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author:("Fang, degu")
1.  Association of MASP-2 Levels and MASP2 Gene Polymorphisms with Rheumatoid Arthritis in Patients and Their Relatives 
PLoS ONE  2014;9(3):e90979.
Background
Mannan-binding lectin-associated serine protease 2 (MASP-2) is a key protein of the lectin pathway of complement. MASP-2 levels have been associated with different polymorphisms within MASP2 gene as well as with the risk for inflammatory disorders and infections. Despite its clinical importance, MASP-2 remains poorly investigated in rheumatoid arthritis (RA).
Methods
In this case-control study, we measured MASP-2 serum levels in 156 RA patients, 44 patient relatives, and 100 controls from Southern Brazil, associating the results with nine MASP2 polymorphisms in all patients, 111 relatives, and 230 controls genotyped with multiplex SSP-PCR.
Results
MASP-2 levels were lower in patients than in controls and relatives (medians 181 vs. 340 or 285 ng/ml, respectively, P<0.0001). Conversely, high MASP-2 levels were associated with lower susceptibility to RA and to articular symptoms independently of age, gender, ethnicity, smoking habit, anti-CCP and rheumatoid factor positivity (OR = 0.05 [95%CI = 0.019–0.13], P<0.0001 between patients and controls; OR = 0.12, [95%CI = 0.03–0.45], P = 0.002 between patients and relatives; OR = 0.06, [95%CI = 0.004–0.73], P = 0.03 between relatives with and without articular symptoms). MASP2 haplotypes *2A1 and *2B1-i were associated with increased susceptibility to RA (OR = 3.32 [95%CI = 1.48–7.45], P = 0.004). Deficiency-causing p.120G and p.439H substitutions were associated with five times increased susceptibility to articular symptoms in relatives (OR = 5.13 [95%CI = 1.36–20.84], P = 0.02). There was no association of MASP-2 levels or MASP2 polymorphisms with autoantibodies, Sjögren's syndrome, nodules and functional class.
Conclusions
In this study, we found the first evidence that MASP-2 deficiency might play an important role in the development of RA and articular symptoms among relatives of RA patients.
doi:10.1371/journal.pone.0090979
PMCID: PMC3954616  PMID: 24632598
2.  Regulation of T Cell Differentiation and Alloimmunity by the Cyclin-Dependent Kinase Inhibitor p18ink4c 
PLoS ONE  2014;9(3):e91587.
Cellular proliferation in response to mitogenic stimuli is negatively regulated by the Cip/Kip and the Ink4 families of cyclin-dependent kinase (CDK) inhibitors. Several of these proteins are elevated in anergic T cells, suggesting a potential role in the induction or maintenance of tolerance. Our previous studies showed that p27kip1 is required for the induction of T cell anergy and transplantation tolerance by costimulatory blockade, but a role for Ink4 proteins in these processes has not been established. Here we show that CD4+ T cells from mice genetically deficient for p18ink4c divide more rapidly than wild-type cells in response to antigenic, costimulatory and growth factor signals. However, this gain of proliferative function was accompanied by a moderate increase in the rate of cell death, and was accompanied by an overall defect in the generation of alloreactive IFNγ-producing effector cells. Consistent with this, p18ink4c-deficient T cells were unable to induce graft-vs-host disease in vivo, and p18ink4c deficiency cooperated with costimulatory blockade to significantly increase the survival of fully mismatched allografts in a cardiac transplantation model. While both p18ink4c and p27kip1 act to restrict T cell proliferation, p18ink4c exerts an opposite effect from p27kip1 on alloimmunity and organ transplant rejection, most likely by sustaining T cell survival and the development of effector function. Our studies point to additional important links between the cell cycle machinery and the processes of T cell differentiation, survival and tolerance.
doi:10.1371/journal.pone.0091587
PMCID: PMC3948896  PMID: 24614758
3.  Myeloid Deletion of SIRT1 Aggravates Serum Transfer Arthritis in Mice via Nuclear Factor-κB Activation 
PLoS ONE  2014;9(2):e87733.
Objective
SIRT1 modulates the acetylation of the p65 subunit of nuclear factor-κB (NF-κB) and plays a pivotal role in the inflammatory response. This study sought to assess the role of SIRT1 in rheumatoid arthritis (RA) using a myeloid cell-specific SIRT1 knockout (mSIRT1 KO) mouse.
Methods
mSIRT1 KO mice were generated using the loxP/Cre recombinase system. K/BxN serum transfer arthritis was induced in mSIRT1 KO mice and age-matched littermate loxP control mice. Arthritis severity was assessed by clinical and pathological scoring. The levels of inflammatory cytokines in the serum and joints were measured by ELISA. Migration, M1 polarization, cytokine production, osteoclastogenesis, and p65 acetylation were assessed in bone marrow-derived monocytes/macrophages (BMMs).
