Liyun Zou

Altered microRNA expression profile with miR-146a upregulation in CD4+ T cells from patients with rheumatoid arthritis

IntroductionIncreasing evidence indicates that microRNAs (miRNAs) play a critical role in the pathogenesis of inflammatory diseases. The aim of the study was to investigate the expression pattern and function of miRNAs in CD4+ T cells from patients with rheumatoid arthritis (RA).MethodsThe expression profile of miRNAs in CD4+ T cells from synovial fluid (SF) and peripheral blood of 33 RA patients was determined by microarray assay and validated by qRT-PCR analysis. The correlation between altered expression of miRNAs and cytokine levels was determined by linear regression analysis. The role of miR-146a overexpression in regulating T cell apoptosis was evaluated by flow cytometry. A genome-wide gene expression analysis was further performed to identify miR-146a-regulated genes in T cells.ResultsmiRNA expression profile analysis revealed that miR-146a expression was significantly upregulated while miR-363 and miR-498 were downregulated in CD4+ T cells of RA patients. The level of miR-146a expression was positively correlated with levels of tumor necrosis factor-alpha (TNF-α), and in vitro studies showed TNF-α upregulated miR-146a expression in T cells. Moreover, miR-146a overexpression was found to suppress Jurkat T cell apoptosis. Finally, transcriptome analysis of miR-146a overexpression in T cells identified Fas associated factor 1 (FAF1) as a miR-146a-regulated gene, which was critically involved in modulating T cell apoptosis.ConclusionsWe have detected increased miR-146a in CD4+ T cells of RA patients and its close correlation with TNF-α levels. Our findings that miR-146a overexpression suppresses T cell apoptosis indicate a role of miR-146a in RA pathogenesis and provide potential novel therapeutic targets.

Oral immunization with rotavirus VP7 expressed in transgenic potatoes induced high titers of mucosal neutralizing IgA.

Rotaviruses (RV) are a common cause of severe diarrhea in young children, resulting in nearly one million deaths worldwide annually. Rotavirus VP7 was the rotavirus neutralizing protein. Previous study reported that VP7 DNA vaccine can induce high levels of IgG in mice but cannot protect mice against challenge (Choi, A.H., Basu, M., Rae, M.N., McNeal, M.M., Ward, R.L., 1998. Virology 250, 230-240). We found that rotavirus VP7 could maintain its neutralizing immunity when it was transformed into the potato genome. Mice immunized with the transformed tubers successfully elicited serum IgG and mucosal IgA specific for VP7. The mucosal IgA titer was as high as 1000, while serum IgG titer was only 600. Neutralizing assays indicated that IgA could neutralize rotavirus. These results indicate the potential usefulness of plants for production and delivery of edible rotavirus vaccines.

Proteomics-based identification of HSP60 as a tumor-associated antigen in colorectal cancer

Patients with cancer frequently develop autoantibodies. The identification of tumor autoantigens may have utility in early cancer diagnosis and immunotherapy. In this study, we used serological proteomics analysis (SERPA) to identify tumor proteins that elicit humoral response in colorectal cancer (CRC). The CRC cell line HCT116 was used as a source of proteins for 2‐DE and subsequent Western blot analysis in which individual serum from patients with CRC was analyzed for autoantibodies. An autoantibody against HSP60 identified by MS was detected in 13 out of 25 patients with CRC and 1 out of 15 healthy subjects. In addition, the HSP60 expressions in tumor tissues collected from 40 patients with CRC were assessed by immunohistochemistry, and serum specimens from 100 patients with cancer and 30 healthy controls were screened for antibody titer to HSP60 by ELISA. The results showed that expressions of HSP60 in tumor tissue and serum antibody titer to HSP60 were significantly higher in patients with CRC than in healthy subjects. Thus, we conclude that the SERPA is an excellent assay for the identification of tumor‐associated antigens and tumor markers. The detection of HSP60 may have clinical utility in CRC screening, diagnosis, and immunotherapy.

