Microbiol Immunol 2015; 59: 231–237 doi: 10.1111/1348-0421.12245


Mannan-binding lectin reduces CpG DNA-induced inflammatory cytokine production by human monocytes Yuan Tang1, Di Ma1, Siqi Ming1, Liyun Zhang1, Jia Zhou1, Guiqiu Shan2, Zhengliang Chen1, Xiao Lu1 and Daming Zuo1,3 1

Department of Immunology, Southern Medical University, Guangzhou 510515, 2Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, 510010 and 3State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China

ABSTRACT 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 CpGDNA-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. Key words

CpG oligodeoxynucleotides, mannan binding lectin, Toll like receptor 9.

The first line of host defense in the mammalian innate immune system is the immediate detection of, and response to, infective organisms. This response relies on recognition of the molecular motifs conserved within a class of microbes, termed pathogen-associated molecular patterns, that are recognized by PRRs (1). TLRs, one such family of PRRs, play a vital role in eradicating invading pathogens upon encounter with biochemically diverse pathogen molecules (2, 3). Among them is TLR9, which is essential for recognition of the specific unmethylated CpG motifs that are prevalent in microbial or synthetic oligonucleotides containing a CpG motif (CpG-ODNs) (4). TLR9 is expressed by numerous cells of the immune system, including dendritic cells,

macrophages, B lymphocytes, monocytes and NK cells (5, 6). TLR9-mediated signaling proceeds through myeloid differentiation protein-88, which activates the IRAK1-TRAF6-TAK1 pathway. The signaling cascade culminates in the activation of several transcription factors, including NF-kB, activating protein-1, CCAAT/ enhancer binding protein and cAMP-responsive element-binding protein, which directly upregulate cytokine/chemokine gene expression (4, 7). A wide spectrum of soluble innate immune PRPs has been identified (8); these include soluble complement receptors, collectins, ficolins and pentraxins, all of which have secreted forms. One such collectin is MBL (also called mannose-binding protein), a major soluble PRP of

Correspondence Daming Zuo, Department of Immunology, School of Basic Medicine, Southern Medical University, Guangzhou, 510515, China. Tel: þ86 20 6164 8220; Fax: þ86 20 6164 8220; email: [email protected] Additional correspondence: Xiao Lu, Department of Immunology, School of Basic Medicine, Southern Medical University, Guangzhou, 510515, China. Tel: þ86 20 6164 8220; Fax: þ86 20 6164 8220; email: [email protected] Received 17 October 2014; revised 28 January 2015; accepted 5 February 2015. List of Abbreviations: CLR, collagen-like region; CMV, cytomegalovirus; CpG-ODN, CpG oligodeoxynucleotides; CRD, carbohydrate recognition domain; MBL, mannan binding lectin; NF-kB, nuclear factor-kappa B; poly(I;C), polyriboinosinic-polyribocytidylic acid; PRP, pattern recognition protein; PRR, pattern recognition receptor.

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the innate immune system that binds to oligosaccharide structures on the surfaces of microorganisms, leading to the killing of bound microbes by complement activation and phagocytosis (9). In addition, accumulating studies suggest that MBL is able to modulate inflammatory responses during bacterial and viral infection (10–12). Our previous study showed that MBL can suppress LPSinduced TNF-a and IL-12 production in THP-1 cells and monocyte-derived dendritic cells by inhibiting LPSinduced NF-kB DNA binding and translocation (13, 14). In addition, we found that MBL attenuates allogeneic T cell proliferation induced by LPS-primed monocytederived dendritic cells (14). Ip et al. have also observed a novel collaboration between MBL and TLR2/6 that is initiated upon engulfment of Staphylococcus aureus (11). MBL traffics into early phagosomes and colocalizes with recruited TLR2, promoting an inflammatory response (11). In our recent study, we found that MBL binds to poly(I:C), after which MBL may carry poly(I:C) to phagosomes, resulting in reduced TLR3 activation (15). Palaniyar et al. reported that surfactant protein D, a member of the collectin family that is similar to MBL, has inhibitory effects on the generation of antiDNA autoantibody (16). However, the immunomodulatory role of MBL in CpG-ODN induced TLR9 activation needs to be further investigated. In the present study, we showed that MBL downregulates CpG-ODN 2006-triggered TLR9 activation and that MBL binds to CpG-ODN 2006 in calciumdependent manner. MBL protein suppresses activation of NF-kB signaling and subsequent production of proinflammatory cytokines from human monocytes cells induced by CpG-ODN 2006. The data indicate that MBL may cross-talk with TLR9 agonist in the innate immune response, which broadens our understanding of the cooperation between MBL and TLR signaling in host defense.