Results
mSIRT1 KO mice showed more severe inflammatory arthritis and aggravated pathological findings than control mice. These effects were paralleled by increases in IL-1, TNF-α, TRAP-positive osteoclasts, and F4/80+ macrophages in the ankles of mSIRT1 KO mice. In addition, BMMs from mSIRT1 KO mice displayed hyperacetylated p65 and increased NF-κB binding activity when compared to control mice, which resulted in increased M1 polarization, migration, pro-inflammatory cytokine production, and osteoclastogenesis.
Conclusion
Our study provides in vivo evidence that myeloid cell-specific deletion of SIRT1 exacerbates inflammatory arthritis via the hyperactivation of NF-κB signaling, which suggests that SIRT1 activation may be beneficial in the treatment of inflammatory arthritis.
doi:10.1371/journal.pone.0087733
PMCID: PMC3912001  PMID: 24498364
4.  CD8+ Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria 
PLoS ONE  2014;9(1):e81344.
We focus on the role of CD8+ Treg cell in Intravenous methyl-prednisolone (IVMP) pulse therapy in forty patients with active Class III/IV childhood lupus nephritis (LN) with heavy proteinuria. IVMP therapy for five days. From peripheral blood mononuclear cells (PBMCs) and renal tissues, we saw IVMP therapy definitely restoring both CD4+CD25+FoxP3+ and CD8+CD25+Foxp3+ Treg cell number plus greater expression with intracellular IL-10 and granzyme B in CD8+FoxP3+ Treg from PBMCs. IVMP-treated CD8+CD25+ Treg cells directly suppressed CD4+ T proliferation and induced CD4+CD45RO+ apoptosis. Histologically, CD4+FoxP3+ as well as CD8+FoxP3+ Treg cells appeared in renal tissue of LN patients before IVMP by double immunohistochemical stain. CD8+FoxP3+ Treg cells increased in 10 follow-up renal biopsy specimens after IVMP. Reverse correlation of serum anti-C1q antibody and FoxP3+ Treg cells in PBMNCs (r = −0.714, P<0.01). After IVMP, serum anti-C1q antibody decrease accompanied increase of CD4+FoxP3+ Treg cells. CD8+Treg cells reduced interferon-r response in PBMCs to major peptide autoepitopes from nucleosomes after IVMP therapy; siRNA of FoxP3 suppressed granzyme B expression while decreasing CD8+CD25+Treg-induced CD4+CD45RO+ apoptosis. Renal activity of LN by SLEDAI-2k in childhood LN was significantly higher than two weeks after IVMP (P<0.01). CD8+FoxP3+ Treg cells return in post-IVMP therapy and exert crucial immune modulatory effect to control autoimmune response in LN.
Trial Registration
DMR97-IRB-259
doi:10.1371/journal.pone.0081344
PMCID: PMC3903465  PMID: 24475019
5.  A Higher Frequency of Circulating IL-22+CD4+ T Cells in Chinese Patients with Newly Diagnosed Hashimoto’s Thyroiditis 
PLoS ONE  2014;9(1):e84545.
Background
IL-22 and IL-17A are implicated in the pathogenesis of autoimmune diseases. However, the role of IL-22+ and IL-17A+ CD4+ T cells in the pathogenesis of Hashimoto’s thyroiditis (HT) is not fully understood. This study investigates serum IL-22 and IL-17A levels and determines the frequency of circulating IL-22+ CD4+ T cells in HT patients to understand their roles in the pathogenesis of HT.
Methods
The levels of serum IL-22, IL-17A and IFN-γ and the frequency of circulating IL-22+CD4+ and IL-17A+CD4+ T cells in 17 HT patients and 17 healthy controls (HC) were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The levels of serum free triiodothyronine (FT4), free thyroxine (FT3), thyroid stimulating hormone (TSH), anti-thyroid peroxidase (TPO) and anti-thyroglobulin antibodies (TgAb) by chemiluminescent enzyme immunoassay and radioimmunoassay.
Results
The percentages of circulating IL-22+CD4+ and IL-17+CD4+ T cells (p<0.0001, p<0.0001) and the levels of serum IL-22, IL-17A and IFN-γ (p<0.0001, p<0.0001, p = 0.0210) in the HT patients were significantly higher than that in the HC. The percentages of IL-22+CD4+ T cells were positively correlated with Th17 cells (r = 0.8815, p<0.0001) and IL-17A+IL-22+CD4+ T cells (r = 0.8914, p<0.0001), but were negatively correlated with Th1 cells (r = −0.6110, p<0.0092) in the HT patients. The percentages of Th22 cells, Th17 cells and IL-17A+IL-22+CD4+ T cells were negatively correlated with the levels of serum TSH in the HT patients (r = −0.8402, p<0.0001; r = −0.8589, p<0.0001; r = −0.8289 p<0.0001, respectively).
