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1.  Sodium Butyrate Promotes the Differentiation of Rat Bone Marrow Mesenchymal Stem Cells to Smooth Muscle Cells through Histone Acetylation 
PLoS ONE  2014;9(12):e116183.
Establishing an effective method to improve stem cell differentiation is crucial in stem cell transplantation. Here we aimed to explore whether and how sodium butyrate (NaB) induces rat bone marrow mesenchymal stem cells (MSCs) to differentiate into bladder smooth muscle cells (SMCs). We found that NaB significantly suppressed MSC proliferation and promoted MSCs differentiation into SMCs, as evidenced by the enhanced expression of SMC specific genes in the MSCs. Co-culturing the MSCs with SMCs in a transwell system promoted the differentiation of MSCs into SMCs. NaB again promoted MSC differentiation in this system. Furthermore, NaB enhanced the acetylation of SMC gene-associated H3K9 and H4, and decreased the expression of HDAC2 and down-regulated the recruitment of HDAC2 to the promoter regions of SMC specific genes. Finally, we found that NaB significantly promoted MSC depolarization and increased the intracellular calcium level of MSCs upon carbachol stimulation. These results demonstrated that NaB effectively promotes MSC differentiation into SMCs, possibly by the marked inhibition of HDAC2 expression and disassociation of HDAC2 recruitment to SMC specific genes in MSCs, which further induces high levels of H3K9ace and H4ace and the enhanced expression of target genes, and this strategy could potentially be applied in clinical tissue engineering and cell transplantation.
PMCID: PMC4280132  PMID: 25548915
2.  Transgenic expression of survivin compensates for OX40-deficiency in driving Th2 development and allergic inflammation 
European journal of immunology  2013;43(7):10.1002/eji.201243081.
Survivin, an inhibitor of apoptosis family molecule, has been proposed as a crucial intermediate in the signaling pathways leading to T-cell development, proliferation, and expansion. However, the importance of survivin to T-cell-driven inflammatory responses has not been demonstrated. Here, we show that survivin transgenic mice exhibit an increased antigen-driven Th2 lung inflammation and that constitutive expression of survivin reversed the defective lung inflammation even in the absence of OX40 costimulation. We found that OX40-deficient mice were compromised in generating Th2 cells, airway eosinophilia, and IgE responses. In contrast, OX40-deficient/survivin transgenic mice generated normal Th2 responses and exhibited strong lung inflammation. These results suggested that OX40 costimulation crucially engaged survivin during antigen-mediated Th2 responses. These findings also promote the notion that OX40 costimulation regulated allergic responses or lung inflammation by targeting survivin thereby enhancing T-cell proliferation and resulting in more differentiated Th2 cells in the allergic inflammatory response.
PMCID: PMC3876962  PMID: 23616302
costimulation; lung inflammation; murine model; survivin; Th2 cells
3.  Erythropoietin-Derived Nonerythropoietic Peptide Ameliorates Experimental Autoimmune Neuritis by Inflammation Suppression and Tissue Protection 
PLoS ONE  2014;9(3):e90942.
Experimental autoimmune neuritis (EAN) is an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system. Erythropoietin (EPO) has been known to promote EAN recovery but its haematopoiesis stimulating effects may limit its clinic application. Here we investigated the effects and potential mechanisms of an EPO-derived nonerythropoietic peptide, ARA 290, in EAN. Exogenous ARA 290 intervention greatly improved EAN recovery, improved nerve regeneration and remyelination, and suppressed nerve inflammation. Furthermore, haematopoiesis was not induced by ARA 290 during EAN treatment. ARA 290 intervention suppressed lymphocyte proliferation and altered helper T cell differentiation by inducing increase of Foxp3+/CD4+ regulatory T cells and IL-4+/CD4+ Th2 cells and decrease of IFN-γ+/CD4+ Th1 cells in EAN. In addition, ARA 290 inhibited inflammatory macrophage activation and promoted its phagocytic activity. In vitro, ARA 290 was shown to promote Schwann cell proliferation and inhibit its inflammatory activation. In summary, our data demonstrated that ARA 290 could effectively suppress EAN by attenuating inflammation and exerting direct cell protection, indicating that ARA 290 could be a potent candidate for treatment of autoimmune neuropathies.
PMCID: PMC3946253  PMID: 24603865
4.  Hepatitis B Virus X Protein-Induced Aberrant Epigenetic Modifications Contributing to Human Hepatocellular Carcinoma Pathogenesis 
Molecular and Cellular Biology  2013;33(15):2810-2816.