MATERIALS AND METHODS Cell isolation Peripheral blood mononuclear cells were isolated from heparinized whole blood by use of Ficoll density gradient centrifugation and monocytes were purified from the PBMCs by immunomagnetic negative selection using a human monocyte isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). Protein and oligodeoxynucleotides Mannan-binding lectin was isolated from human plasma by affinity chromatography on a mannan-agarose column (Sigma, Poole, UK) and subsequent anion232

exchange chromatography using Mono-Q HR 5/5 column (Pharmacia Biotech Europe, Orsay, France) as previously described (13). Possible residual endotoxin in the purified protein was removed by a Detoxi-Gel endotoxin removing column (Pierce, Rockford, IL, USA) and amounts of endotoxin in the protein preparations were detected by a limulus amebocyte lysate kit (Biowhittaker, Walkersville, MD, USA). Recombinant CLR and CRD of MBL were expressed in Escherichia coli using a pET expression system (Novagen, Madison, WI, USA) and purified by nickel-chelating resins (GE Healthcare, Piscataway, NJ, USA) according to the protocols. CpG-ODN 2006 (50 -TCGTCGT TTTGTC GTTTTGTCGTT-30 ) was synthesized with a phosphorothioate backbone (Takara Biotechnology, Dalian, China). Biotin-labeled CpG-ODN 2006 was purchased from Invivogen (San Diego, CA, USA). Direct binding of MBL to CpG-ODN 2006 Mannan-binding lectin (2 mg) was incubated at room temperature with different concentrations of biotinlabeled CpG-ODN 2006 for 30 min in TBS/Ca2þ buffer (50 mM Tris, 150 mM NaCl, 20 mM CaCl2, pH 7.2). For competition studies, MBL was incubated with biotin-CpG-ODN 2006 in the presence of increasing concentrations of unlabeled CpG-ODN 2006. MBL-biotin-CpG-ODN 2006 complex was run on 10% SDS–PAGE under non-reducing conditions and probed with HRP–streptavidin after transferring to a polyvinylidene fluoride membrane (Millipore, Temecula, CA, USA). Reactions were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific, Fremont, CA, USA) for immunoblotting. For immunoprecipitation, MBL-biotin-CpG-ODN 2006 complex was incubated with anti-MBL antibody (HY-13-101) and protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. Eluted immunoprecipitate was run on SDS–sPAGE and examined by HRP–streptavidin and anti-MBL antibody after membrane transferring. For ELISA assays, microtiter wells (Nunc, Kamstrup, Denmark) were coated overnight at 4 °C with 20 mg/ mL of CpG-ODN 2006. Plates were incubated at RT for 1 hr with different concentrations of MBL in TBS/ Ca2þ or TBS/EDTA buffer (50 mM Tris, 150 mM NaCl, 20 mM EDTA, pH 7.2) followed by anti-MBL antibody incubation. The amounts of bound CpGODN 2006 were determined using colorimetric assays after incubation with HRP-conjugated secondary antibody. For the ligand competition assay, CpGODN 2006 was incubated with MBL protein in the presence of increasing concentrations of mannan. © 2015 The Societies and Wiley Publishing Asia Pty Ltd