Conclusions
A higher frequency of circulating IL-22+CD4+ and IL-17A+CD4+ T cells may be associated with the development of HT in Chinese patients.
doi:10.1371/journal.pone.0084545
PMCID: PMC3880314  PMID: 24404171
6.  Suppression of Molecular Inflammatory Pathways by Toll-Like Receptor 7, 8, and 9 Antagonists in a Model of IL-23-Induced Skin Inflammation 
PLoS ONE  2013;8(12):e84634.
Psoriasis is a complex inflammatory disease resulting from the activation of T helper (Th) 1 and Th17 cells. Recent evidence suggests that abnormal activation of Toll-like receptors (TLRs) 7, 8 and 9 contributes to the initiation and maintenance of psoriasis. We have evaluated the effects of TLR antagonists on the gene expression profile in an IL-23-induced skin inflammation model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the dorsum. Two TLR antagonists were compared: IMO-3100, an antagonist of TLRs 7 and 9, and IMO-8400, an antagonist of TLRs 7, 8 and 9, both of which previously have been shown to reduce epidermal hyperplasia in this model. Skin gene expression profiles of IL-23-induced inflammation were compared with or without TLR antagonist treatment. IL-23 injection resulted in alteration of 5100 gene probes (fold change ≥ 2, FDR < 0.05) including IL-17 pathways that are up-regulated in psoriasis vulgaris. Targeting TLRs 7, 8 and 9 with IMO-8400 resulted in modulation of more than 2300 mRNAs while targeting TLRs 7 and 9 with IMO-3100 resulted in modulation of more than 1900 mRNAs. Both agents strongly decreased IL-17A expression (>12-fold reduction), normalized IL-17 induced genes such as beta-defensin and CXCL1, and normalized aberrant expression of keratin 16 (indicating epidermal hyperplasia). These results suggest that IL-23-driven inflammation in mouse skin may be dependent on signaling mediated by TLRs 7, 8, and 9 and that these receptors represent novel therapeutic targets in psoriasis vulgaris and other diseases with similar pathophysiology.
doi:10.1371/journal.pone.0084634
PMCID: PMC3874038  PMID: 24386404
7.  Does Nrf2 Contribute to p53-Mediated Control of Cell Survival and Death? 
Antioxidants & Redox Signaling  2012;17(12):1670-1675.
Abstract
In response to oxidative stress, the transcription factor Nrf2 is upregulated and controls activation of many genes that work in concert to defend cells from damages and to maintain cellular redox homeostasis. p53 has been regarded as the guardian of the genome through its pro-oxidant and antioxidant functions. Under low levels of reactive oxygen species (ROS), “normal” amounts of p53 upregulates expression of antioxidant genes, protecting macromolecules from ROS-induced damage. However, at high levels or extended exposure of ROS, p53 expression is enhanced, activating pro-oxidant genes and resulting in p53-dependent apoptosis. We observed a two-phase Nrf2 expression controlled by p53. (i) The induction phase: when p53 expression is relatively low, p53 enhances the protein level of Nrf2 and its target genes to promote cell survival in a p21-dependent manner. (ii) The repression phase: when p53 expression is high, the Nrf2-mediated survival response is inhibited by p53. Our observation leads to the hypothesis that the p53-mediated biphasic regulation of Nrf2 may be key for the tumor-suppressor function of p53 by coordinating cell survival and death pathways. Antioxid. Redox Signal. 17, 1670–1675.
doi:10.1089/ars.2012.4674
PMCID: PMC3474188  PMID: 22559194
8.  Ginkgo biloba Extract Individually Inhibits JNK Activation and Induces c-Jun Degradation in Human Chondrocytes: Potential Therapeutics for Osteoarthritis 
PLoS ONE  2013;8(12):e82033.
Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation. The ideal anti-OA drug should have immunomodulatory effects while at the same time having limited or no toxicity. We examined the anti-inflammatory effects of Ginkgo biloba extract (EGb) in interleukin-1 (IL-1)-stimulated human chondrocytes. Chondrocytes were prepared from cartilage specimens taken from patients with osteoarthritis who had received total hip or total knee replacement. The concentrations of chemokines and the degree of cell migration were determined by ELISA and chemotaxis assays, respectively. The activation of inducible nitric oxide synthase (iNOS), mitogen-activated protein kinases (MAPKs), activator protein-1 (AP-1), and nuclear factor-kappaB (NF-κB) was determined by immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay. We found that EGb inhibited IL-1-induced production of chemokines, which in turn resulted in attenuation of THP-1 cell migration toward EGb-treated cell culture medium. EGb also suppressed IL-1-stimulated iNOS expression and release of nitric oxide (NO). The EGb-mediated suppression of the iNOS-NO pathway correlated with the attenuation of activator protein-1 (AP-1) but not nuclear factor-kappaB (NF-κB) DNA-binding activity. Of the mitogen-activated protein kinases (MAPKs), EGb inhibited only c-Jun N-terminal kinase (JNK). Unexpectedly, EGb selectively caused degradation of c-Jun protein. Further investigation revealed that EGb-mediated c-Jun degradation was preceded by ubiquitination of c-Jun and could be prevented by the proteosome inhibitor MG-132. The results imply that EGb protects against chondrocyte degeneration by inhibiting JNK activation and inducing ubiquitination-dependent c-Jun degradation. Although additional research is needed, our results suggest that EGb is a potential therapeutic agent for the treatment of OA.
