Daming Zuo

Unfolding the Role of Large Heat Shock Proteins: New Insights and Therapeutic Implications.

Heat shock proteins (HSPs) of eukaryotes are evolutionarily conserved molecules present in all the major intracellular organelles. They mainly function as molecular chaperones and participate in maintenance of protein homeostasis in physiological state and under stressful conditions. Despite their relative abundance, the large HSPs, i.e., Hsp110 and glucose-regulated protein 170 (Grp170), have received less attention compared to other conventional HSPs. These proteins are distantly related to the Hsp70 and belong to Hsp70 superfamily. Increased sizes of Hsp110 and Grp170, due to the presence of a loop structure, result in their exceptional capability in binding to polypeptide substrates or non-protein ligands, such as pathogen-associated molecules. These interactions that occur in the extracellular environment during tissue injury or microbial infection may lead to amplification of an immune response engaging both innate and adaptive immune components. Here, we review the current advances in understanding these large HSPs as molecular chaperones in proteostasis control and immune modulation as well as their therapeutic implications in treatment of cancer and neurodegeneration. Given their unique immunoregulatory activities, we also discuss the emerging evidence of their potential involvement in inflammatory and immune-related diseases.

Suppression of antigen-specific CD4+ T cell activation by SRA/CD204 through reducing the immunostimulatory capability of antigen-presenting cell

Pattern recognition scavenger receptor SRA/CD204, primarily expressed on specialized antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages, has been implicated in multiple physiological and pathological processes, including atherosclerosis, Alzheimer’s disease, endotoxic shock, host defense, and cancer development. SRA/CD204 was also recently shown to function as an attenuator of vaccine response and antitumor immunity. Here, we, for the first time, report that SRA/CD204 knockout (SRA−/−) mice developed a more robust CD4+ T cell response than wild-type mice after ovalbumin immunization. Splenic DCs from the immunized SRA−/− mice were much more efficient than those from WT mice in stimulating naïve OT-II cells, indicating that the suppressive activity of SRA/CD204 is mediated by DCs. Strikingly, antigen-exposed SRA−/− DCs with or without lipopolysaccharide treatment exhibited increased T-cell-stimulating activity in vitro, which was independent of the classical endocytic property of the SRA/CD204. Additionally, absence of SRA/CD204 resulted in significantly elevated IL12p35 expression in DCs upon CD40 ligation plus interferon gamma (IFN-γ) stimulation. Molecular studies reveal that SRA/CD204 inhibited the activation of STAT1, mitogen activated protein kinase p38, and nuclear factor-kappa B signaling activation in DCs treated with anti-CD40 antibodies and IFN-γ. Furthermore, splenocytes from the generated SRA−/− OT-II mice showed heightened proliferation upon stimulation with OVA protein or MHC-II-restricted OVA323–339 peptide compared with cells from the SRA+/+ OT-II mice. These results not only establish a new role of SRA/CD204 in limiting the intrinsic immunogenicity of APCs and CD4+ T cell activation but also provide additional insights into the molecular mechanisms involved in the immune suppression by this molecule.

Protective role of mouse MBL-C on intestinal mucosa during Shigella flexneri invasion

Abstract Mannan-binding lectin (MBL) is a C-type serum lectin, which is believed to play an important role in the innate immunity against a variety of pathogens. MBL can bind to sugar determinants of a wide variety of microorganisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Given that small intestine is a predominant site of extrahepatic expression of MBL, here we addressed the question whether MBL is involved in mucosal innate immunity. The carbohydrate recognition domain (CRD) genes of mouse MBL-C (mMBL-C) were cloned and expressed in Escherichia coli. Recombinant mMBL-C-CRD binds to Shigella flexneri 2a in a calcium-dependent manner and that interaction could be blocked by the anti-mMBL-C-CRD antibody. mMBL-C-CRD protein could inhibit the adhesion of S. flexneri 2a to intestinal mucosa, while administration of anti-mMBL-C-CRD antibody caused an increased level of bacteria adhesion in vitro. Administration of recombinant mMBL-C-CRD protein reduced the secretion of IL-6 and monocyte chemoattractant protein 1 from primary intestinal epithelial cells stimulated with S. flexneri 2a. Furthermore, neutralization of MBL activity by anti-MBL-C-CRD resulted in a significant increase in the number of S. flexneri 2a that colonized the intestines of BALB/c mice and attenuated the severity of inflammation seen in the areas of bacterial invasion. These findings suggest that mMBL-C may protect host intestinal mucosa by directly binding to the bacteria.

