Yingming Jiang

PECAM-1 Ligation Negatively Regulates TLR4 Signaling in Macrophages

Uncontrolled TLR4 signaling may induce excessive production of proinflammatory cytokines and lead to harmful inflammation; therefore, negative regulation of TLR4 signaling attracts much attention now. PECAM-1, a member of Ig-ITIM family, can mediate inhibitory signals in T cells and B cells. However, the role and the mechanisms of PECAM-1 in the regulation of TLR4-mediated LPS response in macrophages remain unclear. In this study, we demonstrate that PECAM-1 ligation with CD38-Fc fusion protein negatively regulates LPS-induced proinflammatory cytokine TNF-α, IL-6, and IFN-β production by inhibiting JNK, NF-κB, and IFN regulatory factor 3 activation in macrophages. In addition, PECAM-1 ligation-recruited Src homology region 2 domain-containing phosphatase 1 (SHP-1) and Src homology region 2 domain-containing phosphatase 2 (SHP-2) may be involved in the inhibitory effect of PECAM-1 on TLR4 signaling. Consistently, silencing of PECAM-1 enhances the macrophage response to LPS stimulation. Taken together with the data that PECAM-1 is constitutively expressed in macrophages and its expression is up-regulated by LPS stimulation, PECAM-1 might function as a feedback negative regulator of LPS inflammatory response in macrophages. This study may provide a potential target for intervention of inflammatory diseases.

Methyltransferase SETD2-Mediated Methylation of STAT1 Is Critical for Interferon Antiviral Activity

Interferon-α (IFNα) signaling is essential for antiviral response via induction of IFN-stimulated genes (ISGs). Through a non-biased high-throughput RNAi screening of 711 known epigenetic modifiers in cellular models of IFNα-mediated inhibition of HBV replication, we identified methyltransferase SETD2 as a critical amplifier of IFNα-mediated antiviral immunity. Conditional knockout mice with hepatocyte-specific deletion of Setd2 exhibit enhanced HBV infection. Mechanistically, SETD2 directly mediates STAT1 methylation on lysine 525 via its methyltransferase activity, which reinforces IFN-activated STAT1 phosphorylation and antiviral cellular response. In addition, SETD2 selectively catalyzes the tri-methylation of H3K36 on promoters of some ISGs such as ISG15, leading to gene activation. Our study identifies STAT1 methylation on K525 catalyzed by the methyltransferase SETD2 as an essential signaling event for IFNα-dependent antiviral immunity and indicates potential of SETD2 in controlling viral infections.

Nerve Growth Factor Promotes TLR4 Signaling-Induced Maturation of Human Dendritic Cells In Vitro through Inducible p75NTR 1

Nerve growth factor (NGF) has been shown to play important roles in the differentiation, function, and survival of immune cells, contributing to immune responses and pathogenesis of autoimmune diseases. Dendritic cells (DCs) are a potent initiator for immune and inflammatory responses upon recognition of pathogens via Toll-like receptors (TLR). However, expression of NGF and its receptors on human monocyte-derived DCs (MoDCs) and the role of NGF in the response of DCs to TLR ligands remain to be investigated. In the present study, we demonstrate that there were weak expressions of NGF and no expression of NGF receptors p140TrkA and p75NTR on human immature MoDCs, however, the expression of NGF and p75NTR on MoDCs could be significantly up-regulated by LPS in a dose- and time-dependent manner. NGF could markedly promote LPS-induced expression of HLA-DR, CD40, CD80, CD83, CD86, CCR7, secretion of IL-12p40 and proinflammatory cytokines IL-1, IL-6, TNF-α, and the T cell-stimulating capacity of MoDCs, indicating that NGF can promote LPS-induced DC maturation. The promoting effect of NGF on LPS-induced MoDCs maturation could be completely abolished by pretreatment of MoDCs with p75NTR antagonist, suggesting that LPS-induced p75NTR mediates the effect. Furthermore, increased activation of the p38MAPK and NF-κB pathways has been shown to be responsible for the NGF-promoted DC maturation. Therefore, NGF facilitates TLR4 signaling-induced maturation of human DCs through LPS-up-regulated p75NTR via activation of p38 MAPK and NF-κB pathways, providing another mechanism for the involvement of NGF in the immune responses and pathogenesis of autoimmune diseases.

