Weijuan Zhang

Blockade of Notch1 Signaling Alleviates Murine Lupus via Blunting Macrophage Activation and M2b Polarization

Patients with systemic lupus erythematosus (SLE) are found to be accompanied with innate immunity dysregulation including abnormally macrophage activation. But the functional polarization of the activated macrophages and its underlying molecular mechanism during the pathogenesis of SLE remains unknown. As an important local cellular interaction mechanism responsible for cell fate determination, Notch signaling is reported to exert crucial functions in the development and differentiation of various immunocytes, whereas its role in macrophage polarization is not fully understood. In this study, in the SLE murine model generated by immunization with activated lymphocyte-derived DNA (ALD-DNA), infiltrated macrophages in the nephritic tissues were found to exhibit activation and M2b functional polarization. Notch1 signaling activity was significantly upregulated in the ALD-DNA–induced M2b macrophages in vitro and in vivo. Furthermore, ALD-DNA–induced M2b polarization was found to be dependent on enhanced Notch1 signaling through accelerating NF-κB p50 translocation into the nucleus mediated by PI3K and MAPK pathways. Moreover, blockade of Notch1 signaling with γ-secretase inhibitor treatment before or after the disease initiation could ameliorate murine lupus through impeding macrophage M2b polarization. Our results implied that Notch1 signaling-dependent macrophage M2b polarization might play a pivotal role in the pathogenesis of SLE, which could provide Notch1 signaling blockade as a potential therapeutic approach for SLE disease.

Macrophage Differentiation and Polarization via Phosphatidylinositol 3-Kinase/Akt–ERK Signaling Pathway Conferred by Serum Amyloid P Component

Macrophage differentiation and polarization is influenced by, and act on, many processes associated with autoimmunity. However, the molecular mechanisms underlying macrophage polarization in systemic lupus erythematosus (SLE) remain largely debated. We previously demonstrated that macrophage M2b polarization conferred by activated lymphocyte-derived (ALD)-DNA immunization could initiate and propagate murine lupus nephritis. Serum amyloid P component (SAP), a conserved acute-phase protein in mice, has been reported to bind to DNA and modulate immune responses. In this study, murine SAP was shown to promote macrophage-mediated ALD-DNA uptake through binding to ALD-DNA (SAP/ALD-DNA). Moreover, macrophage phenotypic switch from a proinflammatory M2b phenotype induced by ALD-DNA alone to an anti-inflammatory M2a phenotype stimulated with SAP/ALD-DNA were found because of PI3K/Akt–ERK signaling activation. Both in vivo SAP supplements and adoptive transfer of ex vivo programmed M2a macrophages induced by SAP/ALD-DNA into SLE mice could efficiently alleviate lupus nephritis. Importantly, increased IL-10 secretion, accompanied by anti-inflammatory effect exerted by M2a macrophages, was found to predominantly impede macrophage M2b polarization. Furthermore, neutralization of IL-10 notably reduced the suppressive effect of M2a macrophages. Our results demonstrate that binding of SAP to ALD-DNA could switch macrophage phenotypic polarization from proinflammatory M2b to anti-inflammatory M2a via PI3K/Akt–ERK signaling activation, thus exerting protective and therapeutic interventions on murine lupus nephritis. These data provide a possible molecular mechanism responsible for modulation of macrophage polarization in the context of lupus nephritis and open a new potential therapeutic avenue for SLE.

AIM2 Facilitates the Apoptotic DNA-induced Systemic Lupus Erythematosus via Arbitrating Macrophage Functional Maturation

PurposeLupus nephritis, a major cause of morbidity in patients with systemic lupus erythematosus (SLE), is generally thought to be induced by macrophage-mediated inflammation following deposition of various autoantibodies in kidneys. We previously reported that macrophage aberrant activation induced by activated lymphocyte-derived apoptotic DNA (apopDNA) have been found to play pathogenic roles in the immunodysregulation in lupus nephritis. However, DNA sensor(s) involved in apopDNA-induced macrophage activation and lupus nephritis remains largely undefined. Herein, we aimed to reveal the DNA sensor(s) involved in SLE disease.MethodsCorrelation between the level of absent in melanoma 2 (AIM2), a cytoplasmic DNA receptor in the inflammasome pathway, and the clinical severity of SLE disease were analyzed in SLE patients as well as in lupus mice. Activated macrophages induced by apopDNA were analyzed by real-time PCR and western blot for AIM2 expression. After silencing of AIM2 via siRNA-mediated knockdown in vitro and in vivo, macrophage activation, inflammatory response, and SLE syndrome were assessed.ResultsAIM2 expression was closely correlated with the severity of disease in SLE patients and in lupus mice. Importantly, AIM2 expression was significantly increased in apopDNA-induced macrophages and closely correlated with macrophage activation. Knockdown of AIM2 significantly blunted apopDNA-induced macrophage activation. Furthermore, blockade of AIM2 expression notably ameliorated SLE syndrome via impeding macrophage activation and dampening inflammatory response in apopDNA-induced lupus mice.ConclusionsOur results implied that AIM2 might act as an important DNA sensor and a potential biomarker for apopDNA-induced macrophage functional maturation and SLE disease.