Polysaccharide extracted from Chinese white wax scale ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis-like symptoms in BALB/c mice

Atopic dermatitis (AD) is a common inflammatory skin disease with high rates of morbidity and is associated with erythema, pruritus, scaling of affected areas of skin. It is extremely important to introduce a therapeutic agent which has significant anti-inflammatory effect with less side-effect for treatment of AD. This study evaluated the effect of a natural compound from herbal extracts, the crude polysaccharide extracted from the white wax scale (CWPS), on AD-like mice. Repeated applications of 2,4-dinitrochlorobenzene (DNCB) were performed on ear and dorsal skin of BALB/c mice to induce AD-like symptoms and skin lesions. Oral administration of CWPS decreased serum IgE level and limited the infiltration of mast cells and eosinophils to the dermal tissues in the DNCB-induced AD mice. In addition, CWPS reduced Th1 and Th17 responses, leading to an attenuated cutaneous inflammatory response. Furthermore, in vitro study also demonstrated that CWPS limited T cell activation and cytokines (i.e. IFN-γ and IL-17) production induced by DNCB. We conclude that CWPS attenuates DNCB-induced AD-like skin lesion through modulating T cell-elicited immune responses and CD4+ T cell polarization, and could be exploited as a new therapeutic approach for AD.

Mannan‐binding lectin reduces CpG DNA‐induced inflammatory cytokine production by human monocytes

Mannan‐binding lectin (MBL) belongs to the collectin family and functions as an opsonin that can also initiate complement activation. Our previous study showed that MBL serves as a double‐stranded RNA binding protein that attenuates polyriboinosinic‐polyribocytidylic acid‐induced TLR3 activation. Prompted by these findings, in the present study cross‐talk between MBL and CpG‐DNA‐induced TLR9 activation was investigated. Here, it was found that MBL also interacts with the TLR9 agonist, CpG oligodeoxynucleotide (CpG‐ODN), in a calcium‐dependent manner. Purified MBL protein suppressed activation of nuclear factor‐kappa B signaling and subsequent production of proinflammatory cytokines from human monocytes induced by CpG‐ODN 2006. These observations indicate that MBL can down‐regulate CpG DNA‐induced TLR9 activation, emphasizing the importance of understanding the interaction of MBL with TLR agonist in host immune defense.

Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A) was administered intravenously to wild-type (WT) and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase activity, and inhibited production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-17A, and IFN-γ). In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism and could be exploited as a new therapeutic approach for autoimmune hepatitis.

Location of MBL-Associated Serine Proteases Binding Motifs on Human Mannan-Binding Lectin (MBL)

The lectin pathway provides an antibody independent route of complement activation. Mannan-binding lectin (MBL) can form compound with MBL-associated serine proteases (MASPs) through its collagen-like region (CLR) to initiate complement fixation. In this study, we designed and synthesized a range of peptides according to the sequence of CLR in human MBL, which were assumed to block the MBL-MASP interaction, in order to locate the serine protease binding motifs on human MBL. It was demonstrated that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. In addition, Arg32Cys, Gly35Asp and Gly37Glu mutant proteins have the similar serine protease binding characteristic with wild type MBL, but the binding between mutated MBL proteins and MASPs is much weaker than that between wild type MBL protein and MASPs.

Fucoidan from Fucus vesiculosus suppresses hepatitis B virus replication by enhancing extracellular signal-regulated Kinase activation.