doi:10.1371/journal.pone.0082033
PMCID: PMC3859542  PMID: 24349175
9.  MCPIP1 Deficiency in Mice Results in Severe Anemia Related to Autoimmune Mechanisms 
PLoS ONE  2013;8(12):e82542.
Autoimmune gastritis is an organ-specific autoimmune disease of the stomach associated with pernicious anemia. The previous work from us and other groups identified MCPIP1 as an essential factor controlling inflammation and immune homeostasis. MCPIP1-/- developed severe anemia. However, the mechanisms underlying this phenotype remain unclear. In the present study, we found that MCPIP1 deficiency in mice resulted in severe anemia related to autoimmune mechanisms. Although MCPIP1 deficiency did not affect erythropoiesis per se, the erythropoiesis in MCPIP1-/- bone marrow erythroblasts was significantly attenuated due to iron and vitamin B12 (VB12) deficiency, which was mainly resulted from autoimmunity-associated gastritis and parietal cell loss. Consistently, exogenous supplement of iron and VB12 greatly improved the anemia phenotype of MCPIP1-/- mice. Finally, we have evidence suggesting that autoimmune hemolysis may also contribute to anemia phenotype of MCPIP1-/- mice. Taken together, our study suggests that MCPIP1 deficiency in mice leads to the development of autoimmune gastritis and pernicious anemia. Thus, MCPIP1-/- mice may be a good mouse model for investigating the pathogenesis of pernicious anemia and testing the efficacy of some potential drugs for treatment of this disease.
doi:10.1371/journal.pone.0082542
PMCID: PMC3855756  PMID: 24324805
10.  A Plant Kavalactone Desmethoxyyangonin Prevents Inflammation and Fulminant Hepatitis in Mice  
PLoS ONE  2013;8(10):e77626.
Alpinia pricei Hayata is a Formosan plant which has been popularly used as nutraceutical or folk medicine for inflammation and various disorders. An active compound of the plant rhizomes, desmethoxyyangonin (DMY), was identified in this study for its novel effect against endotoxin lipopolysaccharide (LPS)-stimulated inflammation in murine macrophages and LPS/D-galactosamine (LPS/D-GalN)-induced fulminant hepatitis in mice. DMY was observed to significantly inhibit proliferation and activation of T cells ex vivo and the activity of several pro-inflammatory mediators in vitro. DMY also protected LPS/D-GalN−induced acute hepatic damages in mice through inhibiting aminotransferases activities and infiltrations of inflammatory macrophages, neutrophils and pathogenic T cells into the liver tissues. In addition, pretreatment with DMY significantly improved the survival rate of LPS/D-GalN−treated mice to 90% (9/10), compared to LPS/D-GalN−treated group (40%, 4/10). UPLC/MS platform-based comparative metabolomics approach was used to explore the serum metabolic profile in fulminant hepatic failure (FHF) mice with or without the DMY pretreatment. The results showed that LPS/D-GalN−induced hepatic damage is likely through perturbing amino acid metabolism, which leads to decreased pyruvate formation via catalysis of aminotransferases, and DMY treatment can prevent to a certain degree of these alterations in metabolic network in mouse caused by LPS/D-GalN. Mechanistic investigation demonstrated that DMY protects LPS or LPS/D-GalN−induced damages in cell or liver tissues mainly through de-regulating IKK/NFκB and Jak2/STAT3 signaling pathways. This report provides evidence-based knowledge to support the rationale for the use of A. pricei root extract in anti-inflammation and also its new function as hepatoprotetive agent against fulminant hepatitis.
doi:10.1371/journal.pone.0077626
PMCID: PMC3797050  PMID: 24143247
11.  Resveratrol Inhibits CD4+ T Cell Activation by Enhancing the Expression and Activity of Sirt1 
PLoS ONE  2013;8(9):e75139.
