Shuxun Liu

Involvement of ERK, p38 and NF‐κB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells

Toll‐like receptors (TLR) are sentinel receptors capable of recognizing pathogen‐associated molecule patterns (PAMP) such as lipopolysaccharide (LPS) and CpG‐containing oligonucleotides (CpG ODN). TLR2 and TLR4 are major receptors for Gram‐positive and Gram‐negative bacterial cell wall components, respectively. TLR9 is necessary for CpG signalling. LPS or CpG ODN can activate immature dendritic cells (DC) and induce DC maturation characterized by production of cytokines, up‐regulation of co‐stimulatory molecules, and increased ability to activate T cells. However, little is known regarding the regulation of TLR gene expression in mouse DC. In this study, we investigated the regulation of TLR2, TLR4 and TLR9 gene expression by LPS in murine immature DC. TLR2, TLR4 and TLR9 mRNA were up‐regulated following LPS stimulation. The up‐regulation of TLR9 expression coincided with significantly increased production of tumour necrosis factor‐α induced by LPS plus CpG ODN. While inhibition of extracellular signal‐related kinase and NF‐κB activation suppressed the up‐regulation of the expression of TLR2, TLR4 and TLR9 mRNA, inhibition of p38 kinase prevented the up‐regulation of TLR2 and TLR4 mRNA expression but enhanced the up‐regulation of TLR9 expression. These results demonstrated that TLR2, TLR4 and TLR9 gene expression was differently regulated by LPS in mouse immature DC. Up‐regulation of TLR2, TLR4 and TLR9 expression by LPS might promote the overall responses of DC to bacteria and help to explain the synergy between LPS and other bacterial products in the induction of cytokine production.

Phosphatase SHP-1 promotes TLR- and RIG-I-activated production of type I interferon by inhibiting the kinase IRAK1

Unbalanced production of proinflammatory cytokines and type I interferons in immune responses may lead to immunopathology; thus, the mechanisms that ensure the beneficial production of proinflammatory cytokines and type I interferons are of particular importance. Here we demonstrate that the phosphatase SHP-1 negatively regulated Toll-like receptor–mediated production of proinflammatory cytokines by inhibiting activation of the transcription factor NF-κB and mitogen-activated protein kinase. Simultaneously, SHP-1 increased the production of type I interferon mediated by Toll-like receptors and the helicase RIG-I by directly binding to and inhibiting activation of the kinase IRAK1. Our data demonstrate that SHP-1 contributes to immune homeostasis by balancing the production of proinflammatory cytokines and type I interferons in the innate immune response.

The Involvement of TNF-α-Related Apoptosis-Inducing Ligand in the Enhanced Cytotoxicity of IFN-β-Stimulated Human Dendritic Cells to Tumor Cells

TNF-α-related apoptosis-inducing ligand (TRAIL) is characterized by its preferential induction of apoptosis of tumor cells but not normal cells. Dendritic cells (DCs), besides their role as APCs, now have been demonstrated to exert cytotoxicity or cytostasis on some tumor cells. Here, we report that both human CD34+ stem cell-derived DCs (CD34DCs) and human CD14+ monocyte-derived DCs (MoDCs) express TRAIL and exhibit cytotoxicity to some types of tumor cells partially through TRAIL. Moderate expression of TRAIL appeared on CD34DCs from the 8th day of culture and was also seen on freshly isolated monocytes. The level of TRAIL expression remained constant until DC maturation. TRAIL expression on immature CD34DCs or MoDCs was greatly up-regulated after IFN-β stimulation. Moreover, IFN-β could strikingly enhance the ability of CD34DCs or MoDCs to kill TRAIL-sensitive tumor cells, but LPS did not have such an effect. The up-regulation of TRAIL on IFN-β-stimulated DCs partially contributed to the increased cytotoxicity of DCs. Pretreatment of TRAIL-sensitive tumor cells with caspase-3 inhibitor could significantly increase their resistance to the cytotoxicity of IFN-β-stimulated DCs. In contrast, NF-κB inhibitor could significantly increase the sensitivity of tumor cells to the killing by nonstimulated or LPS-stimulated DCs. Our studies demonstrate that IFN-β-stimulated DCs are functionally cytotoxic. Thus, an innate mechanism of DC-mediated antitumor immunity might exist in vivo in which DCs act as effectors to directly kill tumor cells partially via TRAIL. Subsequently, DCs act as APCs involved in the uptake, processing, and presentation of apoptotic tumor Ags to cross-prime CD8+ CTL cells.