Hepatocellular carcinoma (HCC) remains one of the most prevalent malignant diseases worldwide, and the majority of cases are related to hepatitis B virus (HBV) infection. Interactions between the HBV-encoded X (HBx) protein and host factors are known to play major roles in the onset and progression of HBV-related HCC. These dynamic molecular mechanisms are extremely complex and lead to prominent changes in the host genetic and epigenetic architecture. This review summarizes the current knowledge about HBx-induced epigenetic changes, including aberrations in DNA methylation, histone modifications, and microRNA expression, and their roles in HBV-infected liver cells and HBV-related HCC. Moreover, the HBx-mediated epigenetic control of HBV covalently closed circular DNA (cccDNA) is also discussed. Although this field of study is relatively new, the accumulated evidence has indicated that the epigenetic events induced by HBx play important roles in the development of HBV-related HCC. Ongoing research will help to identify practical applications of the HBV-related epigenetic signatures as biomarkers for early HCC detection or as potential targets to prevent and treat HBV-related HCC.
PMCID: PMC3719687  PMID: 23716588
5.  The influence of cathelicidin LL37 in human anti-neutrophils cytoplasmic antibody (ANCA)-associated vasculitis 
Arthritis Research & Therapy  2013;15(5):R161.
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterised by the autoinflammation and necrosis of blood vessel walls. The renal involvement is commonly characterised by a pauci-immune crescentic glomerulonephritis (PiCGN) with a very rapid decline in renal function. Cathelicidin LL37, an endogenous antimicrobial peptide, has recently been implicated in the pathogenesis of autoimmune diseases. To assess whether serum LL37 reflects renal crescentic formation, we measured the serum levels of LL37 in AAV patients with and without crescentic glomerulonephritis (crescentic GN) as compared to healthy controls (HCs). We also analysed the correlation of the serum levels of LL37 and interferon-α (IFN-α) with the clinical characteristics of the patients.
The study population consisted of 85 AAV patients and 51 HCs. In 40 ANCA-positive patients, a parallel analysis was performed, including the assessment of LL37 and IFN-α levels in the serum and renal biopsies. Of those studied, 15 AAV patients had biopsy-proven crescentic GN, and 25 AAV patients lacked crescent formation. The serum levels of cathelicidin LL37 and IFN-α were both measured by ELISA, and the clinical and serological parameters were assessed according to routine procedures. Immunofluorescence staining was performed on frozen sections of kidney needle biopsies from AAV patients with crescentic GN.
The serum levels of LL37 and IFN-α were significantly increased in AAV patients with crescentic GN compared to AAV patients without crescentic formation and HCs, and patients with high LL37 and IFN-α levels were more likely to be in the crescentic GN group. The LL37 levels were positively correlated with the IFN-α levels, and both LL37 and IFN-α levels showed a positive correlation with serum creatinine and no correlation with complement C3. The renal tissue of crescentic GN patients showed expression of LL37 and IFN-α at the Bowman’s capsule and extracellular sites, suggesting the active release of LL37 and IFN-α.
Significantly higher levels of LL-37 and IFN-α were observed in AAV patients, particularly those with crescentic formation, and LL37 and IFN-α were expressed in the renal tissue of patients with crescentic GN. These data suggest that serum levels of LL37 and IFN-α may reflect both local renal inflammation and systemic inflammation.
PMCID: PMC3979017  PMID: 24286516
6.  Cyclosporin A Inhibits Rotavirus Replication and Restores Interferon-Beta Signaling Pathway In Vitro and In Vivo 
PLoS ONE  2013;8(8):e71815.
Rotavirus (RV) is the most common cause of severe diarrhea among infants and young children. Currently, there is no specific drug available against rotavirus, largely due to the lack of an ideal target molecule which has hampered drug development. Our previous studies have revealed that cyclosporin A (CsA) might be potentially useful as an anti-RV drug. We therefore used both cellular and mouse models to study the immunological safety and effectiveness of CsA as an anti-RV drug. We found that CsA treatment of HT-29 cells before, during, and after viral infection efficiently inhibited Wa strain RV replication and restored IFN-β expression in a HT-29 cell line model. Exploring the underlying mechanisms showed that CsA promoted Interferon Regulatory Factor-5 (IRF-5) expression (a key positive regulator of the type I IFN signaling pathway), but not IRF-1, IRF-3, or IRF-7. Additionally, CsA inhibited SOCS-1 expression (the key negative regulator of IFN-α/β), but not SOCS-2 or SOCS-3. The antiviral effect of CsA was confirmed in an RV-infected neonatal mouse model by evaluation of antigen clearance and assessment of changes in intestinal tissue pathology. Also, no differences in T cell frequency or proliferation between the CsA- and vehicle-treated groups were observed. Thus, both our in vitro and in vivo findings suggest that CsA, through modulating the expression of key regulators in IFN signaling pathway, promote type I IFN-based intracellular innate immunity in RV host cells. These findings suggest that CsA may be a useful candidate to develop a new anti-RV strategy, although further evaluation and characterization of CsA on RV-induced diarrhea are warranted.