Apoptotic cell administration enhances pancreatic islet engraftment by induction of regulatory T cells and tolerogenic dendritic cells

Apoptotic cell transfer has been found to be able to facilitate engraftment of allograft. However, the underlying mechanisms remain to be fully understood. Here we demonstrate that intravenous administration of donor apoptotic splenocytes can promote pancreatic islet engraftment by inducing generation of tolerogenic dendritic cells (Tol-DCs) and expansion of CD4+Foxp3+ regulatory T cells (Tregs). In vivo clearance of either dendritic cells (DCs) or Tregs prevented the induction of immune tolerance by apoptotic cell administration. Transient elimination of Tregs using anti-CD25, monoclonal antibody (mAb) abrogated the generation of Tol-DCs after administration of apoptotic splenocytes. Reciprocally, depletion of DCs within CD11c-DTR mice using diphtheria toxin (DT) prevented the generation of Tregs in the recipients with administration of apoptotic splenocytes. Induction of Tregs by Tol-DCs required direct cell contact between the two cell types, and programmed death 1 ligand (PD-L1) played important role in the Tregs expansion. Apoptotic cell administration failed to induce Tol-DCs in IL-10-deficient and Smad3-deficient mice, suggesting that IL-10 and transforming growth factor-β (TGF-β) are needed to maintain DCs in the tolerogenic state. Therefore, we demonstrate that Tol-DCs promote the expansion of Tregs via PD-L1 on their surface and reciprocally Tregs facilitate Tol-DCs to maintain transplantation tolerance induced by apoptotic cells via secreting IL-10 and TGF-β.

Regulation of hepatic lipogenesis by the zinc finger protein Zbtb20

Hepatic de novo lipogenesis (DNL) converts carbohydrates into triglycerides and is known to influence systemic lipid homoeostasis. Here, we demonstrate that the zinc finger protein Zbtb20 is required for DNL. Mice lacking Zbtb20 in the liver exhibit hypolipidemia and reduced levels of liver triglycerides, along with impaired hepatic lipogenesis. The expression of genes involved in glycolysis and DNL, including that of two ChREBP isoforms, is decreased in livers of knockout mice. Zbtb20 binds to and enhances the activity of the ChREBP-α promoter, suggesting that altered metabolic gene expression is mainly driven by ChREBP. In addition, ChREBP-β overexpression largely restores hepatic expression of genes involved in glucose and lipid metabolism, and increases plasma and liver triglyceride levels in knockout mice. Finally, we show that Zbtb20 ablation protects from diet-induced liver steatosis and improves hepatic insulin resistance. We suggest ZBTB20 is an essential regulator of hepatic lipogenesis and may be a therapeutic target for the treatment of fatty liver disease.

DC-CLM, a cadherin-like molecule cloned from human dendritic cells, inhibits growth of breast cancer cells