EZH2-mediated loss of miR-622 determines CXCR4 activation in hepatocellular carcinoma

The CXC chemokine receptor 4 (CXCR4) exerts a variety of functions at different steps of hepatocellular carcinoma (HCC) progression. The molecular mechanisms and therapeutic value of CXCR4 in the development of HCC remain undefined. Here we show that aberrant CXCR4 overexpression is associated with poor prognosis and aggressive characteristics of HCC. Suppression of CXCR4 activity via CXCR4 knockdown, AMD3100 or neutralizing antibody administration inhibits hepatoma cell tumorigenesis in vitro and in vivo. CXCR4 overexpression displays the opposite effects. Using Mir library screening we identify miR-622 as a regulator of CXCR4. Further studies show that miR-622 directly target the 3′ untranslated region of CXCR4 and is transcriptionally repressed by EZH2-induced H3K27 trimethylation and promoter methylation. EZH2/miR-622 promotes tumorigenesis through CXCR4. EZH2-mediated loss of miR-622 is found to correlate with CXCR4 overexpression and unfavourable prognosis in HCC patients. This study establishes EZH2/miR-622/CXCR4 as a potential adverse prognostic factor and therapeutic target for HCC patients.

Decreased Expression of Hepatocyte Nuclear Factor 4α (Hnf4α)/MicroRNA-122 (miR-122) Axis in Hepatitis B Virus-associated Hepatocellular Carcinoma Enhances Potential Oncogenic GALNT10 Protein Activity

Background: GALNT, the initial enzyme in mucin-type O-glycosylation, plays critical roles in cancer etiology. Results: GALNT10-induced cellular proliferation was associated with EGFR activation mediated by down-regulation of miR-122 in HBV-associated HCC. Conclusion: A regulatory pathway of Hnf4α/miR-122/GALNT10/EGFR may represent a possible mechanism underlying HBV-associated hepatocarcinogenesis. Significance: This finding provides a novel role for O-glycosylation in HCC pathogenesis. MicroRNA-122 (miR-122), a mammalian liver-specific miRNA, has been reported to play crucial roles in the control of diverse aspects of hepatic function and dysfunction, including viral infection and hepatocarcinogenesis. In this study, we explored the clinical significance, transcriptional regulation, and direct target of miR-122 in hepatitis B virus (HBV)-associated hepatocellular carcinoma. Reduced expression of miR-122 in patients with HBV-associated hepatocellular carcinoma was correlated with venous invasion and poor prognosis. Furthermore, UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase-10 (GALNT10) was identified as a bona fide target of miR-122 in hepatoma cells. Ectopic expression and knockdown studies showed that GALNT10 indeed promotes proliferation and apoptosis resistance of hepatoma cells in a glycosyltransferase-dependent manner. Critically, adverse correlation between miR-122 and GALNT10, a poor prognosticator of clinical outcome, was demonstrated in hepatoma patients. Hepatocyte nuclear factor 4α (Hnf4α), a liver-enriched transcription factor that activates miR-122 gene transcription, was suppressed in HBV-infected hepatoma cells. Chromatin immunoprecipitation assay showed significantly reduced association of Hnf4α with the miR-122 promoter in HBV-infected hepatoma cells. Moreover, GALNT10 was found to intensify O-glycosylation following signal activation of the epidermal growth factor receptor. In addition, in a therapeutic perspective, we proved that GALNT10 silencing increases sensitivity to sorafenib and doxorubicin challenge. In summary, our results reveal a novel Hnf4α/miR-122/GALNT10 regulatory pathway that facilitates EGF miR-122 activation and hepatoma growth in HBV-associated hepatocarcinogenesis.