PMCID: PMC3749198  PMID: 23990993
7.  Programming of regulatory T cells from pluripotent stem cells and prevention of autoimmunity* 
Regulatory T (Treg) cells are being used to treat autoimmunity and prevent organ rejection; however, Treg cell-based therapies have been hampered by the technical limitation in obtaining a high number of functional Treg cells. Here we show how to generate functional Treg cells from induced pluripotent stem (iPS) cells, and to determine the potential role of such cells for Treg-based immunotherapy against autoimmunity in a therapeutic setting. Ligation of a Notch ligand and transduction of the gene of forkhead box P3 (FoxP3) induce iPS cells to differentiate into Treg cells. Expression of FoxP3 and co-culture on Notch ligand-expressing stromal cells augment expression of CD3, TCR, CD4, CD25, and CTLA-4 on iPS cell-differentiated Treg cells, which are able to secrete TGF-β and IL-10 both in vivo and in vitro. Importantly, adoptive transfer of iPS cell-derived Treg cells expressing large amounts of FoxP3 and Bcl-xL significantly suppresses host immune responses and reduces arthritis development within murine models. These data suggest that Notch signaling and FoxP3 regulate the development and function of Treg cells derived from iPS cells. Our results provide a novel approach for generating potentially therapeutic Treg cells for the treatment of autoimmune diseases.
PMCID: PMC3401327  PMID: 22732595
8.  Hepatitis B Virus Induces IL-23 Production in Antigen Presenting Cells and Causes Liver Damage via the IL-23/IL-17 Axis 
PLoS Pathogens  2013;9(6):e1003410.
IL-23 regulates myriad processes in the innate and adaptive immune systems, and is a critical mediator of the proinflammatory effects exerted by Th17 cells in many diseases. In this study, we investigated whether and how hepatitis B virus (HBV) causes liver damage directly through the IL-23 signaling pathway. In biopsied liver tissues from HBV-infected patients, expression of both IL-23 and IL-23R was remarkably elevated. In vivo observations also indicated that the main sources of IL-23 were myeloid dendritic cells (mDCs) and macrophages. Analysis of in vitro differentiated immature DCs and macrophages isolated from healthy donors revealed that the HBV surface antigen (HBsAg) efficiently induces IL-23 secretion in a mannose receptor (MR)-dependent manner. Culture with an endosomal acidification inhibitor and the dynamin inhibitor showed that, upon binding to the MR, the HBsAg is taken up by mDCs and macrophages through an endocytosis mechanism. In contrast, although the HBV core antigen (HBcAg) can also stimulate IL-23 secretion from mDCs, the process was MR- and endocytosis-independent. In addition, IL-23 was shown to be indispensible for HBsAg-stimulated differentiation of naïve CD4+ T cells into Th17 cells, which were determined to be the primary source of IL-17 in HBV-infected livers. The cognate receptor, IL-17R, was found to exist on the hepatic stellate cells and mDCs, both of which might represent the potential target cells of IL-17 in hepatitis B disease. These data provide novel insights into a yet unrecognized mechanism of HBV-induced hepatitis, by which increases in IL-23 expression, through an MR/endocytosis-dependent or -independent manner, produce liver damage through the IL-23/IL-17 axis.
Author Summary
While it is known that IL-23 plays a pivotal role in maintenance of the Th17 phenotype and their production of the IL-17 cytokine, the mechanisms by which HBV induces particular immune cell types to produce IL-23 remain unknown. In the study of human hepatitis B described herein, we demonstrated that IL-23 is principally derived from the liver myeloid dendritic cells (mDCs) and macrophages. In vitro assay showed that mDCs produce large amounts of IL-23 upon stimulation with HBV surface antigen (HBsAg) through the mannose receptor (MR) and an endocytosis mechanism. In contrast, although the HBV core antigen (HBcAg) was also capable of stimulating IL-23 secretion from mDCs, the process occurs in an MR- and endocytosis-independent manner. IL-23 was also shown to efficiently stimulate the differentiation of naïve CD4+ T cells into Th17 cells in the presence of HBsAg or HBcAg; furthermore, the Th17 cells were shown to be the primary source of IL-17. The results also indicated that both hepatic satellite cells and mDCs might be the potential target cells of IL-17 in hepatitis B disease. Therefore, our study not only provides further insights into the mechanisms underlying HBV pathogenesis, but also suggests the potential intervening targets for patient treatment.
PMCID: PMC3694858  PMID: 23825942
9.  The role of microRNAs in B-cell development and function 
Cellular and Molecular Immunology  2013;10(2):107-112.
MicroRNA (miRNA)-mediated gene silencing at the translational level has led to novel discoveries for numerous biological processes. Recently, there has been increasing evidence to indicate that miRNAs are involved in normal immune functions and inflammation. In this review, we focus on recent advances that have elucidated the role of miRNAs in B-cell development, differentiation, apoptosis and function. While the regulatory mechanisms of miRNAs in controlling and maintaining B-cell fate remain largely uncharacterized, further studies on miRNAs and their targets will increase our understanding of B-cell development and function. Such studies may be able to provide new therapeutic strategies for treating autoimmune diseases.