PurposeTo identify the characteristics and function of a cadherin-like molecule, cloned from a human dendritic cell (DC) cDNA library and designated DC-derived cadherin-like molecule (DC-CLM).MethodsThe mRNA expression of DC-CLM in tissues and cells was analyzed by Northern blot and RT-PCR, respectively. In order to express DC-CLM in target cells, we constructed a pcDNA3.1/DC-CLM expression vector and transfected it into MCF-7 human breast cancer cells. Tumor growth was demonstrated by cell proliferation and colony formation.ResultsDC-CLM cDNA encoded a protein of 260 amino acids and the gene was localized to chromosome 5q31. The predicted protein possessed a definitive cadherin-specific sequence motif and shared homology with classical cadherin. However, no transmembrane segment was observed in DC-CLM. Northern blot revealed the ubiquitous nature of DC-CLM transcripts in human tissues, with high expression in heart, brain, prostate, testis and ovary. RT-PCR demonstrated that DC-CLM was widely expressed in hematopoietic and epithelial tumor cell lines, but was not expressed in MCF-7. Interestingly, DC-CLM expression was upregulated in DC activated by lipopolysaccharides. DC-CLM expression in the stable transfectant (MCF-7/DC-CLM) was confirmed by RT-PCR and Western blot. DC-CLM protein was found to be secreted by MCF-7/DC-CLM but not expressed on the membrane of MCF-7/DC-CLM. DC-CLM transfection resulted in significant inhibition of in vitro growth and colony formation of MCF-7 cells.ConclusionsA cadherin-like molecule DC-CLM was cloned from human DC and it may be a soluble cadherin-like molecule for tumor suppression. DC-CLM was upregulated in activated DC and may be involved in the effector function of activated DC.

Lipopolysaccharide Promotes Nerve Growth Factor (NGF)-Induced Maturation and Activation of Dendritic Cells Via Up-Regulation of NGF Receptors P75

Lipopolysaccharide Promotes Nerve Growth Factor (NGF)Induced Maturation and Activation of Dendritic Cells Via UpRegulation of NGF Receptors P75 Yingming Jiang, Guoyou Chen, Lin Lu, Huazhang An, Xuetao Cao. Institute of Immunology, Second Military Medical University, Shanghai, China. Nerve growth factor (NGF) plays important roles in survival, development, and maintenance of peripheral and central nervous neurons. The biological activities of NGF are mediated by two receptors: the low-affinity NGF receptor p75 and the tyrosine protein kinase receptor TrkA. In addition to its role in the nervous system, NGF and its receptors are also expressed in the immune system, and NGF may regulate immune response, thus suggesting a possible autocrine manner of NGF to regulate immune system. However, there are few reports about regulation of dendritic cells (DCs), the most potent antigenpresenting cells, by NGF. In this study, we investigated the expression of NGF and its receptors in monocytes-derived DCs (MoDCs) and regulation of DCs by NGF. Flow cytometry showed that MoDCs did not express NGF receptor p75 and TrkA under normal conditions. So almost no response was observed when MoDCs were incubated with NGF even at concentration of 1000 ng/ml. When MoDCs were stimulated by various concentrations of Lipopolysaccharide (LPS) (1,10,100, or 1000 ng/ml) for different time (6,12,24,48 hours), expression of NGF receptors on MoDCs was detected by flow cytometry and western blot. It was found that p75 was up-regulated after 10 ng/ml LPS stimulation for 12h. Moreover, p75 expression was dose-dependently increased with LPS stimulation, with maximal effect at 100 ng/ml LPS. No expression of TrkA was found on MoDCs. It was found that NF-kB activation was involved in the up-regulation of p75 by LPS because increased p75 expression could be blocked by NF-kB inhibitor PDTC.We went further to investigate the biological functions of the increased p75 expression on MoDCs induced by LPS stimulation. After preincubation with 10 ng/ml LPS for 12 hours, MoDCs were washed with PBS buffer and treated with various concentrations of NGF. NGF could dose-dependently up-regulate the surface markers of CD40, CD80, CD83, CD86 and HLA-DR on MoDCs, and maximal effect was observed with 100ng/ml NGF for 24 hours. The secretion of IL-12 p40, TNF-a and IL-1 was dramatically enhanced by NGF in a dosedependent manner. Allostimulatory activity of MoDCs was increased significantly after NGF stimulation. All the above responses could be inhibited by preincubation of MoDCs by p75 receptor antagonist 30 min before NGF stimulation. Our data demonstrate that LPS promotes NGFinduced maturation and activation of MoDCs via up-regulation of NGF receptor p75. Data presented here indicate that inflammatory stumuli could result in increased expression of p75 receptor on MoDCs, leading to more sensitive of DCs to NGF, thus helpful to investigation of DC immunobiology and immunotherapy.