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Keywords:

  • DC;
  • Immunomodulation;
  • Th2;
  • Triterpenoid

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Dendritic cells (DC) play a central role in the initiation and regulation of immune responses. Increasing evidence has indicated that manipulation of DC can serve as a therapeutic mechanism for immunomodulation. In this study we tested some unique compounds isolated from Antrodia cinnamomea, a medicinal fungus in Taiwan, on mouse bone marrow-derived DC activation. A triterpenoid methyl antcinate K (me-AntK) promoted DC maturation by enhancing the expression of MHC class II, CD86, and reducing the endocytosis. TNF-α, MCP-1, and MIP-1β were secreted by DC after me-AntK treatment, indicating augmentation of innate immunity by me-AntK. Interestingly, the me-AntK-activated DC induced Ag-specific T-cell proliferation and facilitated Th2 differentiation. Examining signaling responses, we found that me-AntK treatment uniquely activated JNK and ERK in DC. Our results demonstrate that me-AntK is the first natural triterpenoid to promote the ability of DC to prime Th2 responses. This suggests that me-AntK can potentially be applied to enhance immune responses and modulate DC function in immunotherapy.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

DC are specialized leukocytes which take up and process Ag in peripheral tissues and then transport and present Ag to activate T cells in secondary lymphoid organs 1, 2. DC are heterogeneous but all subsets have intrinsic and cooperative Ag-presenting function 3, 4. Exposure of DC to pathogen products or pro-inflammatory stimuli induces DC maturation, which dramatically enhances the ability of DC to activate Ag-specific T cells 5. TLR are important receptors involved in detection of PAMP in DC. TLR ligation induces the activation of NF-κB and MAP kinase pathways, resulting in DC maturation, which is characterized by upregulation of MHC class II and costimulatory molecule expression as well as cytokine and chemokine production 6. Befitting the key regulatory role of DC in immune responses, DC are being developed for the treatment of cancers, allergies, and viral infections, and as adjuvants for potent new vaccines to prevent or treat cancers and infectious diseases 7, 8. Thus, DC are manipulated as pharmacological targets for discovering novel biological modifiers of immune responses 9–11.

Natural products and their derivatives have historically been a rich source of therapeutic agents and lead molecules in drug discovery 12. A number of biologically active natural products have been isolated from Aphyllophorales, many of which are known as polypores. Polypores are a large group of terrestrial fungi of the phylum Basidiomycota (basidiomycetes), and they are a major source of pharmacologically active substances 13. For example, Ganoderma lucidum (Ling Zhi or Reishi), a well-known fungus that has been used for medicinal purposes for centuries, has been studied extensively 14, 15. A precious basidiomycete Antrodia cinnamomea [syn. A. camphorata] (Ac) restrictively grows in a unique host, the endemic and endangered tree Cinnamomum kanehirai Hay. (Lauraceae) in Taiwan 16, 17. Ac is a folk medicine and has been used to treat abdominal pain, diarrhea, hypertension, tumor, and to improve immunity and liver function 18. Recently, researchers have studied the biological effects of bioactive components extracted or purified from its fruiting bodies, pure culture mycelia, and culture filtrate (cultured broth) 19–22. However, it has been shown that the cultured mycelia did not produce certain bioactive compounds, which were only isolated from fruiting bodies of Ac 23, 24. Thus, the pharmacological and medical application of Ac remains to be explored by more convincing investigations.

Many bioactive components have been isolated from Ac, such as triterpenoids, polysaccharides, proteins, fatty acids, and phenyl derivatives 18. The triterpenoids, a large and structurally diverse group of natural products derived from squalene or related acyclic 30-carbon precursors, are uniquely abundant in Ac, especially in its fruiting bodies. This large group of natural products displays well over 100 distinct skeletons and has well-characterized biological activities 25, 26. Studies have shown that triterpenoids can modulate the function of immune cells 27–29. Although the effect of some triterpenoids on DC has been reported 30–32, many triterpenoid compounds still remain to be examined. Several triterpenoids have been isolated from Ac previously 33, 34. We have recently purified a number of triterpenoids from fruiting bodies of Ac and evaluated their anti-inflammatory activity 23, 35–37. In this study, we tested the effect of these triterpenoids on mouse bone marrow-derived DC (BMDC). Methyl antcinate K (me-AntK), a recently identified triterpenoid isolated from Ac (unpublished data) or another mushroom Antrodia salmonea (As) 38, was shown to display potent stimulating activity on DC.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Activation of DC by a triterpenoid me-Ant K

We have identified some unique triterpenoids isolated from the fruiting bodies of Ac and As and evaluated their biological effects 23, 35–38. However, their immunomodulatory activity is not known. DC play a central role in immune system. Thus, DC are selected for evaluating the immunomodulatory effect of these compounds. We tested some reported triterpenoid compounds, including eburicoic acid, dehydroeburicoic acid, sulphurenic acid, dehydrosulphurenic acid, 3-keto-dehydrosulphurenic acid, 15α-acetyl-dehydrosulphurenic acid, versisponic acid D, zhankuic acid A, B, C, antcin A, C, K, and me-Ant K. In addition, we included two newly identified triterpenoids from Ac, antcin N (3α, 7β, 12α-trihydroxy-4α-methylergosta-8, 24(29)-dien-11-on-26-oic acid) and me-Ant N (unpublished data), which have the same molecular weight as antcin K (3α, 4β, 7β-trihydroxy-4α-methylergosta-8, 24(29)-dien-11-on-26-oic acid) and me-Ant K, respectively (Fig. 1A). To determine the effect of these triterpenoids on DC, we first examined the BMDC cultures by microscopy after treatment of these compounds for 16 h. All compounds had >90–95% purity and were used at 50 μM as the maximal bioactivity was shown at this concentration for some compounds 23, 36, 38. In addition, all compounds had no significant cytotoxicity at this dose as measured by PI staining (data not shown). DC aggregation was observed after the compound me-Ant K treatment (data not shown), indicating that me-Ant K can potentially activate DC. Using flow cytometry, we found that me-Ant K, and to a lesser extent me-Ant N, induced upregulation of MHC class II and CD86 on DC while other triterpernoids had no effect (Fig. 1B). The degree of MHC class II and CD86 upregulation with me-Ant K approximates, but is less than, that seen with LPS stimulation.