PMCID: PMC4003047  PMID: 23314697
microRNA; B cell; development; function
10.  A trial of patient-oriented problem-solving system for immunology teaching in China: a comparison with dialectic lectures 
BMC Medical Education  2013;13:11.
The most common teaching method used in China is lecturing, but recently, efforts have been widely undertaken to promote the transition from teacher-centered to student-centered education. The patient-oriented problem-solving (POPS) system is an innovative teaching-learning method that permits students to work in small groups to solve clinical problems, promotes self-learning, encourages clinical reasoning and develops long-lasting memory. To our best knowledge, however, POPS has never been applied in teaching immunology in China. The aim of this study was to develop POPS in teaching immunology and assess students’ and teachers’ perception to POPS.
321 second-year medical students were divided into two groups: I and II. Group I, comprising 110 students, was taught by POPS, and 16 immunology teachers witnessed the whole teaching process. Group II including the remaining 211 students was taught through traditional lectures. The results of the pre- and post-test of both groups were compared. Group I students and teachers then completed a self-structured feedback questionnaire for analysis before a discussion meeting attended only by the teachers was held.
Significant improvement in the mean difference between the pre- and post-test scores of those in Groups I and II was seen, demonstrating the effectiveness of POPS teaching. Most students responded that POPS facilitates self-learning, helps them to understand topics and creates interest, and 88.12% of students favored POPS over simple lectures. Moreover, while they responded that POPS facilitated student learning better than lectures, teachers pointed out that limited teaching resources would make it difficult for wide POPS application in China.
While POPS can break up the monotony of dialectic lectures and serve as a better teaching method, it may not be feasible for the current educational environment in China. The main reason for this is the relative shortage of teaching resources such as space, library facilities and well-trained teachers.
PMCID: PMC3570451  PMID: 23356717
Patient-oriented problem-solving; Medical education; Immunology; China
11.  The Intrahepatic Expression and Distribution of BTLA and its Ligand HVEM in patients with HBV-related acute-on-chronic liver failure 
Diagnostic Pathology  2012;7:142.
It has been demonstrated that signals from the inhibitory receptor B and T lymphocyte attenuator (BTLA) are involved in regulating the pathogenesis of infectious diseases. However, the expression and anatomical distribution of BTLA and its ligand, the herpes virus entry mediator (HVEM), have not yet been determined in cases of HBV-related acute-on-chronic liver failure (HBV-ACLF) patients.
In this study, the expression of BTLA and HVEM in liver tissues from HBV-ACLF, chronic hepatitis B (CHB) patients and healthy individuals was analyzed by immunohistochemistry.
The results of this analysis demonstrated that both molecules were observed in the HBV-ACLF samples and that their expression was chiefly in the infiltrating inflammatory cells and the damaged bile ducts. However, they were absent in liver sections from CHB patients and healthy controls. Immunofluorescence double-staining indicated that BTLA was found on CK-18+ epithelial cells, CD31+ endothelial cells, CD68+ macrophages, CD56+ NK cells, CD16+ monocytes, CD3+ , CD8+ T cells, and Foxp3+ regulatory T cells (Treg). By contrast, HVEM expression was restricted to CK18+ epithelial cells and CD68+ macrophages. Moreover, the expression of several members of the B7 superfamily, including PD-L1, PD-L2, B7-H3 and B7-H4, was also detected in these liver tissues, and these proteins were co-expressed with HVEM. Interestingly, the expression of fibrinogen-like protein 2 (FGL2), a virus-induced procoagulant molecule, was also found in liver sections from HBV-ACLF, this molecule also co-expresses with BTLA and HVEM.
These results suggest that BTLA-HVEM signaling is likely to affect the pathogenesis of HBV-ACLF, a clear understanding of the functional roles of these proteins should further elucidate the disease process.
Virtual slides
The virtual slide(s) for this article can be found here:
PMCID: PMC3488509  PMID: 23067542
BTLA; HBV-ACLF; HVEM; Immunohistochemistry; B7 superfamily
12.  Quetiapine, an Atypical Antipsychotic, Is Protective against Autoimmune-Mediated Demyelination by Inhibiting Effector T Cell Proliferation 
PLoS ONE  2012;7(8):e42746.