Efficient induction of antitumor T cell immunity by exosomes derived from heat‐shocked lymphoma cells

Exosomes secreted by tumor cells could serve as a promising immunotherapeutic tumor vaccine. Heat shock proteins (HSP) induced in tumor cells by heat shock are molecular chaperones with potent adjuvant activity in the induction of antigen‐specific T cell responses. To improve exosome‐based tumor vaccines, we have investigated the efficacy of exosomes derived from heat‐shocked mouse B lymphoma cells (HS‐Exo) in the induction of antitumor immune responses. We found that HS‐Exo, compared with control exosomes derived from the same cells (Exo), contain more HSP60 and HSP90 and increased amounts of molecules involved in immunogenicity including MHC class I, MHC class II, CD40, CD86, RANTES and IL‐1β. Furthermore, HS‐Exo induce both phenotypic and functional maturation of dendritic cells more efficiently. HS‐Exo immunization activates T cell responses more potently. Importantly, HS‐Exo induce dramatically increased antitumor immune responses compared to control exosomes from the same cells in prophylaxis and therapeutic in vivo lymphoma models. We further demonstrate that CD8+ T cells are the predominant T cell subset responsible for the antitumor effect of HS‐Exo and that CD4+ T cells are necessary in the induction phase of tumor rejection in a prophylaxis model. These findings provide a novel strategy to improve the efficacy of exosome‐based tumor vaccines.

Up-regulation of TLR9 gene expression by LPS in mouse macrophages via activation of NF-κB, ERK and p38 MAPK signal pathways

Toll-like receptors (TLR) are critical in the activation of macrophages by bacterial products. It has been shown that TLR2 and TLR4 mediate lipopolysaccharide (LPS) and lipoproteins signal transduction, respectively. Regulation of TLR2 and TLR4 expression by LPS was considered to be one of the mechanisms to control the overall responses of immune cells to bacteria. However, little is known about whether the other members of TLR family are regulated by LPS. Recently, TLR9 was demonstrated to be essential for CpG DNA signaling. Given the effective immune modulation by CpG DNA, regulation of TLR9 expression might play important role in controlling the overall responses of immune cells to bacteria. In this study, regulation of TLR9 gene expression in mouse macrophage cell line RAW264.7 by LPS was investigated. Semiquantitative RT-PCR was performed to determine gene expression of TLR9. Following LPS stimulation, TLR9 gene expression was upregulated within 1 h and reached peak level at about 3 h. LPS stimulation activated NF-kappaB, ERK and p38 MAPK signal pathways. Pretreatment of macrophages with inhibitors of NF-kappaB, ERK and p38 MAPK signal pathways inhibited LPS-induced upregulation of TLR9 mRNA expression. Our results demonstrated that LPS stimulation could upregulate gene expression of TLR9 via NF-kappaB, ERK, and p38 MAPK signal pathways in macrophages, indicating that macrophages with increased TLR9 expression induced by LPS might respond to invading bacteria more effectively.

Involvement of leptin signaling in the survival and maturation of bone marrow-derived dendritic cells.

Previous studies demonstrated that lymphocyte development is impaired in leptin receptor (Ob‐R)‐deficient db/db mice. However, it remains unclear whether or not leptin signaling plays a physiological role in dendritic cell (DC) development and function. In this study, we first detected Ob‐R expression in murine DC. Using db/db mice at a pre‐diabetic stage, we demonstrate that the total number of DC generated from bone marrow (BM) cultures is significantly lower than in WT controls. Similarly, selective blockade of leptin with a soluble mouse Ob‐R chimera (Ob‐R:Fc) inhibited DC generation in wild‐type BM cultures. The reduced DC yield in db/db BM culture was attributed to significantly increased apoptosis, which was associated with dysregulated expression of Bcl‐2 family genes. Moreover, db/db DC displayed markedly reduced expression of co‐stimulatory molecules and a Th2‐type cytokine profile, with a poor capacity to stimulate allogeneic T cell proliferation. Consistent with their impaired DC phenotype and function, db/db DC showed significantly down‐regulated activities of the PI3K/Akt pathway as well as STAT‐3 and IκB‐α. In conclusion, our findings demonstrate the involvement of leptin signaling in DC survival and maturation.

Histone Methyltransferase Ash1l Suppresses Interleukin-6 Production and Inflammatory Autoimmune Diseases by Inducing the Ubiquitin-Editing Enzyme A20

Histone modifications play important roles in multiple physiological processes by regulating gene expression. However, the roles of histone modifications in immunity remain poorly understood. Here we report that Ash1l, a H3K4 methyltransferase, suppressed interleukin-6 (IL-6), and tumor necrosis factor (TNF) production in Toll-like receptor (TLR)-triggered macrophages, protecting mice from sepsis. Ash1l-silenced mice were more susceptible to autoimmune disease as a result of enhanced IL-6 production. Ash1l enhanced A20 expression through induction of H3K4 modification at the Tnfaip3 promoter via H3K4 methyltransferase activity of Ash1l SET (Su[var]3-9, E[z] and trithorax) domain. Ash1l suppressed NF-κB, mitogen-activated protein kinase (MAPK) pathways, and subsequent IL-6 production via facilitating A20-mediated NF-κB signal modulator NEMO and transducer TRAF6 deubiquitination. Therefore, Ash1l-mediated H3K4 methylation at the Tnfaip3 promoter is required for controlling innate IL-6 production and suppressing inflammatory autoimmune diseases, providing mechanistic insight into epigenetic modulation of immune responses and inflammation.