Resveratrol, a natural polyphenol compound, has broad effects on critical events, including inflammation, oxidation, cancer and aging. However, the function and molecular mechanisms of resveratrol on T cell activation are controversial. In the present study, we found that resveratrol significantly inhibits the activation and cytokine production of T cells in vitro and in vivo. Sirt1 expression was up-regulated in resveratrol-treated T cells. Once Sirt1 was down-regulated in the T cells, the resveratrol-induced inhibition of T cell activation noticeably diminished. The acetylation of c-Jun decreased and its translocation was impeded in the resveratrol-treated T cells. The incidence and severity of collagen-induced arthritis in the resveratrol-treated mice were considerably reduced.
doi:10.1371/journal.pone.0075139
PMCID: PMC3779207  PMID: 24073240
12.  Regulation of FOXOs and p53 by SIRT1 Modulators under Oxidative Stress 
PLoS ONE  2013;8(9):e73875.
Excessive reactive oxygen species (ROS) induce apoptosis and are associated with various diseases and with aging. SIRT1 (sirtuin-1), an NAD+-dependent protein deacetylase, decreases ROS levels and participates in cell survival under oxidative stress conditions. SIRT1 modulates the transcription factors p53, a tumor suppressor and inducer of apoptosis, and the forkhead O (FOXO) family, both of which play roles for cell survival and cell death. In this study, we aimed to know which is working greatly among p53 and FOXOs transcription factors in SIRT1’s cell protective functions under oxidative stress conditions. The antimycin A-induced increase in ROS levels and apoptosis was enhanced by SIRT1 inhibitors nicotinamide and splitomicin, whereas it was suppressed by a SIRT1 activator, resveratrol, and a SIRT1 cofactor, NAD+. SIRT1-siRNA abolished the effects of splitomicin and resveratrol. p53-knockdown experiment in C2C12 cells and experiment using p53-deficient HCT116 cells showed that splitomicin and resveratrol modulated apoptosis by p53-dependent and p53-independent pathways. In p53-independent cell protective pathway, we found that FOXO1, FOXO3a, and FOXO4 were involved in SOD2’s upregulation by resveratrol. The knockdown of these three FOXOs by siRNAs completely abolished the SOD2 induction, ROS reduction, and anti-apoptotic function of resveratrol. Our results indicate that FOXO1, FOXO3a and FOXO4, are indispensable for SIRT1-dependent cell survival against oxidative stress, although deacetylation of p53 has also some role for cell protective function of SIRT1.
doi:10.1371/journal.pone.0073875
PMCID: PMC3770600  PMID: 24040102
13.  EGF-Induced Bronchial Epithelial Cells Drive Neutrophil Chemotactic and Anti-Apoptotic Activity in Asthma 
PLoS ONE  2013;8(9):e72502.
Chronic damage and repair of the bronchial epithelium are features of asthma. We have previously reported that ex vivo stimulation of normal bronchial epithelial cells with epidermal growth factor (EGF), a key factor of epithelial repair, enhances the mechanisms of neutrophil accumulation, thereby promoting neutrophil defences during acute injury but potentially enhancing inflammation in chronic airway diseases. We have now sought to (i) determine whether this EGF-dependent pro-neutrophil activity is increased in asthma, where EGF and its epithelial receptor are over-expressed, and (ii) elucidate some of the mechanisms underlying this asthmatic epithelial-neutrophil interaction. Primary bronchial epithelial cells (PBEC) from healthy subjects, mild asthmatics and moderate-to-severe asthmatics (Mod/Sev) were stimulated with EGF, a model that mimics a repairing epithelium. Conditioned culture media (EGF-CM) were assessed for neutrophil chemotactic and anti-apoptotic activities and inflammatory mediator production. EGF induced the epithelium to produce soluble mediators with neutrophil chemotactic (p<0.001) and pro-survival (p = 0.021) activities which were related to the clinical severity of asthma (trend p = 0.010 and p = 0.009, respectively). This was associated with enhanced IL-6, IL-8, GM-CSF and TNF-α release, and cytokine-neutralising experiments using EGF-CM from Mod/Sev asthmatics demonstrated a role for GM-CSF in neutrophil survival (p<0.001). Pre-treatment of neutrophils with specific inhibitors of the myeloid-restricted class I phosphatidylinositol-3-OH kinase (PI(3)K) isoforms showed that the EGF-CM from Mod/Sev asthmatics depended on the γ (p<0.021) but not δ isoforms, while neutrophil survival required multiple class I PI(3)Ks. The EGF-induced chemotactic, but not pro-survival activity, involved RhoA signaling in neutrophils (p = 0.012). EGF whose activity is upregulated in asthma induces ex vivo the epithelium from asthmatic patients to produce pro-neutrophil activities; these are related to asthma severity and, in moderate-to-severe asthmatics, involves class IB PI(3)Kγ signaling, providing a potential therapeutic target for neutrophilic forms of asthma.
doi:10.1371/journal.pone.0072502
PMCID: PMC3770689  PMID: 24039773
14.  Trametes versicolor Protein YZP Activates Regulatory B Lymphocytes – Gene Identification through De Novo Assembly and Function Analysis in a Murine Acute Colitis Model 
PLoS ONE  2013;8(9):e72422.