Quetiapine (Que), a commonly used atypical antipsychotic drug (APD), can prevent myelin from breakdown without immune attack. Multiple sclerosisis (MS), an autoimmune reactive inflammation demyelinating disease, is triggered by activated myelin-specific T lymphocytes (T cells). In this study, we investigated the potential efficacy of Que as an immune-modulating therapeutic agent for experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. Que treatment was initiated on the onset of MOG35–55 peptide induced EAE mice and the efficacy of Que on modulating the immune response was determined by Flow Cytometry through analyzing CD4+/CD8+ populations and the proliferation of effector T cells (CD4+CD25−) in peripheral immune organs. Our results show that Que dramatically attenuates the severity of EAE symptoms. Que treatment decreases the extent of CD4+/CD8+ T cell infiltration into the spinal cord and suppresses local glial activation, thereby diminishing the loss of mature oligodendrocytes and myelin breakdown in the spinal cord of EAE mice. Our results further demonstrate that Que treatment decreases the CD4+/CD8+ T cell populations in lymph nodes and spleens of EAE mice and inhibits either MOG35–55 or anti-CD3 induced proliferation as well as IL-2 production of effector T cells (CD4+CD25−) isolated from EAE mice spleen. Together, these findings suggest that Que displays an immune-modulating role during the course of EAE, and thus may be a promising candidate for treatment of MS.
PMCID: PMC3418290  PMID: 22912731
13.  Effects of Telbivudine Treatment on the Circulating CD4+ T-Cell Subpopulations in Chronic Hepatitis B Patients 
Mediators of Inflammation  2012;2012:789859.
CD4+ T cells serve as master regulators of the adaptive immune response to HBV. However, CD4+ T-cell subsets are heterogeneous, and it remains unknown how the antiviral agents affect the different CD4+ T cell subtypes. To this end, the expressions of signature transcription factors and cytokines of CD4+ T-cell subtypes were examined in hepatitis B patients before and after treatment with telbivudine. Results showed that, upon the rapid HBV copy decrease induced by telbivudine treatment, the frequencies and related cytokines of Th17 and Treg cells were dramatically decreased, while those for Th2 cells were dramatically increased. No obvious changes were observed in Th1 cell frequencies; although, IFN-γ expression was upregulated in response to telbivudine treatment, suggesting another cell source of IFN-γ in CHB patients. Statistical analyses indicated that Th17 and Tr1 (a Treg subtype) cells were the most sensitive subpopulations of the peripheral blood CD4+ T cells to telbivudine treatment over 52 weeks. Thus, Th17 and Tr1 cells may represent a suitable and effective predictor of responsiveness during telbivudine therapy. These findings not only improve our understanding of hepatitis pathogenesis but also can aid in future development of appropriate therapeutic strategies to control viral hepatitis.
PMCID: PMC3337496  PMID: 22570512
14.  Expression of CD39 on FoxP3+ T regulatory cells correlates with progression of HBV infection 
BMC Immunology  2012;13:17.
Although it is known that regulatory T cells (Tregs) can suppress the function of effector T cells, and may contribute to impaired immune response, the precise role of Tregs during the course of hepatitis B virus (HBV) infection remains to be elucidated. A newly identified subset of the CD4+Foxp3+ Tregs, the CD39+ Tregs, has been associated with viral infections and autoimmune diseases. Therefore, we hypothesized that this discrete Treg subset may contribute to the chronic infection of HBV.
Initial characterization studies of healthy peripheral CD39+FoxP3+CD4+ T cells revealed that the majority were CD45RA- Treg cells. Subsequent analysis of HBV-infected patients (38 asymptomatic HBV carriers (AsCs), 37 chronic active hepatitis B (CAH), 29 HBV-associated acute-on-chronic liver failure (ACLF)) and healthy individuals (25 controls) was conducted to assess association with HBV copy number and the liver injury marker alanine aminotransferase (ALT). A higher percentage of CD39+ Tregs was detected within the population of FoxP3+CD4+ T cells in peripheral blood of AsCs patients. Moreover, the percentage of CD39+ Tregs was significantly less in CAH and ACLF patients. The increased proportions of circulating CD39+ Tregs were positively correlated with serum viral load, but inversely correlated with serum ALT level.
These findings not only suggest that CD39+ Treg cells may be involved in HBV disease progression but also identify CD39+ Tregs as a dynamic immune regulatory cell population that may represent a new target of immunomodulatory therapeutic interventions.
PMCID: PMC3364870  PMID: 22489829
Hepatitis B; CD39; Regulatory T lymphocyte
16.  Global Mapping of H3K4me1 and H3K4me3 Reveals the Chromatin State-Based Cell Type-Specific Gene Regulation in Human Treg Cells 
PLoS ONE  2011;6(11):e27770.
Regulatory T cells (Treg) contribute to the crucial immunological processes of self-tolerance and immune homeostasis. Genomic mechanisms that regulate cell fate decisions leading to Treg or conventional T cells (Tconv) lineages and those underlying Treg function remain to be fully elucidated, especially at the histone modification level. We generated high-resolution genome-wide distribution maps of monomethylated histone H3 lysine 4 (H3K4me1) and trimethylated H3K4 (H3K4me3) in human CD4+CD25+FOXP3+ Tregs and CD4+CD25+FOXP3− activated (a)Tconv cells by DNA sequencing-by-synthesis. 2115 H3K4me3 regions corresponded to proximal promoters; in Tregs, the genes associated with these regions included the master regulator FOXP3 and the chemokine (C-C motif) receptor 7 (CCR7). 41024 Treg-specific H3K4me1 regions were identified. The majority of the H3K4me1 regions differing between Treg and aTconv cells were located at promoter-distal sites, and in vitro reporter gene assays were used to evaluate and identify novel enhancer activity. We provide for the first time a comprehensive genome-wide dataset of lineage-specific H3K4me1 and H3K4me3 patterns in Treg and aTconv cells, which may control cell type-specific gene regulation. This basic principle is likely not restricted to the two closely-related T cell populations, but may apply generally to somatic cell lineages in adult organisms.