HIF-dependent induction of adenosine receptor A2b skews human dendritic cells to a Th2-stimulating phenotype under hypoxia

Hypoxia is a common characteristic of many pathological and physiological conditions that can markedly change cellular metabolism and cause the accumulation of extracellular adenosine. Recent studies have shown that adenosine can modulate the function of certain immune cell types through binding with different adenosine receptors. Our previous studies have shown that hypoxia has an effect on the biological activity of dendritic cells (DCs) by inducing their differentiation towards a Th2 polarising phenotype. However, the mechanisms underlying this suppression remain unclear. In this study, we have demonstrated that hypoxic mDCs predominantly express adenosine receptor A2b. The A2b receptor antagonist MRS1754 was able to increase the production of IL‐12p70 and TNF‐α by hypoxic mDCs and elevate the amount of Th1 cytokine IFN‐γ production in a mDCs‐T‐cell co‐culture system. We also found that the effect of hypoxia on IL‐12p70 production was mediated via increased intracellular cAMP levels through the up‐regulation of A2b adenosine receptor and the preferential expression of adenosine A2b receptors in hypoxic mDCs was HIF‐1α dependent. Therefore, the hypoxic mDCs could provide a useful tool for researching the function of A2bR in human DCs. Our results offer new insights into understanding the molecular mechanisms underlying the biological activities of DCs in local‐tissue hypoxic microenvironments.

Rhbdd3 controls autoimmunity by suppressing the production of IL-6 by dendritic cells via K27-linked ubiquitination of the regulator NEMO

Excessive activation of dendritic cells (DCs) leads to the development of autoimmune and inflammatory diseases, which has prompted a search for regulators of DC activation. Here we report that Rhbdd3, a member of the rhomboid family of proteases, suppressed the activation of DCs and production of interleukin 6 (IL-6) triggered by Toll-like receptors (TLRs). Rhbdd3-deficient mice spontaneously developed autoimmune diseases characterized by an increased abundance of the TH17 subset of helper T cells and decreased number of regulatory T cells due to the increase in IL-6 from DCs. Rhbdd3 directly bound to Lys27 (K27)-linked polyubiquitin chains on Lys302 of the modulator NEMO (IKKγ) via the ubiquitin-binding–association (UBA) domain in endosomes. Rhbdd3 further recruited the deubiquitinase A20 via K27-linked polyubiquitin chains on Lys268 to inhibit K63-linked polyubiquitination of NEMO and thus suppressed activation of the transcription factor NF-κB in DCs. Our data identify Rhbdd3 as a critical regulator of DC activation and indicate K27-linked polyubiquitination is a potent ubiquitin-linked pattern involved in the control of autoimmunity.

Heat shock up-regulates expression of Toll-like receptor-2 and Toll-like receptor-4 in human monocytes via p38 kinase signal pathway.

Heat stress can alert innate immunity by inducing stress proteins such as heat‐shock proteins (HSPs). However, it remains unclear whether heat stress affects the activation of antigen‐presenting cell (APC) in response to pathogen‐associated molecule patterns (PAMPs) by directly regulating pathogen recognition receptors (PRRs). As an important kind of PRRs, Toll‐like receptors (TLRs) play critical roles in the activation of immune system. In this study, we demonstrated that heat shock up‐regulated the expression of HSP70 as well as TLR2 and TLR4 in monocytes. The induction of TLRs was prior to that of HSP70, which suggesting the up‐regulation of TLR2 and TLR4 might be independent of the induction of HSP70. Heat shock activated p38 kinase, extracellular signal‐related kinase (ERK) and nuclear factor‐kappa B (NF‐κB) signal pathways in monocytes. Pretreatment with specific inhibitor of p38 kinase, but not those of ERK and NF‐κB, inhibited heat shock‐induced up‐regulation of TLR2 and TLR4. This indicates that p38 pathway takes part in heat shock‐induced up‐regulation of TLR2 and TLR4. Heat shock also increased lipoteichoic acid‐ or lipopolysaccharide‐induced interleukin‐6 production by monocytes. These results suggest that the p38 kinase‐mediated up‐regulation of TLR2 and TLR4 might be involved in the enhanced response to PAMP in human monocytes induced by heat shock.