Background
Trametes versicolor (Yun-Zhi) is a medicinal fungus used as a chemotherapy co-treatment to enhance anti-tumor immunity. Although the efficacies of T. versicolor extracts have been documented, the active ingredients and mechanisms underlying the actions of these extracts remain uncharacterized.
Results
We purified a new protein, YZP, from the fruiting bodies of T. versicolor and identified the gene encoding YZP using RNA-seq and de novo assembly technologies. YZP is a 12-kDa non-glycosylated protein comprising 139 amino acids, including an 18-amino acids signal peptide. YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d+ B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d. YZP-induced B cells suppressed approximately 40% of the LPS-activated macrophage production of inflammatory cytokines in a mixed leukocyte reaction and significantly alleviated the disease activity and colonic inflammation in a DSS-induced acute colitis murine model. Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.
Conclusions
We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene. We demonstrated that YZP activated CD1d+ Breg differentiation through TLR2/4-mediated signaling pathway, and the YZP-stimulated B cells exhibited anti-inflammatory efficacies in vitro and in murine acute colitis models.
doi:10.1371/journal.pone.0072422
PMCID: PMC3760908  PMID: 24019869
15.  Pathogenic Intestinal Bacteria Enhance Prostate Cancer Development via Systemic Activation of Immune Cells in Mice 
PLoS ONE  2013;8(8):e73933.
A role for microbes has been suspected in prostate cancer but difficult to confirm in human patients. We show here that a gastrointestinal (GI) tract bacterial infection is sufficient to enhance prostate intraepithelial neoplasia (PIN) and microinvasive carcinoma in a mouse model. We found that animals with a genetic predilection for dysregulation of wnt signaling, ApcMin/+ mutant mice, were significantly susceptible to prostate cancer in an inflammation-dependent manner following infection with Helicobacter hepaticus. Further, early neoplasia observed in infected ApcMin/+ mice was transmissible to uninfected mice by intraperitoneal injection of mesenteric lymph node (MLN) cells alone from H. hepaticus-infected mutant mice. Transmissibility of neoplasia was preventable by prior neutralization of inflammation using anti-TNF-α antibody in infected MLN donor mice. Taken together, these data confirm that systemic inflammation triggered by GI tract bacteria plays a pivotal role in tumorigenesis of the prostate gland.
doi:10.1371/journal.pone.0073933
PMCID: PMC3753256  PMID: 23991210
16.  Grape Seed Procyanidin Reversal of P-glycoprotein Associated Multi-Drug Resistance via Down-regulation of NF-κB and MAPK/ERK Mediated YB-1 Activity in A2780/T Cells 
PLoS ONE  2013;8(8):e71071.
The expression and function of P-glycoprotein (P-gp) is associated with the phenotype of multi-drug resistance (MDR), leading chemotherapy failure of patients suffered with cancer. Grape seed procyanidin(GSP) is a natural polyphenol supplement with anti-inflammatory effect. Present study assessed a new use of GSP on the MDR reversal activity and its possible molecular mechanisms in MDR1-overpressing paclitaxel resistant ovarian cancer cells. Our results showed GSP significantly enhanced the cytotoxicity of paclitaxel and adriamycin in paclitaxel resistant A2780/T cells but its parental A2780 cells. Furthermore, GSP strongly inhibited P-gp expression by blocking MDR1 gene transcription, as well as, increased the intracellular accumulation of the P-gp substrate rhodamine-123 in A2780/T cells. Nuclear factor-κB(NF-κB) activity, IκB degradation level and NF-κB/p65 nuclear translocation induced by lipopolysaccharide (LPS) and receptor activator for nuclear factor-κB ligand (RANKL) were markedly inhibited by pre-treatment with GSP. Meanwhile, GSP inhibited MAPK/ERK pathway by decreasing the phosphorylation of ERK1/2, resulting in reduced the Y-box binding protein 1 (YB-1) activation with blocking its nuclear translocation. Moreover, the up-regulation of P-gp expression, the activation of AKT/NF-κB and MAPK/ERK pathway induced by LPS was attenuated by GSP administration. Compared with PDTC and U1026, inhibitor of NF-κB and MAPK/ERK respectively, GSP showed the same tendency of down-regulating NF-κB and MAPK/ERK mediated YB-1 activities. Thus, GSP reverses P-gp associated MDR by inhibiting the function and expression of P-gp through down-regulation of NF-κB activity and MAPK/ERK pathway mediated YB-1 nuclear translocation, offering insight into the mechanism of reversing MDR by natural polyphenol supplement compounds. GSP could be a new potential MDR reversal agent used for combination therapy with chemotherapeutics in clinic.
doi:10.1371/journal.pone.0071071
PMCID: PMC3744527  PMID: 23967153
17.  Regulation of A1 by OX40 Contributes to CD8+ T Cell Survival and Anti-Tumor Activity 
PLoS ONE  2013;8(8):e70635.