PMCID: PMC3223197  PMID: 22132139
17.  Complement and the Alternative Pathway Play an Important Role in LPS/D-GalN-Induced Fulminant Hepatic Failure 
PLoS ONE  2011;6(11):e26838.
Fulminant hepatic failure (FHF) is a clinically severe type of liver injury with an extremely high mortality rate. Although the pathological mechanisms of FHF are not well understood, evidence suggests that the complement system is involved in the pathogenesis of a variety of liver disorders. In the present study, to investigate the role of complement in FHF, we examined groups of mice following intraperitoneal injection of LPS/D-GalN: wild-type C57BL/6 mice, wild-type mice treated with a C3aR antagonist, C5aR monoclonal antibody (C5aRmAb) or CR2-Factor H (CR2-fH, an inhibitor of the alternative pathway), and C3 deficient mice (C3−/− mice). The animals were euthanized and samples analyzed at specific times after LPS/D-GalN injection. The results show that intraperitoneal administration of LPS/D-GalN activated the complement pathway, as evidenced by the hepatic deposition of C3 and C5b-9 and elevated serum levels of the complement activation product C3a, the level of which was associated with the severity of the liver damage. C3a receptor (C3aR) and C5a receptor (C5aR) expression was also upregulated. Compared with wild-type mice, C3−/− mice survived significantly longer and displayed reduced liver inflammation and attenuated pathological damage following LPS/D-GalN injection. Similar levels of protection were seen in mice treated with C3aR antagonist,C5aRmAb or CR2-fH. These data indicate an important role for the C3a and C5a generated by the alternative pathway in LPS/D-GalN-induced FHF. The data further suggest that complement inhibition may be an effective strategy for the adjunctive treatment of fulminant hepatic failure.
PMCID: PMC3206060  PMID: 22069473
18.  Programmed Death (PD)-1-Deficient Mice Are Extremely Sensitive to Murine Hepatitis Virus Strain-3 (MHV-3) Infection 
PLoS Pathogens  2011;7(7):e1001347.
The inhibitory receptor programmed death-1 (PD-1) has the capacity to maintain peripheral tolerance and limit immunopathological damage; however, its precise role in fulminant viral hepatitis (FH) has yet to be described. Here, we investigated the functional mechanisms of PD-1 as related to FH pathogenesis induced by the murine hepatitis virus strain-3 (MHV-3). High levels of PD-1-positive CD4+, CD8+ T cells, NK cells and macrophages were observed in liver, spleen, lymph node and thymus tissues following MHV-3 infection. PD-1-deficient mice exhibited significantly higher expression of the effector molecule which initiates fibrinogen deposition, fibrinogen-like protein 2 (FGL2), than did their wild-type (WT) littermates. As a result, more severe tissue damage was produced and mortality rates were higher. Fluorescence double-staining revealed that FGL2 and PD-1 were not co-expressed on the same cells, while quantitative RT-PCR demonstrated that higher levels of IFN-γ and TNF-α mRNA transcription occurred in PD-1-deficient mice in response to MHV-3 infection. Conversely, in vivo blockade of IFN-γ and TNF-α led to efficient inhibition of FGL2 expression, greatly attenuated the development of tissue lesions, and ultimately reduced mortality. Thus, the up-regulation of FGL2 in PD-1-deficient mice was determined to be mediated by IFN-γ and TNF-α. Taken together, our results suggest that PD-1 signaling plays an essential role in decreasing the immunopathological damage induced by MHV-3 and that manipulation of this signal might be a useful strategy for FH immunotherapy.
Author Summary
The principal characteristic of fulminant viral hepatitis (FH) induced by the murine hepatitis virus strain-3 (MHV-3) is severe hepatocellular necrosis, which is mediated by the fibrinogen-like protein 2 (FGL2), a molecule that has the capacity to promote fibrinogen deposition and activate the coagulation cascades. Here, we report that MHV-3 infection of program death-1 (PD-1)-deficient mice results in tissue damage throughout multiple organs, including the liver, spleen, thymus and lymph nodes. The liver damage, in particular, occurred earlier and was more severe in PD-1-deficient mice than in their wild type (WT) littermates. Further investigation determined that MHV-3 infection was associated with high levels of IFN-γ and TNF-α in the damaged organs of PD-1-deficient mice. Conversely, intraperitoneal injection of a combination of anti-IFN-γ and anti-TNF-α blocking mAbs led to inhibition of FGL2 expression, greatly attenuated tissue lesions and reduced mortality. Our results demonstrate that PD-1 signaling controls immunopathological damage following MHV-3 infection, indicating that manipulation of the PD-1 signal might represent a useful strategy for FH immunotherapy.