MBL reduces inflammatory cytokines

Cell stimulation Human monocytes were incubated with CpG-ODN 2006 (5 mg/mL) in the presence or absence of MBL (10 mg/mL). MBL protein was pre-incubated with CpGODN 2006 for 30 min at room temperature for complex formation prior to incubation. Culture supernatants were collected 24 hr after stimulation and cleared of debris by centrifugation. Degree of expression of TNF-a and IL-6 in the culture supernatants was assayed with ELISA kits (eBiosciences, San Diego, CA, USA) according to the manufacturer's instructions. Cell signaling Human monocytes were incubated with CpG-ODN 2006 (5 mg/mL) in the presence or absence of MBL (10 mg/mL) at the indicated times. Protein lysates prepared using radioimmunoprecipitation assay buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, pH 7.4) were separated by SDS–PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked in Tris-buffered saline containing 0.05% Tween20 and 5% BSA for 1 hr at room temperature, then incubated overnight at 4 °C with the appropriate primary antibodies against phospho-p65 (Cell Signaling Technology, Danvers, MA, USA) or b-actin (Sigma–Aldrich, St Louis, MO, USA). After washing with Tris-buffered saline containing 0.05% Tween-20, the blots were incubated with appropriate HRP-conjugated secondary antibody (Multisciences, Hangzhou, China). Visualization was completed with enhanced chemiluminescence reagents (Thermo Fisher Scientific). Statistical analysis Data are presented as mean  SD. One-way anova followed by Bonferroni adjustment was used for comparisons among multiple groups. Student's t-test was used for comparisons between two groups. A probability of P < 0.05 was considered significant.

RESULTS MBL directly binds with CpG-ODN 2006 Our previous study showed that MBL binds to TLR4 ligand LPS (13) and TLR3 ligand poly(I:C) (15). Ip et al. have also observed that MBL can interact with both lipoteichoic acid and peptidoglycan in a calciumdependent manner (11). We therefore assessed the interaction between MBL and TLR9 ligand, CpG-ODN 2006 by incubating purified MBL protein with biotin labeled CpG-ODN 2006 and determining MBL association with biotin-CpG-ODN 2006 by western blotting © 2015 The Societies and Wiley Publishing Asia Pty Ltd

using streptavidin-HRP and anti-MBL antibodies (Fig. 1a). A competition study showed that MBLbiotin-CpG-ODN 2006 complex formation was blocked by the presence of excessive unlabeled CpG-ODN 2006 (Fig. 1b). We confirmed the MBL-biotin-CpG-ODN 2006 complex by co-immunoprecipitation assay (Fig. 1c). Additional study demonstrated that MBL was able to bind to CpG-ODN 2006 in solid phase in a dose-dependent manner (Fig. 1d). It is also important to note that the interaction between MBL and CpG-ODN 2006 is calcium dependent and can be blocked by EDTA (Fig. 1d). Our findings also indicate that MBL interacts with CpG-ODN 2006 using its CRD (Fig. 1e). A ligand competition study showed that MBL-CpG-ODN 2006 association is blocked by the presence of excessive mannan, a novel ligand for MBL (Fig. 1f). MBL attenuates CpG-ODN 2006-stimulated expression of inflammatory cytokines in monocytes The observation of MBL binding with CpG-ODN 2006 prompted us to determine whether and how MBL regulates inflammatory cytokine responses upon CpGODN 2006 stimulation. We treated isolated monocytes with CpG-ODN 2006 either alone or in complex with MBL that had been generated by preincubation for 30 min at room temperature. CpG-ODN 2006 complexed with MBL attenuated production of TNF-a and IL-6 compared with CpG-ODN 2006 alone, as shown by ELISA assays (Fig. 2a,b). Additionally, we also saw decreased IL-6 production by monocytes when we complexed CpG-ODN 2006 with titrated amounts of MBL (Fig. 2c). MBL attenuates CpG-ODN 2006-induced activation of NF-kB signaling Unmethylated CpG DNA binding to the TLR9 initiates cascades of phosphorylation and transcriptional activation of NF-kB that result in the production of inflammatory cytokines (17). We found that MBLCpG-ODN 2006 complex induced less phosphorylation of NF-kB p65 than did CpG-ODN 2006 stimulation alone (Fig. 3).

