-6 in HBMECs. The downregulation of secreted chemokines or cytokines was not observed in SubAA272B-treated cells, indicating a requirement for BiP cleavage. The downregulation of secreted chemokines and cytokines by SubAB was not reflected at the mRNA and cell-associated protein levels, suggesting a SubAB-induced export defect.

Cutting edge: Endoplasmic reticulum stress licenses macrophages to produce mature IL-1ß in response to TLR4 stimulation through a caspase-8- and TRIF-dependent pathway.

The accumulation of improperly folded proteins within the endoplasmic reticulum (ER) generates perturbations known as ER stress that engage the unfolded protein response. ER stress is involved in many inflammatory pathologies that are also associated with the production of the proinflammatory cytokine IL-1ß. In this study, we demonstrate that macrophages undergoing ER stress are able to drive the production and processing of pro-IL-1ß in response to LPS stimulation in vitro. Interestingly, the classical NLRP3 inflammasome is dispensable, because maturation of pro-IL-1ß occurs normally in the absence of the adaptor protein ASC. In contrast, processing of pro-IL-1ß is fully dependent on caspase-8. Intriguingly, we found that neither the unfolded protein response transcription factors XBP1 and CHOP nor the TLR4 adaptor molecule MyD88 is necessary for caspase-8 activation. Instead, both caspase activation and IL-1ß production require the alternative TLR4 adaptor TRIF. This pathway may contribute to IL-1-driven tissue pathology in certain disease settings.

Differential responses of hepatic endoplasmic reticulum stress and inflammation in diet-induced obese rats with high-fat diet rich in lard oil or soybean oil.

SCOPES: To investigate the effects of high-fat diet enriched with lard oil or soybean oil on liver endoplasmic reticulum (ER) stress and inflammation markers in diet-induced obese (DIO) rats and estimate the influence of following low-fat diet feeding. METHODS AND RESULTS: Male SD rats were fed with standard low-fat diet (LF, n = 10) and two isoenergentic high-fat diets enriched with lard (HL, n = 45) or soybean oil (HS, n = 45) respectively for 10 weeks. Then DIO rats from HL and HS were fed either high-fat diet continuously (HL/HL, HS/HS) or switched to low-fat diet (HL/LF, HS/LF) for another 8 weeks. Rats in control group were maintained with low-fat diet. Body fat, serum insulin level, HOMA-IR and ectopic lipid deposition in liver were increased in HL/HL and HS/HS compared to control, but increased to a greater extent in HL/HL compared to HS/HS. Markers of ER stress including PERK and CHOP protein expression and phosphorylation of eIF2α were significantly elevated in HL/HL group while phosphorylation of IRE1α and GRP78 protein expression were suppressed in both HL/HL and HS/HS. Besides, inflammatory signals (OPN, TLR2, TLR4 and TNF-α) expressions significantly increased in HL/HL compared to others. Switching to low-fat diet reduced liver fat deposition, HOMA-IR, mRNA expression of TLR4, TNF-α, PERK in both HL/LF and HS/LF, but only decreased protein expression of OPN, PERK and CHOP in HL/LF group. In addition, HL/LF and HS/LF exhibited decreased phosphorylation of eIF2α and increased phosphorylation of IRE1α and GRP78 protein expression when compared with HL/HL and HS/HS respectively. CONCLUSIONS: Lard oil was more deleterious in insulin resistance and hepatic steatosis via promoting ER stress and inflammation responses in DIO rats, which may be attributed to the enrichment of saturated fatty acid. Low-fat diet was confirmed to be useful in recovering from impaired insulin sensitivity and liver fat deposition in this study.

Repression of peroxisome proliferator-activated receptor gamma by mucosal ribotoxic insult-activated CCAAT/enhancer-binding protein homologous protein.

CCAAT/enhancer-binding protein homologous protein (CHOP) is a crucial stress-responsive factor in various mucosal injuries, including cellular translational stress conditions. In this study, chemical ribosome-inactivating stresses were assessed for their effects on stress-inducible CHOP expression and its association with epithelial inflammatory cytokine production. Several representative ribotoxic agents (deoxynivalenol, anisomycin, and 15-acetyldeoxynivalenol) enhanced CHOP expression and its nuclear translocation in human intestinal epithelial cells. Moreover, CHOP was a strong positive regulator of IL-8 production, but CHOP-mediated IL-8 production was inversely associated with expression of the mucosal regulatory factor peroxisome proliferator-activated receptor γ (PPARγ). Based on our recent report that PPARγ is a negative regulator of mRNA stability of IL-8, PPARγ was linked to a notable mRNA stabilizing protein, HuR, since ribotoxin-induced IL-8 mRNA is stabilized by HuR protein. Expression of exogenous PPARγ suppressed ribotoxin-triggered cytoplasmic translocation of HuR. In contrast, PPARγ-regulating CHOP was a positive modulator of HuR protein export from nuclei. Taken together, the results indicate that ribotoxin-induced CHOP protein is positively associated with production of proinflammatory cytokine IL-8, but it downregulates PPARγ action, subsequently allowing the cytosolic translocation of HuR protein and stabilization of IL-8 mRNA in gut epithelial cells. CHOP and PPARγ may represent critical mechanistic links between ribotoxic stress and proinflammatory cytokine production, and they may have a broader functional significance with regard to gastrointestinal stresses by toxic mucosal insults.

C/EBPzeta (CHOP/Gadd153) is a negative regulator of LPS-induced IL-6 expression in B cells.