PMCID: PMC3131267  PMID: 21750671
19.  Activated IL-23/IL-17 pathway closely correlates with increased Foxp3 expression in livers of chronic hepatitis B patients 
BMC Immunology  2011;12:25.
Foxp3 protein plays a critical role in mediating the inflammatory response and can inhibit the proinflammatory IL-23/IL-17 pathway. However, the molecular interplay of Foxp3 and the IL-23/IL-17 pathway in patients with chronic hepatitis B (CHB) remains unclear. To this end, we analyzed the expression patterns of Foxp3- and IL-23/IL-17 pathway-related proinflammatory cytokines in 39 patients with acute-on-chronic liver failure, 71 patients with CHB and 32 healthy controls.
Foxp3 expression was found to be elevated in and mainly expressed by the CD4+ T cell sub-population of peripheral blood mononuclear cells and liver tissues of patients with hepatitis B. The intrahepatic expression of Foxp3 strongly correlated with the copies of HBV DNA and the concentration of surface antigen, HBsAg. IL-23/IL-17 pathway-related proinflammatory cytokines were also found to be significantly increased in patients' liver tissues, as compared to healthy controls. Moreover, Foxp3 expression was strikingly correlated with the production of these cytokines in liver tissues of CHB patients.
The closely-correlated increase of Foxp3 and IL-23/IL-17 pathway activity in HBV-infected livers suggests that the proinflammatory IL-23/IL-17 pathway had not been effectively suppressed by the host immune machinery, such as Treg (Foxp3) cells. Constitutive activation of the IL-23/17 pathway, thus, may support the chronic hepatitis B state.
PMCID: PMC3094328  PMID: 21489307
Treg; Foxp3; Th17; IL-23; IL-17; Hepatitis B
Costimulation regulates multiple cellular processes of T cells inducing proliferation, expansion and survival. The molecular targets of costimulation might then be useful to augment T cell activities. Two defined targets of costimulatory signals in primary T cells are the anti-apoptotic bcl-2 family molecule Bcl-xL, and Survivin, an Inhibitor of Apoptosis family member that might regulate both cell division and survival. However, the relative importance of, and relationship between, these molecules in primary T cells is not clear. To understand whether they have overlapping or co-operative functions, we used retrovirus-mediated transduction to introduce Bcl-xL and Survivin separately, or together linked by a 2A picornavirus self-cleaving peptide, into antigen (Ag)-responding CD8+ T cells. We found that CD8+ effector T cells expressing both Bcl-xL and Survivin strongly expanded at an early stage and had a long-term survival advantage over cells transduced with either molecule alone. In vivo, with response to tumor-expressed Ag following adoptive T cell transfer, Ag-reactive CD8+ T cells expressing both Bcl-xL and Survivin displayed greatly enhanced tumor protective activity compared to CD8+ T cells expressing either molecule introduced separately. These results indicate that Bcl-xL and Survivin can critically contribute in a co-operative, non-redundant, manner to augment the accumulation and persistence of CD8+ T cells following encounter with Ag. The data provide new insights into why costimulatory signals might need to be sustained over time and suggest a potential novel approach to augment cellular immunotherapy for cancer.
PMCID: PMC2748393  PMID: 19454669
Mouse; CD8 T cells; Survivin; Bcl-xL; Antigens/peptides; Costimulation; Cell activation
21.  Altered microRNA expression profile with miR-146a upregulation in CD4+ T cells from patients with rheumatoid arthritis 
Increasing 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).
The 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.
miRNA 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.
We 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.
PMCID: PMC2911863  PMID: 20459811
22.  FoxP3 and Bcl-xL cooperatively promote regulatory T cell persistence and prevention of arthritis development 
Forkhead box p3 (FoxP3)-expressing regulatory T cells (Tregs) have been clearly implicated in the control of autoimmune disease in murine models. In addition, ectopic expression of FoxP3 conveys a Treg phenotype to CD4+ T cells, lending itself to therapeutic use in the prevention of rheumatoid arthritis (RA). In this study, we generated therapeutically active Tregs with an increased life span and hence greater therapeutic potential.
We used retrovirus-mediated transduction to introduce FoxP3 or FoxP3 with anti-apoptotic Bcl-2 family molecule Bcl-xL linked by a 2A picornavirus self-cleaving peptide into CD4+ T cells to generate Tregs. In addition, by using in vitro functional analyses and adoptive immunotherapy in a murine model of RA, we demonstrated that these Tregs were highly reactive.