The TNFR family member OX40 (CD134) is critical for optimal clonal expansion and survival of T cells. However, the intracellular targets of OX40 in CD8 T cells are not fully understood. Here we show that A1, a Bcl-2 family protein, is regulated by OX40 in effector CD8 T cells. In contrast to wild-type T cells, OX40-deficient CD8 T cells failed to maintain A1 expression driven by antigen. Conversely, enforced OX40 stimulation promoted A1 expression. In both situations, the expression of A1 directly correlated with CD8 T cell survival. In addition, exogenous expression of A1 in OX40-deficient CD8 T cells reversed their survival defect in vitro and in vivo. Moreover, forced expression of A1 in CD8 T cells from OX40-deficient mice restored the ability of these T cells to suppress tumor growth in a murine model. These results indicate that OX40 signals regulate CD8 T cell survival at least in part through maintaining expression of the anti-apoptotic molecule A1, and provide new insight into the mechanism by which OX40 may impact anti-tumor immunity.
doi:10.1371/journal.pone.0070635
PMCID: PMC3731243  PMID: 23936461
18.  Sulforaphane prevents pulmonary damage in response to inhaled arsenic by activating the Nrf2-defense response 
Toxicology and applied pharmacology  2012;265(3):292-299.
Exposure to arsenic is associated with an increased risk of lung disease. Novel strategies are needed to reduce the adverse health effects associated with arsenic exposure in the lung. Nrf2, a transcription factor that mediates an adaptive cellular defense response, is effective in detoxifying environmental insults and prevents a broad spectrum of diseases induced by environmental exposure to harmful substances. In this report, we tested whether Nrf2 activation protects mice from arsenic-induced toxicity. We used an in vivo arsenic inhalation model that is highly relevant to low environmental human exposure to arsenic-containing dusts. Two-week exposure to arsenic-containing dust resulted in pathological alterations, oxidative DNA damage, and mild apoptotic cell death in the lung; all of which were blocked by sulforaphane (SF) in an Nrf2-dependent manner. Mechanistically, SF-mediated activation of Nrf2 alleviated inflammatory responses by modulating cytokine production. This study provides strong evidence that dietary intervention targeting Nrf2 activation is a feasible approach to reduce adverse health effects associated with arsenic exposure.
doi:10.1016/j.taap.2012.08.028
PMCID: PMC3725323  PMID: 22975029
Nrf2; Keap1; Arsenic; Antioxidant response
19.  A High Frequency of Circulating Th22 and Th17 Cells in Patients with New Onset Graves’ Disease 
PLoS ONE  2013;8(7):e68446.
T-helper (Th) 22 and Th17 cells are involved in the pathogenesis of autoimmune diseases. However, their roles in the pathogenesis of Graves’disease (GD) are unclear. This study is aimed at examining the frequency of peripheral blood Th22, Th17, and Th1 cells and the levels of plasma IL-22, IL-17, and IFN-γ in patients with GD. A total of 27 patients with new onset GD and 27 gender- and age-matched healthy controls (HC) were examined for the frequency of peripheral blood Th22, Th17, and IFN-γ cells by flow cytometry. The concentrations of plasma IL-22, IL-17, and IFN-γ were examined by enzyme-linked immunosorbent assay. The levels of serum TSHR antibodies (A-TSHR), free triiodothyronine (FT3), free thyroxine (FT4), and thyroid stimulating hormone (TSH) were examined by radioimmunoassay and chemiluminescent assay, respectively. The levels of serum TSAb were examined by enzyme-linked immunosorbent assay. In comparison with those in the HC, significantly elevated percentages of Th22 and Th17 cells, but not Th1 cells, and increased levels of plasma IL-22 and IL-17, but not IFN-γ, were detected in GD patients (P<0.0001, for both). The percentages of both Th22 and Th17 cells and the levels of plasma IL-22 and IL-17 were correlated positively with the levels of serum TSAb in GD patients (r = 0.7944, P<0.0001; r = 0.8110, P<0.0001; r = 0.7101, p<0.0001; r = 0.7407, p<0.0001, respectively). Th22 and Th17 cells may contribute to the pathogenesis of GD.
doi:10.1371/journal.pone.0068446
PMCID: PMC3708941  PMID: 23874630
20.  Policy Trap and Optimal Subsidization Policy under Limited Supply of Vaccines 
PLoS ONE  2013;8(7):e67249.
