Zhengliang Chen

Mannan-binding lectin regulates dendritic cell maturation and cytokine production induced by lipopolysaccharide

BackgroundMannan-binding lectin (MBL) is a pattern-recognition molecule present in serum, which is involved in the innate immune defense by activating complement and promoting opsonophagocytosis. Dendritic cells (DCs) are professional antigen presenting cells (APCs) that are crucial for the initiation of adaptive immunity. Lipopolysaccharide (LPS) has been shown to be a strong activator of the inflammatory response and immune regulation. We first examined whether MBL modulated LPS-induced cellular responses, then investigated possible mechanisms of its inhibitory effect.ResultsMBL at higher concentrations (10-20 μg/ml) significantly attenuated LPS-induced maturation of monocyte-derived DCs (MDCs) and production of proinflammatory cytokines (e.g., IL-12 and TNF-α), and inhibited their ability to activate allogeneic T lymphocytes. It bound to immature MDCs at physiological calcium concentrations, and was optimal at supraphysiological calcium concentrations. MBL also bound directly to immature MDCs and attenuated the binding of LPS to the cell surfaces, resulting in decreased LPS-induced nuclear factor-κB (NF-κB) activity in these cells.ConclusionAll these data suggest that MBL could affect the functions of DCs by modifying LPS-induced cellular responses. This study supports an important role for MBL in the regulation of adaptive immune responses and inflammatory responses.

Mannan-binding lectin directly interacts with Toll-like receptor 4 and suppresses lipopolysaccharide-induced inflammatory cytokine secretion from THP-1 cells

Mannan-binding lectin (MBL) plays a key role in the lectin pathway of complement activation and can influence cytokine expression. Toll-like receptor 4 (TLR4) is expressed extensively and has been demonstrated to be involved in lipopolysaccharide (LPS)-induced signaling. We first sought to determine whether MBL exposure could modulate LPS-induced inflammatory cytokine secretion and nuclear factor-κB (NF-κB) activity by using the monocytoid cell line THP-1. We then investigated the possible mechanisms underlying any observed regulatory effect. Using ELISA and reverse transcriptase polymerase chain reaction (RT-PCR) analysis, we found that at both the protein and mRNA levels, treatment with MBL suppresses LPS-induced tumor-necrosis factor (TNF)-α and IL-12 production in THP-1 cells. An electrophoretic mobility shift assay and western blot analysis revealed that MBL treatment can inhibit LPS-induced NF-κB DNA binding and translocation in THP-1 cells. While the binding of MBL to THP-1 cells was evident at physiological calcium concentrations, this binding occurred optimally in response to supraphysiological calcium concentrations. This binding can be partly inhibited by treatment with either a soluble form of recombinant TLR4 extracellular domain or anti-TLR4 monoclonal antibody (HTA125). Activation of THP-1 cells by LPS treatment resulted in increased MBL binding. We also observed that MBL could directly bind to the extracellular domain of TLR4 in a dose-dependent manner, and this interaction could attenuate the binding of LPS to cell surfaces. Taken together, these data suggest that MBL may affect cytokine expression through modulation of LPS-/TLR-signaling pathways. These findings suggest that MBL may play an important role in both immune regulation and the signaling pathways involved in cytokine networks.

Scavenger receptor a restrains T‐cell activation and protects against concanavalin A‐induced hepatic injury

Negative feedback immune mechanisms are essential for maintenance of hepatic homeostasis and prevention of immune‐mediated liver injury. We show here that scavenger receptor A (SRA/CD204), a pattern recognition molecule, is highly up‐regulated in the livers of patients with autoimmune or viral hepatitis, and of mice during concanavalin A (Con A)‐induced hepatitis (CIH). Strikingly, genetic SRA ablation strongly sensitizes mice to Con A‐induced liver injury. SRA loss, increased mortality and liver pathology correlate with excessive production of IFN‐γ and heightened activation of T cells. Increased liver expression of SRA primarily occurs in mobilized hepatic myeloid cells during CIH, including CD11b+Gr‐1+ cells. Mechanistic studies establish that SRA on these cells functions as a negative regulator limiting T‐cell activity and cytokine production. SRA‐mediated protection from CIH is further validated by adoptive transfer of SRA+ hepatic mononuclear cells or administration of a lentivirus‐expressing SRA, which effectively ameliorates Con A‐induced hepatic injury. Also, CIH and clinical hepatitis are associated with increased levels of soluble SRA. This soluble SRA displays a direct T‐cell inhibitory effect and is capable of mitigating Con A‐induced liver pathology. Conclusion: Our findings demonstrate an unexpected role of SRA in attenuation of Con A‐induced, T‐cell‐mediated hepatic injury. We propose that SRA serves as an important negative feedback mechanism in liver immune homeostasis, and may be exploited for therapeutic treatment of inflammatory liver diseases. (HEPATOLOGY 2013)

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.

Evaluation of the effects of homologous platelet gel on healing lower extremity wounds in patients with diabetes.