BackgroundHepatitis B virus (HBV) infection is a serious public health problem leading to cirrhosis and hepatocellular carcinoma. As the clinical utility of current therapies is limited, the development of new therapeutic approaches for the prevention and treatment of HBV infection is imperative. Fucoidan is a natural sulfated polysaccharide that extracted from different species of brown seaweed, which was reported to exhibit various bioactivities. However, it remains unclear whether fucoidan influences HBV replication or not.MethodsThe HBV-infected mouse model was established by hydrodynamic injection of HBV replicative plasmid, and the mice were treated with saline or fucoidan respectively. Besides, we also tested the inhibitory effect of fucoidan against HBV infection in HBV-transfected cell lines.ResultsThe result showed that fucoidan from Fucus vesiculosus decreased serum HBV DNA, HBsAg and HBeAg levels and hepatic HBcAg expression in HBV-infected mice. Moreover, fucoidan treatment also suppressed intracellular HBcAg expression and the secretion of the HBV DNA as well as HBsAg and HBeAg in HBV-expressing cells. Furthermore, we proved that the inhibitory activity by fucoidan was due to the activation of the extracellular signal-regulated kinase (ERK) pathway and the subsequent production of type I interferon. Using specific inhibitor of ERK pathway abrogated the fucoidan-mediated inhibition of HBV replication.ConclusionThis study highlights that fucoidan might be served as an alternative therapeutic approach for the treatment of HBV infection.

Mannan-binding lectin at supraphysiological concentrations inhibits differentiation of dendritic cells from human CD14+ monocytes

Mannan‐binding lectin (MBL), a circulating C‐type lectin, is an important member of the defense collagen family. It exhibits a high potential for recognizing broad categories of pathogen‐associated molecular patterns and initiating complement cascade responses. DCs are well‐known specialist antigen‐presenting cells that significantly trigger specific T cell‐mediated immune responses. In our previous study, it was observed that high concentrations of MBL significantly attenuate LPS‐induced maturation of monocyte‐derived DCs (MoDCs). In the current study, it was postulated that MBL at similar supraphysiological concentrations would affect early differentiation of MoDCs in some way. CD14+ monocytes from human peripheral blood mononuclear cells were cultured with granulocyte‐macrophage colony‐stimulating factor and IL‐4 in the presence or absence of physiological (1 µg/mL) and supraphysiological concentrations (20 µg/mL) of MBL protein, respectively. Phenotypic analysis indicated that the differentiated DCs incubated with high concentrations of MBL expressed MHC class II and costimulatory molecules (e.g., CD80 and CD40) more weakly than did control groups. The secretion of IL‐10 and IL‐6 increased markedly, whereas their mixed lymphocyte reaction‐stimulating capacity decreased. Members of the signal transducer and activator of transcription family were also found to be differentially regulated. Thus, beyond the role of MBL as an opsonin, our data reveal a possible inhibitory effect of MBL at high concentrations in monocyte‐DC transition, which probably provides one way of regulating adaptive immune responses by strict regulation of DCs, making MBL a better prospect for controlling relevant pathological events such as autoimmune diseases.

Mannan-binding lectin, a serum collectin, suppresses T-cell proliferation via direct interaction with cell surface calreticulin and inhibition of proximal T-cell receptor signaling

Mannan binding lectin (MBL), initially reported to activate the complement pathway, is also known to be involved in the pathogenesis of autoimmune diseases. We report a thus far unknown function of MBL as a suppressor of T‐cell activation. MBL markedly inhibited T‐cell proliferation induced by anti‐CD3 and anti‐CD28 antibodies. Moreover, the presence of MBL during T‐cell priming interfered with proximal T‐cell receptor signaling by decreasing phosphorylation of Lck, ZAP‐70, and LAT. MBL bound to T cells through interaction between the collagen‐like region of MBL and calreticulin (CRT) expressed on the T‐cell surface. The neutralizing antibody against CRT abrogated MBL‐mediated suppression of T‐cell proliferation, suggesting that MBL down‐modulates T‐cell proliferation via cell surface CRT. We further demonstrated that the feature of MBL‐mediated T‐cell suppression is shared by other serum collectins (e.g., C1q and collectin 11). The concentrations of MBL correlated negatively with in vivo T‐cell activation status in patients with early‐stage silicosis. Furthermore, MBL efficiently inhibited activation and proliferation of autoreactive T cells derived from patients with silicosis, indicating that MBL serves as a negative feedback control of the T‐cell responses.—Zhao, N., Wu, J., Xiong, S., Zhang, L., Lu, X., Chen, S., Wu, Q., Wang, H., Liu, Y., Chen, Z., Zuo, D. Mannan‐binding lectin, a serum collectin, suppresses T‐cell proliferation via direct interaction with cell surface calreticulin and inhibition of proximal T‐cell receptor signaling. FASEB J. 31, 2405–2417 (2017). www.fasebj.org