We found that CD4+ T cells expressing both FoxP3 and Bcl-xL were able to differentiate into functional Tregs, which have a long-term survival advantage over cells transduced with FoxP3 alone. In an in vivo murine model, adoptive transfer of Tregs expressing both FoxP3 and Bcl-xL demonstrated more effective suppression of RA than CD4+ T cells expressing FoxP3 alone.
FoxP3 and Bcl-xL can cooperatively promote the differentiation and persistence of Tregs, with the capacity to prevent arthritis. Our results provide a novel approach for generating highly reactive Tregs for augmenting cellular immunotherapy for autoimmune disease.
PMCID: PMC2888221  PMID: 20384988
23.  Identification of a novel conserved HLA-A*0201-restricted epitope from the spike protein of SARS-CoV 
BMC Immunology  2009;10:61.
The spike (S) protein is a major structural glycoprotein of coronavirus (CoV), the causal agent of severe acute respiratory syndrome (SARS). The S protein is a potent target for SARS-specific cell-mediated immune responses. However, the mechanism CoV pathogenesis in SARS and the role of special CTLs in virus clearance are still largely uncharacterized. Here, we describe a study that leads to the identification of a novel HLA-A*0201-restricted epitope from conserved regions of S protein.
First, different SARS-CoV sequences were analyzed to predict eight candidate peptides from conserved regions of the S protein based upon HLA-A*0201 binding and proteosomal cleavage. Four of eight candidate peptides were tested by HLA-A*0201 binding assays. Among the four candidate peptides, Sp8 (S958-966, VLNDILSRL) induced specific CTLs both ex vivo in PBLs of healthy HLA-A2+ donors and in HLA-A2.1/Kb transgenic mice immunized with a plasmid encoding full-length S protein. The immunized mice released IFN-γ and lysed target cells upon stimulation with Sp8 peptide-pulsed autologous dendritic cells in comparison to other candidates.
These results suggest that Sp8 is a naturally processed epitope. We propose that Sp8 epitope should help in the characterization of mechanisms of virus control and immunopathology in SARS-CoV infection.
PMCID: PMC2792222  PMID: 19958537
24.  High-level expression and large-scale preparation of soluble HBx antigen from Escherichia coli 
Biotechnology and Applied Biochemistry  2009;54(Pt 3):141-147.
The HBx (hepatitis B virus X protein) is a multifunctional regulator of cellular signal transduction and transcription pathways in host-infected cells. Evidence suggests that HBx has a critical role in the pathogenesis of hepatocellular carcinoma. However, the lack of efficient large-scale preparation methods for soluble HBx has hindered studies on the structure and function of HBx. Here, a new pMAL-c2x protein fusion and purification system was used for high-level expression of soluble HBx fusion protein. The high-purity fusion protein was obtained via amylose resin chromatography and Q-Sepharose chromatography. The untagged HBx was efficiently and rapidly purified by Sephadex G-75 chromatography after cleavage by Factor Xa at 23 °C. The purity of active HBx protein was >99% with a very stable secondary structure dominated by α-helix, β-sheet and random structure. The purified HBx protein can be analysed to determine its crystal structure and function and its capabilities as an effective immunogen.
PMCID: PMC2782320  PMID: 19607648
amylose resin chromatography; Escherichia coli strain JM109; hepatitis B virus X protein (HBx); hepatocellular carcinoma; Q-Sepharose chromatography; Sephadex G-75 chromatography; CAT, chloramphenicol acetyltransferase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HBV, hepatitis B virus; HBx, HBV X protein; HCC, hepatocellular carcinoma; IPTG, isopropyl β-D-thiogalactoside; LB, Luria–Bertani; LTR, long terminal repeat; MBP, maltose-binding protein; TF, transcription factor
25.  Protective Immunity Elicited by a Divalent DNA Vaccine Encoding Both the L7/L12 and Omp16 Genes of Brucella abortus in BALB/c Mice  
Infection and Immunity  2006;74(5):2734-2741.
This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. Intramuscular injection of this divalent DNA vaccine into BALB/c mice elicited markedly both humoral and cellular immune responses. The specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the dual-gene DNA vaccine elicited a strong T-cell proliferative response and induced a large amount of gamma interferon-producing T cells upon restimulation in vitro with recombinant fusion protein L7/L12-Omp16, suggesting the induction of a typical T-helper-1-dominated immune response in vivo. This divalent DNA vaccine could also induce a significant level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice. Furthermore, the protection level induced by the divalent DNA vaccine was significantly higher than that induced by the univalent DNA vaccines pcDNA3.1-L7/L12 or pcDNA3.1-Omp16. Taken together, the results of this study verify for the first time that the Omp16 gene can be a candidate target for a DNA vaccine against brucellosis. Additionally, a divalent genetic vaccine based on the L7/L12 and Omp16 genes can elicit a stronger cellular immune response and better immunoprotection than the relevant univalent vaccines can.
PMCID: PMC1459688  PMID: 16622210

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