DISCUSSION Increasing evidence indicates an important role for MBL in modulating inflammatory response (18–20). Studies by Ip et al. have demonstrated that MBL cooperates with TLR2/TLR6 to increase NF-kB activation and cytokine response to S. aureus (11). We previously observed that MBL regulates LPS-induced maturation of human DCs 233

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Fig. 1. MBL interacts with CpG-ODN 2006 directly. (a) Dose-dependent binding of CpG-ODN 2006 to MBL. MBL was incubated with increasing concentrations of biotin-CpG-ODN 2006 at room temperature, followed by immunoblotting analysis using streptavidin. IB, immunoblotting. (b) Competition assays of MBL and CpG-ODN 2006 interaction. MBL was incubated with biotin-CpG-ODN 2006 in the presence of excess unlabeled CpG-ODN 2006, followed by immunoblotting analysis. IB, immunoblotting. (c) MBL interacts with CpG-ODN 2006 by forming a complex. MBL was incubated with biotin-CpG-ODN 2006 and then immunoprecipitated with anti-MBL. The eluted immunoprecipitate was run on SDS–PAGE and blotted with horseradish peroxidase-streptavidin and antibody against MBL. (d) Analysis of MBL binding to CpG-ODN 2006 using ELISA assays. Different concentrations of MBL were incubated with microtiter plates pre-coated with CpG-ODN 2006 in TBS/Ca2þ or TBS/EDTA buffer. The concentrations of bound MBL were determined using colorimetric assays. **, P < 0.01. (e) Analysis of recombinant CRD or CLR of MBL binding to CpG-ODN 2006 by ELISA assays. Different concentrations of recombinant proteins were incubated with microtiter plates pre-coated with CpG-ODN 2006 in TBS/Ca2þ buffer, followed by anti-His antibody incubation and the concentrations of bound protein determined using colorimetric assays. Representative data from three independent experiments with similar results are shown. **, P < 0.01. (f) Competitive binding of MBL and CpG-ODN 2006 with mannan. MBL was incubated with microtiter plates pre-coated with CpG-ODN 2006 in the presence of excess mannan, followed by anti-MBL antibodies incubation and colorimetric assays. **, P < 0.01. Representative data from three independent experiments with similar results are shown.


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MBL reduces inflammatory cytokines

Fig. 2. MBL reduces CpG-ODN 2006-induced inflammatory cytokine production. Human monocytes were stimulated with CpG-ODN 2006 in the presence or absence of MBL. Protein concentrations of (a) TNF-a and(b) IL-6 were evaluated using ELISA assays. **, P < 0.01. The data shown are from three independent experiments with similar results. (c) Effect of titrated MBL complexed with CpG-ODN 2006 on IL-6 production. The data shown represent two independent experiments with similar results. *, P < 0.05; **, P < 0.01, compared to the group without MBL protein.

by interacting with TLR4 and also inhibits TLR3 agonist poly(I:C)-induced innate immune responses (13–15). In the present study, we have reported the contribution of MBL to CpG-ODN 2006-elicited TLR9 activation and demonstrated that MBL is capable of down-regulating TLR9 signaling. We observed that MBL binds to CpGODN 2006 and suppresses activation of NF-kB signaling and subsequent production of proinflammatory cytokines from monocytes induced by CpG-ODN 2006. Mannan-binding lectin belongs to a family of proteins called collectins, which consist of CLRs and C-type lectin domains that are also called CRDs (20, 21). Similar to other collectins, MBL shows selective and calcium© 2015 The Societies and Wiley Publishing Asia Pty Ltd

dependent binding to terminal sugars d-mannose, lfucose and N-acetyl-d-glucosamine, which is present on the surface of many microorganisms (20). Our recent study revealed another recognition property of MBL in interactions with DNA substrate. Indeed, the ability of MBL to bind to TLR ligands such as peptidoglycan, lipoteichoic acid, poly(I:C) and LPS has been observed previously (11, 13–15, 22). Here, we have provided evidence that MBL can also bind to the TLR9 agonist CpG-ODN 2006 in a calcium-dependent manner. In addition, excess mannan interferes with the binding between MBL and CpG-ODN 2006, suggesting that CpG-ODN 2006 and saccharide ligands have several 235