Expression of B7-H1 in Inflammatory Renal Tubular Epithelial Cells

Background: Renal tubular epithelial cells (TECs) function as antigen-presenting cells (APCs) as they constitutively express MHC-II molecules and have the capacity to present peptide antigen to T cells. Nevertheless, co-stimulatory signals provided by TECs for regulating T cell activation have not been fully characterized. We therefore investigated the expression of B7-H1, a member of the B7 superfamily, on TECs under normal or pathologic conditions in vivo and analyzed the regulation and functional role of it after proinflammatory factors treatment in vitro. Methods: Immunohistological staining for B7-H1 on cryostat sections of core needle biopsies from patients with different renal diseases was examined. Furthermore, we also detected B7-H1 protein expression on cultured human TECs stimulated by various inflammatory factors and performed TEC/T-cell co-cultured experiment to determine TEC-associated B7-H1 in regulating CD4+ T cell activation as well as antigen presentation. Results: Significant B7-H1 protein was detected in TECs of diseased renal samples. Although the presence of B7-H1 does not have any correlation with clinicopathological variables, marked B7-H1 expression on sections without interstitial inflammation revealed that B7-H1 has some protective function. In vitro, the expression of B7-H1 on TECs was increased after TECs were stimulated with IL-1α, LPS, TNF-α, IFN-γ or anti-CD40. Co-cultured experiments revealed that TEC-related B7-H1 was identified as a strong inhibitor of CD4+ T-cell activation as assessed by increased cytokine production (interleukin-2 and interferon-γ) and expression levels of the T cell activation marker (CD69) in the presence of a neutralizing antibody against B7-H1 (clone MIH1). Interestingly, IL-2 production by C10 T cells after antigen presentation by murine TECs was also enhanced when the B7-H1/PD-1 pathway was interrupted. Conclusion: This study clearly shows that B7-H1 is an inducible renal tubular epithelial antigen that inhibits T cell activation. It is speculated that B7-H1/PD-1 pathway might play a role in protecting tubular epithelium from immune-mediated damage and active delivery of the B7-H1 inhibitory signal represents a novel therapeutic strategy in autoimmune renal diseases.

Forced miR-146a expression causes autoimmune lymphoproliferative syndrome in mice via downregulation of Fas in germinal center B cells

By inhibiting target gene expression, microRNAs (miRNAs) play major roles in various physiological and pathological processes. miR-146a, a miRNA induced upon lipopolysaccharide (LPS) stimulation and virus infection, is also highly expressed in patients with immune disorders such as rheumatoid arthritis, Sjögrens syndrome, and psoriasis. Whether the high level of miR-146a contributes to any of these pathogenesis-related processes remains unknown. To elucidate the function of miR-146a in vivo, we generated a transgenic (TG) mouse line overexpressing miR-146a. Starting at an early age, these TG mice developed spontaneous immune disorders that mimicked human autoimmune lymphoproliferative syndrome (ALPS) with distinct manifestations, including enlarged spleens and lymph nodes, inflammatory infiltration in the livers and lungs, increased levels of double-negative T cells in peripheral blood, and increased serum immunoglobulin G levels. Moreover, with the adoptive transfer approach, we found that the B-cell population was the major etiological factor and that the expression of Fas, a direct target of miR-146a, was significantly dampened in TG germinal center B cells. These results indicate that miR-146a may be involved in the pathogenesis of ALPS by targeting Fas and may therefore serve as a novel therapeutic target.

The GTPase Rab3b/3c-positive recycling vesicles are involved in cross-presentation in dendritic cells

Antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. In this study, we examined the effect of siRNA-mediated knockdown of 57 Rab GTPases, the key regulators of membrane trafficking, on antigen cross-presentation. Twelve Rab GTPases were identified to be associated with antigen cross-presentation, and Rab3b/3c was indicated to be colocalized with MHC class I molecules at perinuclear tubular structure. Tracing with fluorescence protein-tagged β2-microglobulin demonstrated that the MHC class I molecules were internalized from the plasma membrane to Rab3b/3c-positive compartments, which were also colocalized with the internalized transferrin. Moreover, depletion of Rab3b/3c strongly reduced the fast phase recycling rate of transferrin receptors. Furthermore, the Rab3b/3c-positive compartments were colocalized with a fraction of Rab27a at a juxtaposition of phagosomes. Together, these data demonstrate that Rab3b/3c-positive recycling vesicles are involved in and may constitute one of the recycling compartments in exogenous antigen cross-presentation.

Neutrophil extracellular trap formation is associated with autophagy-related signalling in ANCA-associated vasculitis.