C/EBPzeta was originally identified as a gene induced upon DNA damage and growth arrest. It has been shown to be involved in the cellular response to endoplasmic reticulum stress. Because of sequence divergence from other C/EBP family members in its DNA-binding domain and its consequent inability to bind the C/EBP consensus-binding motif, C/EBPzeta can act as a dominant negative inhibitor of other C/EBPs. C/EBP transactivators are essential to the expression of many proinflammatory cytokines and acute phase proteins, but a role for C/EBPzeta in regulating their expression has not been described. We found that expression of C/EBPzeta is induced in response to LPS treatment of B cells at both the mRNA and protein levels. Correlating with the highest levels of C/EBPzeta expression at 48 h after LPS treatment, there is an increased association of C/EBPzeta with C/EBPbeta, and both the abundance of C/EBP DNA-binding species and IL-6 expression are downregulated. Furthermore, ectopic expression of C/EBPzeta inhibited C/EBPbeta-dependent IL-6 expression from both the endogenous IL-6 gene and an IL-6 promoter-reporter. These results suggest that C/EBPzeta functions as negative regulator of IL-6 expression in B cells and that it contributes to the transitory expression of IL-6 that is observed after LPS treatment.

Many commercially available antibodies for detection of CHOP expression as a marker of endoplasmic reticulum stress fail specificity evaluation.

Endoplasmic reticulum (ER) stress contributes to beta cell death in type 2 diabetes (T2DM). ER stress is characterized by increased level of ER stress markers such as C/EBP homologous protein (CHOP). Activation of CHOP leads to its translocation into the nucleus, where it induces cell death. We previously reported nuclear CHOP in pancreatic sections from T2DM, but not T1DM, and in human islet amyloid polypeptide (IAPP) transgenic rodent pancreatic sections. These studies underscore the importance of studying nuclear CHOP. We have observed inconsistency in the detection of CHOP antibodies reported in the literature and also in our own experiments. To investigate the specificity of CHOP antibodies, we first induced ER stress by tunicamycin in rat insulinoma (INS) cells and prepared nuclear and cytoplasmic fractions. Then we examined CHOP expression by Western blotting and immunocytochemistry using seven commercially available CHOP antibodies in INS cells and human IAPP (h-IAPP) transgenic rodent pancreatic tissue. These studies show that three commercially available CHOP antibodies out of seven tested were non-specific. In conclusion, we give recommendations for CHOP antibody selection and methods to verify CHOP antibody specificity. Also, we propose that the authors report the catalog and lot numbers of the CHOP antibodies used.

Diagnosis of suspicious thyroid nodules using four protein biomarkers.

PURPOSE: Fine-needle aspiration (FNA) cytology, a standard method for thyroid nodule diagnosis, cannot distinguish between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC). Previously, using expression profiling, we found that a combination of transcript expression levels from DDIT3, ARG2, C1orf24, and ITM1 distinguished between FTA and FTC. The goal of this study was to determine if antibody markers used alone or in combination could accurately distinguish between a wider variety of benign and malignant thyroid lesions in fixed sections and FNA samples. EXPERIMENTAL DESIGN: Immunohistochemistry was done on 27 FTA, 25 FTC, and 75 other benign and malignant thyroid tissue sections using custom antibodies for chromosome 1 open reading frame 24 (C1orf24) and integral membrane protein 1 (ITM1) and commercial antibodies for DNA damage-inducible transcript 3 (DDIT3) and arginase II (ARG2). FNA samples were also tested using the same antibodies. RNA expression was measured by quantitative PCR in 33 thyroid lesions. RESULTS: C1orf24 and ITM1 antibodies had an estimated sensitivity of 1.00 for distinguishing FTA from FTC. For the expanded analysis of all lesions studied, ITM1 had an estimated sensitivity of 1.00 for detecting malignancy. Because all four cancer biomarkers did well, producing overlapping confidence intervals, not one best marker was distinguished. Transcript levels also reliably predicted malignancy, but immunohistochemistry had a higher sensitivity. Malignant cells were easily detected in FNA samples using these markers. CONCLUSIONS: We improved this diagnostic test by adding C1orf24 and ITM1 custom antibodies and showing use on a wider variety of thyroid pathology. We recommend that testing of all four cancer biomarkers now be advanced to larger trials. Use of one or more of these antibodies should improve diagnostic accuracy of suspicious thyroid nodules from both tissue sections and FNA samples.

Generation of the novel monoclonal antibody against TLS/EWS-CHOP chimeric oncoproteins that is applicable to one of the most sensitive assays for myxoid and round cell liposarcomas.

The fusion oncoproteins, TLS-CHOP and EWS-CHOP, are characteristic markers for myxoid and round cell liposarcomas (MLS/RCLS). Especially, the peptide sequence of 26 amino acids corresponding to the normally untranslated CHOP exon 2 and parts of exon 3 (5'-UTR) is a unique structure for these chimeric proteins. In this report, we have generated monoclonal antibodies against the unique peptide sequence of TLS/EWS-CHOP oncoproteins. These antibodies reacted with TLS-CHOP fusion protein, but not reacted with normal TLS and CHOP proteins by Western blot analysis. In addition, one of the antibodies also recognized the chimeric oncoprotein in archival paraffin-embedded tissue samples of MLS/RCLS. The oncoprotein was detectable by the antibody even in the paraffin-embedded tissue samples whose mRNAs were too degraded to be detected by a nested reverse transcription-polymerase chain reaction-based assay. Thus, the molecular assay using the novel antibody is expected to be one of the most sensitive diagnostic assays for MLS/RCLS.