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Fig. 3. MBL attenuates CpG-ODN 2006-induced activation of NF-kB signaling. Human monocytes were treated with CpG-ODN 2006 or CpG-ODN 2006 in complex with MBL for the times indicated. Cell lysates were analyzed using immunoblotting for phosphorylation of NF-kB p65. b-actin served as a loading control. The data shown represent two independent experiments with similar results.

similar binding sites on MBL. Considering the inhibitory activity is moderate, it is possible that some other amino acid residues besides mannan recognition sites on CRD are associated with CpG motif binding; this remains to be determined. Palaniyar et al. reported that MBL and other collectins (e.g., surfactant proteins A and D) can interact with DNA from a variety of origins, including bacteria and synthetic oligonucleotides, thus playing an important role in reducing the inflammation caused by DNA in lungs and other tissues (23, 24). Another study has also shown that murine MBL binds to HBsAg in a calcium-dependent manner and inhibits antigen-specific IgM and IgG immune responses to soluble glycoprotein antigen in vivo (25). Toll-like receptor-9-dependent signaling is a vital contributor to innate immune defense against CMV infection (26). Several studies using in vivo patient models have reported an association between MBL gene polymorphisms and risk of CMV infection (27, 28), whereas an in vitro study showed that both recombinant and native human MBL inhibit human CMV infection (29). MBL incubation reportedly reduces phosphorylation of p65 upon CMV stimulation in human embryonic pulmonary fibroblasts (29), which is consistent with our present finding that MBL inhibits phosphorylation activity of p65 initiated by CpG-ODN 2006 in monocytes. It should be noted that in the current study we used a type B CpG-ODN (i.e., CpG-ODN 2006), which is known to preferentially activate Th1-like immune responses and proinflammatory responses (30, 31). In contrast, type A CpG-ODNs are weak stimulators of TLR9 dependent NF-kB signaling and pro-inflammatory cytokine production but strongly induce IFN-a production by plasmacytoid dendritic cells (31, 32). Whether MBL also cross-talks with type A CpG-ODNs in driving type I IFN responses remains of interest. In our previous studies, we have demonstrated direct interactions of MBL with TLR3 (15) and 236

TLR4 (13). The potential interaction between MBL and TLR9 also need to be explored in further studies. Recognition of microbial non-self is based on interactions between pattern recognition molecules, including membrane PRRs and soluble PRPs; cross-talk serves to avoid misinterpretation of signals from non-dangerous sources and to facilitate appropriate immune responses to a particular microbe. Complement is a system of soluble PRPs and plays an important role in innate immune response. C3 suppresses CpG-ODN 2006 uptake into human monocytes, thus blocking CpG motif-induced TNF-a and IL-6 secretion (33). It has also been reported that binding of the complement-derived anaphylatoxins C5a and C3a to their receptors modulates TLR4, TLR2/6 and TLR9 signaling (34). Our present results show that MBL inhibits CpG-ODN 2006-mediated immune responses, suggesting that MBL has an anti-inflammatory effect during bacterial and DNA virus infections. Direct interactions between MBL and TLR ligands may be a decisive factor in MBL-mediated regulation of TLR responses. Consequently, cross-talk between membranebound PRRs localized on the cellular or endosomal membranes and soluble PRPs in the extracellular milieu may play a critical role in innate immunity.

ACKNOWLEDGMENTS This work was supported in part by grants from the Natural Science Foundation of China (No. 81202329 and No. 31370875), Foundation for Distinguished Young Teacher in Higher Education of Guangdong Province Yq2013034 and Medical Scientific Research Foundation of Guangdong Province A2013362. DISCLOSURE The authors declare no financial or commercial conflicts of interest.

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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 stu...
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