Increasing evidence indicates that aberrant neutrophil extracellular trap (NET) formation could contribute to the pathogenesis of anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV). Recent research has provided evidence that a novel type of ANCA autoantibody, anti‐lysosomal membrane protein‐2 (LAMP‐2) antibody, may have a pathogenic role in AAV. We have shown previously that anti‐LAMP‐2 antibody‐stimulated NET formation contains autoantigens and anti‐microbial peptides. The current study sought to determine whether LAMP‐2, as a novel antigen of ANCA, was present on NETs in AAV patients, the influence of the anti‐LAMP‐2 antibody on the neutrophil apoptosis rate and the role of autophagy in anti‐LAMP‐2 antibody‐induced NET formation. NET formation was assessed using immunofluorescence microscopy, scanning electron microscopy or live cell imaging. The neutrophil apoptosis rate was analysed using fluorescence activated cell sorting (FACS). Autophagy was detected using LC3B accumulation and transmission electron microscopy. The results showed that enhanced NET formation, which contains LAMP‐2, was observed in kidney biopsies and neutrophils from AAV patients. The apoptosis rate decreased significantly in human neutrophils stimulated with anti‐LAMP‐2 antibody, and this effect was attenuated by the inhibitors of autophagy 3‐methyladenine (3MA) and 2‐morpholin‐4‐yl‐8‐phenylchromen‐4‐one (LY294002). The anti‐LAMP‐2 antibody‐stimulated NET formation was unaffected by benzyloxycarbonyl‐Val‐ Ala‐Asp (OMe)‐fluoromethylketone (zVAD‐fmk) and necrostatin‐1 (Nec‐1), which are inhibitors of apoptosis and necrosis, respectively, but was inhibited by 3MA and LY294002. Moreover, the proportion of LC3BI that was converted to LC3BII increased significantly (P = 0·0057), and massive vacuolizations that exhibited characteristics typical of autophagy were detected in neutrophils stimulated with anti‐LAMP‐2 antibody. Our results provide further evidence that autophagy is involved in ANCA‐induced NET formation in human neutrophils.

Cross-presentation of phage particle antigen in MHC class II and endoplasmic reticulum marker-positive compartments.

It has been shown that exogenous antigens can access the MHC class I pathway of professional antigen‐processing cells. However, details as to how the MHC class I‐peptide complex forms in the presentation pathway are still poorly understood. Here we used MHC class I‐peptide‐specific antibodies to investigate the formation and intracellular location of class I‐peptide complexes in macrophages. We observed that the formation of class I‐peptide complexes occurs within a few hours and lasts for another few hours on the cell surface of macrophages following loading with filamentous phage particles. The class I‐peptide complexes in the process were co‐localized with MHC class II molecules and endocytic system markers. Moreover, endosomal compartments containing class I‐peptide complexes were found within intracellular organelles stained by DiOC6 and calnexin. In addition, the cross‐presentation of phage particles was transporter associated with antigen processing (TAP)‐dependent and sensitive to proteasome inhibitors and NH4Cl. These data suggest that endocytosed phage particles may be processed and cross‐presented in organelles positive for phagosome and endoplasmic reticulum (ER) markers via a classical ER MHC class I loading mechanism.

Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from CML28

Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. CML28, a recently discovered cancer-testis (CT) antigen from chronic myelogenous leukemia, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A*0201 is one of the most common histocompatibility molecule in Chinese, we aim at identifying CML28 peptides presented by HLA-A*0201. A panel of CML28-derived antigenic peptides was predicted using a computer-based program. Four peptides with highest predicted score were synthesized and tested for their binding affinities to HLA-A*0201 molecule. Then these peptides were assessed for their immunogenicity to elicit specific immune responses mediated by CTLs both in vitro, from PBMCs sourced from four healthy HLA-A*0201+ donors, and in vivo, in HLA-A*0201 transgenic mice. One of the tested peptides, CML28(173–181), induced peptide-specific CTLs in vitro as well as in vivo, which could specifically secrete IFN-γ and lyse major histocompatibility complex (MHC)-matched tumor cell lines endogenously expressing CML28 antigen and CML28(173–181) pulsed Jurkat-A2/Kb cells, respectively. These results demonstrate that CML28(173–181) is a naturally processed and presented CTL epitope with HLA-A*0201 motif and has a promising immunogenicity both in vitro and in vivo. As CML28 is expressed in a large variety of histological tumors besides chronic myelogenous leukemia, we propose that the newly identified epitope, CML28(173–181), would be of potential use in peptide-based, cancer-specific immunotherapy against a broad spectrum of tumors.

Mimovirus: a Novel Form of Vaccine That Induces Hepatitis B Virus-Specific Cytotoxic T-Lymphocyte Responses In Vivo

ABSTRACT CD8+ cytotoxic T lymphocytes (CTLs) are now recognized as important mediators of immunity against intracellular pathogens, including human immunodeficiency virus and tumors. How to efficiently evoke antigen-specific CTL responses in vivo has become a crucial problem in the development of modern vaccines. Here, we developed a completely novel CTL vaccine—mimovirus, which is a kind of virus-size particulate antigen delivery system. It was formed by the self-assembly of a cationic peptide containing 18 lysines and a CTL-epitope peptide of HBsAg28-39, with a plasmid encoding mouse interleukin-12 (IL-12) through electrostatic interactions. We examined the formation of mimovirus by DNA retardation assay, DNase I protection assay, and transmission electron microscopy and demonstrated that mimovirus could efficiently transfer the plasmid encoding IL-12 into mammalian cells such as P815 cells in vitro. Furthermore, it was proved that mimovirus could induce an HBsAg28-39-specific CTL response in vivo. Considering its effectiveness, flexibility, and defined composition, mimovirus is potentially a novel system for vaccination against intracellular pathogens and tumors.