Amelioration of Lupus Nephritis by Serum Amyloid P Component Gene Therapy with Distinct Mechanisms Varied from Different Stage of the Disease

Background Our previous study revealed that administration of syngeneic female BALB/c mice with excessive self activated lymphocyte-derived DNA (ALD-DNA) could induce systemic lupus erythematosus (SLE) disease, indicating that overload of self-DNA might exceed normal clearance ability and comprise the major source of autoantigens in lupus mice. Serum amyloid P component (SAP), an acute-phase serum protein with binding reactivity to DNA in mice, was proved to promote the clearance of free DNA and prevent mice against self-antigen induced autoimmune response. It is reasonable to hypothesize that SAP treatment might contribute to alleviation of SLE disease, whereas its role in ALD-DNA-induced lupus nephritis is not fully understood. Methodology/Principal Findings The ratios of SAP to DNA significantly decreased and were negatively correlated with the titers of anti-dsDNA antibodies in ALD-DNA-induced lupus mice, indicating SAP was relatively insufficient in lupus mice. Herein a pcDNA3-SAP plasmid (pSAP) was genetically constructed and intramuscularly injected into BALB/c mice. It was found that SAP protein purified from the serum of pSAP-treated mice bound efficiently to ALD-DNA and inhibited ALD-DNA-mediated innate immune response in vitro. Treatment of ALD-DNA-induced lupus mice with pSAP in the early stage of SLE disease with the onset of proteinuria reversed lupus nephritis via decreasing anti-dsDNA autoantibody production and immune complex (IC) deposition. Further administration of pSAP in the late stage of SLE disease that had established lupus nephritis alleviated proteinuria and ameliorated lupus nephritis. This therapeutic effect of SAP was not only attributable to the decreased levels of anti-dsDNA autoantibodies, but also associated with the decreased infiltration of lymphocytes and the reduced production of inflammatory markers. Conclusion/Significance These results suggest that SAP administration could effectively alleviated lupus nephritis via modulating anti-dsDNA antibody production and the inflammation followed IC deposition, and SAP-based intervening strategy may provide new approaches for treating SLE disease.

N-acetylglucosaminyltransferase V confers hepatoma cells with resistance to anoikis through EGFR/PAK1 activation

Elevated expression and activity of N-acetylglucosaminyltransferase V (Mgat5) in hepatocellular carcinoma (HCC) is a common early event involved in tumor invasion during hepatocarcinogenesis. A better understanding of the functional role and the molecular mechanism for Mgat5-targeted protein and downstream signaling pathway behind hepatoma invasion and metastasis is urgently needed. Here, we show that Mgat5 overexpression promoted anchorage-independent growth and inhibited anoikis in hepatoma cells. This effect was reversed by glycosyltransferase inactive mutant Mgat5 L188R transfection, α-mannosidase II inhibitor swainsonine treatment and N-acetyl glucosamine (GlcNAc) phosphotransferase (GPT) inhibitor tunicamycin administration. Mgat5 overexpression increased p21-activated kinase 1 (PAK1) expression and shRNA-mediated PAK1 knockdown and kinase inactivation with kinase dead mutant PAK1 K299R coexpression or allosteric inhibitor P21-activated kinase inhibitor III (IPA3) treatment reversed anoikis resistance in Mgat5-overexpressed hepatoma cells. Furthermore, Mgat5 overexpression upregulated β-1-6-GlcNAc branched N-glycosylation and following phosphorylation of epidermal growth factor receptor (EGFR) in hepatoma cells. EGFR tyrosine kinase inhibitors AG1478 and Iressa treatment declined anchorage-independent growth and anoikis resistance, which could be rescued by constitutive active mutant PAK1 T423E coexpression in Mgat5-overexpressed hepatoma cells. Conversely, knockdown of Mgat5 reduced EGFR/PAK1-dependent anoikis resistance, which could be reversed by PAK1 T423E. These results identified Mgat5-mediated β-1-6-GlcNAc branched N-glycosylation and following activation of EGFR as a potential novel upstream molecular event for PAK1-induced anoikis resistance in hepatoma cells, implicating that molecular targeted therapeutics against Mgat5/EGFR/PAK1 might open a new avenue for personalized medicine in advanced-stage HCC patients.

Polycomb-mediated loss of microRNA let-7c determines inflammatory macrophage polarization via PAK1-dependent NF-κB pathway.