We adopt a susceptible-infected-susceptible (SIS) model on a Barabási and Albert (BA) network to investigate the effects of different vaccine subsidization policies. The goal is to control the prevalence of the disease given a limited supply and voluntary uptake of vaccines. The results show a uniform subsidization policy is always harmful and increases the prevalence of the disease, because the lower degree individuals’ demand for vaccine crowds out the higher degree individuals’ demand. In the absence of an effective uniform policy, we explore a targeted subsidization policy which relies on a proxy variable instead of individuals’ connectivity. Findings show a poor proxy-based targeted program can still increase the disease prevalence and become a policy trap. The results are robust to general scale-free networks.
doi:10.1371/journal.pone.0067249
PMCID: PMC3698145  PMID: 23840869
21.  IGHV1-69-Encoded Antibodies Expressed in Chronic Lymphocytic Leukemia React with Malondialdehyde–Acetaldehyde Adduct, an Immunodominant Oxidation-Specific Epitope 
PLoS ONE  2013;8(6):e65203.
The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde–acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells.
doi:10.1371/journal.pone.0065203
PMCID: PMC3688726  PMID: 23840319
22.  Schistosomajaponicum Eggs Induce a Proinflammatory, Anti-Fibrogenic Phenotype in Hepatic Stellate Cells 
PLoS ONE  2013;8(6):e68479.
Hepatic fibrosis induced by egg deposition is the most serious pathology associated with chronic schistosomiasis, in which the hepatic stellate cell (HSC) plays a central role. While the effect of Schistosoma mansoni eggs on the fibrogenic phenotype of HSCs has been investigated, studies determining the effect of eggs of S. japonicum on HSCs are lacking. Disease caused by S. japonicum is much more severe than that resulting from S. mansoni infection so it is important to compare the pathologies caused by these two parasites, to determine whether this phenotype is due to the species interacting differently with the mammalian host. Accordingly, we investigated the effect of S. japonicum eggs on the human HSC cell line, LX-2, with and without TGF-β (Transforming Growth Factor beta) co-treatment, so as to determine the impact on genes associated with fibrogenesis, inflammation and matrix re-organisation. Activation status of HSCs was assessed by αSMA (Alpha Smooth Muscle Actin) immunofluorescence, accumulation of Oil Red O-stained lipid droplets and the relative expression of selected genes associated with activation. The fibrogenic phenotype of HSCs was inhibited by the presence of eggs both with or without TGF-β treatment, as evidenced by a lack of αSMA staining and reduced gene expression of αSMA and Col1A1 (Collagen 1A1). Unlike S. mansoni-treated cells, however, expression of the quiescent HSC marker PPAR-γ (Peroxisome Proliferator-Activated Receptor gamma) was not increased, nor was there accumulation of lipid droplets. In contrast, S. japonicum eggs induced the mRNA expression of MMP-9 (Matrix Metalloproteinase 9), CCL2 (Chemokine (C-C motif) Ligand 2) and IL-6 (Interleukin 6) in HSCs indicating that rather than inducing complete HSC quiescence, the eggs induced a proinflammatory phenotype. These results suggest HSCs in close proximity to S. japonicum eggs in the liver may play a role in the proinflammatory regulation of hepatic granuloma formation.
doi:10.1371/journal.pone.0068479
PMCID: PMC3686876  PMID: 23840855
23.  E3 Ubiquitin Ligase E6AP Negatively Regulates Adipogenesis by Downregulating Proadipogenic Factor C/EBPalpha 
PLoS ONE  2013;8(6):e65330.
CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is a key transcription factor involved in the adipocyte differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase inhibits adipocyte differentiation in 3T3-L1 cells as revealed by reduced lipid staining with oil red. Knock down of E6AP in mouse 3T3L1 preadipocytes is sufficient to convert them to adipocytes independent of external hormonal induction. C/EBPα protein level is drastically increased in E6AP deficient 3T3L1 preadipocytes while inverse is observed when wild type E6AP is over expressed. We show that transient transfection of wild type E6AP downregulates C/EBPα protein expression in a dose dependent manner while catalytically inactive E6AP-C843A rather stabilizes it. In addition, wild type E6AP inhibits expression of proadipogenic genes while E6AP-C843A enhances them. More importantly, overexpression of E6AP-C843A in mesenchymal progenitor cells promotes accumulation of lipid droplets while there is drastically reduced lipid droplet formation when E6AP is over expressed. Taken together, our finding suggests that E6AP may negatively control adipogenesis by inhibiting C/EBPα expression by targeting it to ubiquitin-proteasome pathway for degradation.
doi:10.1371/journal.pone.0065330
PMCID: PMC3676453  PMID: 23762344
24.  Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst 
PLoS ONE  2013;8(6):e64631.
Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the underlying mechanisms by which sCD40L/CD40 pathway contributes to inflammation and vascular diseases.
doi:10.1371/journal.pone.0064631
PMCID: PMC3675111  PMID: 23785403
25.  Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa 
PLoS ONE  2013;8(5):e64664.
Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (−)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05) in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.
doi:10.1371/journal.pone.0064664
PMCID: PMC3669385  PMID: 23741362

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