The treatment of chronic diabetic wounds remains complicated, despite new insight into the cellular and molecular basis of wound healing and cutaneous regeneration. A growing body of clinical trials has shown that platelet release has a notable effectiveness on refractory ulcer healing. However, patients with chronic diabetic ulcers usually have poor general health, and the large-volume blood absence required to produce autologous platelet-rich plasma often causes adverse effects. To overcome the limitation, the homologous platelet gel (PG) from healthy donor was used for the treatment of chronic diabetic lower extremity wound in the study. We show here that homologous derived platelets significantly enhanced EVC304 cell and HaCaT cell proliferation and homologous PG was capable of prompting cell migration. Twenty-one patients with refractory diabetic lower extremity ulcers, who had no response to conventional treatments, were treated in this study. Our data indicated that homologous PG was effective for the enhancement and acceleration of diabetic lower extremity wounds healing. We propose that homologous PG appeared to enhance vascularization and epithelialization, which might induce a quicker healing process and and encourage controlled studies in future.

Mannan binding lectin attenuates double-stranded RNA-mediated TLR3 activation and innate immunity

Mannan binding lectin (MBL) functions as a pattern recognition molecule (PRM) which is able to initiate complement activation. Here, we characterize a previously unrecognized attribute of MBL as a double‐stranded RNA (dsRNA) binding protein capable of modifying Toll like receptor 3 (TLR3) activation. MBL interacts with poly(I:C) and suppresses poly(I:C)‐induced activation of TLR3 pathways and subsequent cytokine production. In addition, MBL binds to TLR3 directly. Surprisingly, disrupting the interaction between MBL and complement receptor 1 (CR1) or restraining the traffic of MBL to phagosome reversed the MBL limited TLR3 activation. We demonstrate the importance of MBL guided ligands intracellular localization, emphasizing the significance of understanding the dynamics of TLR agonists complexed with MBL or other PRMs inside the cell in immune defense.

Molecular chaperoning by glucose-regulated protein 170 in the extracellular milieu promotes macrophage-mediated pathogen sensing and innate immunity

Recognition of pathogen‐associated molecular patterns by innate immune receptors is essential for host defense responses. Although extracellular stress proteins are considered as indicators of the stressful conditions (e.g., infection or cell injury), the exact roles of these molecules in the extracellular milieu remain less defined. We found that glucose‐regulated protein 170 (Grp170), the largest stress protein and molecular chaperone, is highly efficient in binding CpG oligodeoxynucleotides (CpG‐ODN), the microbial DNA mimetic sensed by toll‐like receptor 9 (TLR9). Extracellular Grp170 markedly potentiates the endocytosis and internalization of CpG‐ODN by mouse bone marrow‐derived macrophages and directly interacts with endosomal TLR9 on cell entry. These molecular collaborations result in the synergistic activation of the MyD88‐dependent signaling and enhanced production of proinflammatory cytokines and nitric oxide in mouse primary macrophages as well as human THP‐1 monocyte‐derived macrophages, suggesting that Grp170 released from injured cells facilitates the sensing of pathogen‐associated “danger” signals by intracellular receptors. This CpG‐ODN chaperone complex‐promoted innate immunity confers increased resistance in mice to infection of Listeria monocytogenes compared with CpG‐ODN treatment alone. Our studies reveal a previously unrecognized attribute of Grp170 as a superior DNA‐binding chaperone capable of amplifying TLR9 activation on pathogen recognition, which provides a conceptual advance in understanding the dynamics of ancient chaperoning functions inside and outside the cell.—Zuo, D., Yu, X., Guo, C., Yi, H., Chen, X., Conrad, D. H., Guo, T. L., Chen, Z., Fisher, P. B., Subjeck, J. R., Wang, X.‐Y. Molecular chaperoning by glucose‐regulated protein 170 in the extracellular milieu promotes macrophage‐mediated pathogen sensing and innate immunity. FASEB J. 26, 1493‐1505 (2012). www.fasebj.org

Mannose-binding lectin inhibits monocyte proliferation through transforming growth factor-β1 and p38 signaling pathways.

Mannose-binding lectin (MBL), a plasma C-type lectin, plays an important role in innate immunity. However, the interaction, and the consequences of it, between MBL and the immune system remain ill defined. We have investigated the contributing mechanisms and effects of MBL on the proliferation of human monocytes. At lower concentrations (≤4 μg/ml) MBL was shown to partially enhance monocyte proliferation. By contrast, at higher concentrations (8–20 μg/ml) of MBL, cell proliferation was markedly attenuated. MBL-induced growth inhibition was associated with G0/G1 arrest, down-regulation of cyclin D1/D3, cyclin-dependent kinase (Cdk) 2/Cdk4 and up-regulation of the Cdk inhibitory protein Cip1/p21. Additionally, MBL induced apoptosis, and did so through caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage. Moreover, transforming growth factor (TGF)-β1 levels increased in the supernatants of MBL-stimulated monocyte cultures. We also found that MBL-dependent inhibition of monocyte proliferation could be reversed by the TGF-β receptor antagonist SB-431542, or by anti-TGF-β1 antibody, or by the mitogen-activated protein kinase (MAPK) inhibitors specific for p38 (SB203580), but not ERK (U0126) or JNK (SP600125). Thus, at high concentrations, MBL can affect the immune system by inhibiting monocyte proliferation, which suggests that MBL may exhibit anti-inflammatory effects.

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.