Serine/threonine kinase family members p21-activated kinases (PAKs) are important regulators of cytoskeletal remodeling and cell motility in mononuclear phagocytic system, but their role in macrophage differentiation and polarization remains obscure. We have shown here that inflammatory stimuli induced PAK1 overexpression in human and murine macrophages. Elevated expression of PAK1 contributed to macrophage M1 polarization and lipopolysaccharide (LPS)-induced endotoxin shock. We further observed that epigenetic loss of microRNA let-7c due to enhancer of zeste homolog 2 (EZH2) upregulation determined PAK1 elevation and inflammatory phenotype in M1 macrophages. EZH2/let-7c/PAK1 axis promotes macrophage M1 polarization via NIK-IKK-NF-κB signaling. Moreover, pharmacological and genetic ablation with EZH2/let-7c/PAK1 axis blunted inflammatory phenotype in M1 macrophages. Critically, either myeloid-restricted PAK1 deletion (PAK1Lyz2cre) or pharmacological and genetic ablation with EZH2/let-7c/PAK1 signal resulted in resistance to LPS-induced endotoxin shock via blunting macrophage M1 polarization. PAK1, therefore, is an essential controller of inflammatory macrophage polarization, regulating immune responses against pathogenic stimuli.

DNA-dependent Activator of Interferon-regulatory Factors (DAI) Promotes Lupus Nephritis by Activating the Calcium Pathway

Background: Macrophage M2b polarization conferred by self-DNA immunization initiates and propagates lupus nephritis. Results: Knockdown of DNA-dependent activator of interferon-regulatory factors (DAI) ameliorates SLE syndrome via blunting macrophage M2b polarization. Conclusion: DAI functions as a DNA sensor in self-DNA-induced macrophage M2b polarization and lupus nephritis. Significance: We disclose the mechanism by which self-DNA induces macrophage M2b polarization and lupus nephritis. DNA-dependent activator of interferon-regulatory factors (DAI) functions as a cytoplasmic DNA sensor that activates the innate immune system. We previously found that activated lymphocyte-derived self-apoptotic DNA (ALD-DNA) immunization led to pathological macrophage activation and M2b polarization, which could initiate and propagate murine lupus nephritis. However, the specific DNA sensor(s) as well as underlying molecular mechanisms involved in ALD-DNA-induced macrophage M2b polarization in systemic lupus erythematosus (SLE) disease remains unknown. In this study, we reported that DAI expression was significantly increased in SLE patients as well as in lupus mice. Gain- and loss-of-function studies revealed that DAI was involved in ALD-DNA-induced macrophage activation and M2b polarization. Moreover, ALD-DNA notably induced dimerization/oligomerization of DAI and consequently activation of nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF3) signaling pathways via calcium signaling, resulting in macrophage activation and M2b polarization. More importantly, blockade of DAI in vivo or selective knockdown of DAI in macrophages could ameliorate SLE syndrome via blunting macrophage M2b polarization and inhibiting inflammatory response in lupus mice. Our results suggest that DAI could function as a DNA sensor and a regulator in ALD-DNA-induced macrophage M2b polarization and lupus nephritis, providing the possible molecular mechanisms involved in ALD-DNA-induced macrophage M2b polarization in SLE disease and making DAI as a potential therapeutic target for the treatment of SLE.

Identification of serum CCL15 in hepatocellular carcinoma.

Background:Early serum detection is of critical importance to improve the therapy for hepatocellular carcinoma (HCC), one of the most deadly cancers. Hepatitis infection is a leading cause of HCC.Methods:In the present study, we collected total serum samples with informed consent from 80 HCC patients with HBV (+)/cirrhosis (+), 80 patients with benign diseases (50 liver cirrhosis patients and 30 HBV-infected patients) and 60 healthy controls. Analysis was by using surface-enhanced laser desorption/ionisation-time-of-flight mass spectroscopy (SELDI-TOF-MS) to find new serum markers of HCC. SELDI peaks were isolated by SDS–PAGE, identified by LC-MS/MS and validated by immunohistochemistry (IHC) in liver tissues. Migration and invasion assay were performed to test the ability of cell migration and invasion in vitro.Results:SELDI-TOF-MS revealed a band at 7777 M/Z in the serum samples from HCC patients but not from healthy controls or patients with benign diseases. The protein (7777.27 M/Z) in the proteomic signature was identified as C-C motif chemokine 15 (CCL15) by peptide mass fingerprinting. A significant increase in serum CCL15 was detected in HCC patients. Functional analysis showed that HCC cell expressed CCL15, which in turn promoted HCC cell migration and invasion.Conclusion:CCL15 may be a specific proteomic biomarker of HCC, which has an important role in tumorigenesis and tumour invasion.