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Figure 1. The structures of four triterpenoid compounds and their effects on BMDC activation. (A) The structure of antcin K, antcin N, me-Ant K, and me-Ant N. Antcin N and me-Ant N are newly identified triterpenoids from Ac. (B) BMDC were incubated with compounds (50 μM) (black line) or DMSO (control, gray line) for 16 h. LPS (100 ng/mL) treated cells (black line) or untreated cells are shown in the right panels. Dotted lines represent staining with an isotype-matched control Ab. DC activation was determined by flow cytometry after staining with mAb specific for MHC class II and CD86. The change of MFI from control to treatment was indicated. Data are representative of three independent experiments.

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Promotion of DC maturation by me-Ant K

To expand the study of the effect of me-Ant K on DC, we examined a number of other cell surface markers in resting versus me-Ant K treated cells (Fig. 2A). We found that me-Ant K induced upregulation of CD54 to nearly the extent of LPS stimulation. Likewise, both LPS and me-Ant K caused downregulation of CD119 (IFN-γ receptor), which is the phenotype of mature DC 39. However, me-Ant K failed to upregulate the expression of CD80 and CD40. DC activation is accompanied by reduced endocytosis of large molecules, such as dextran. Indeed, me-AntK treatment reduced uptake of FITC-labeled dextran to a similar degree as seen in LPS treated cells (Fig. 2B). The me-Ant-K-activated DC also showed the morphology of mature DC, which express a lot of dendrites on the surface, when examined by microscopy (data not shown). These results suggest that me-AntK can activate DC and promote DC maturation. Although me-AntN can weakly activate DC (Fig. 1B), we did not observe a significant effect of me-Ant N on CD54 or CD119 expression, or on endocytosis (unpublished data).

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Figure 2. Me-Ant K promotes DC maturation. DC were incubated with me-Ant K (50 μM) (black line) or DMSO (gray line) for 16 h. LPS (100 ng/mL) stimulated cells are shown in lower panels. Dotted lines represent staining with an isotype-matched control Ab. DC maturation was determined by flow cytometry. (A) Stains with mAb specific for CD54, CD119, CD80, and CD40. The change of MFI from control to treatment was indicated. (B) Uptake of Dextran-FITC. Cells were incubated with Dextran-FITC at 4°C (gray line) or 37°C (black line) and harvested after 1 h. The percentage of Dextran-FITC+ cells is shown above the regional marker. All data shown are gated on CD11c+ cells. Data are representative of three independent experiments.

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Cytokine and chemokine production by DC after me-AntK stimulation

Activated DC secrete pro-inflammatory cytokines and chemokines, which can consequently regulate immune responses. To determine whether the stimulation of me-AntK induced DC pro-inflammatory cytokine production, intracellular cytokines were detected by flow cytometry. Me-AntK treatment induced significant production of TNF-α (Fig. 3A), however, IL-6 and IL-12 p40 could not be detected (data not shown). Again, me-AntN had no effect on cytokine production (data not shown). We also measured the cytokine production of me-Ant-K-treated DC by ELISA. As shown in Fig. 3B, DC produced TNF-α after me-AntK treatment in a dose-dependent manner. No IL-6 or IL-12 could be detected, and IL-1α, IL-1β, IL-2, IL-10, and IFN-γ production was not significant (data not shown). In addition to pro-inflammatory cytokines, chemokines MCP-1 (CCL2) and MIP-1β (CCL4) were secreted by me-Ant-K-stimulated DC (Fig. 3C). However, MIP-1α and RANTES were not significantly generated (data not shown). These results indicate that me-AntK can enhance innate immunity.

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Figure 3. DC produce TNF-α and chemokines in response to me-Ant K stimulation. (A) DC were incubated with me-Ant K (50 μM) (black line) or DMSO (gray line) for 6 h in the presence of Brefeldin A for the last 4 h. Intracellular TNF-α was determined by flow cytometry after staining with mAb specific for TNF-α. Dotted line represents staining with an isotype-matched control Ab. The percentage of TNF-α-producing CD11c+ cells was shown above the regional marker. (B, C) DC were treated with indicated concentration of me-Ant K for 24 h (or 6 h for TNF-α), and supernatants were collected. The production of TNF-α (B), MCP-1, and MIP-1β (C) were determined by ELISA. Error bars indicated mean+SD of two to four independent experiments. The significances NSp>0.05, *p<0.05, **p<0.01 (Student's t-test) were obtained as compared me-Ant-K-treated to DMSO-treated DC. Data shown are representative of two separate experiments.

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Induction of T-cell proliferation by me-Ant-K-treated DC

The primary function of mature DC is to induce Ag-specific T-cell activation and proliferation. We cultured CD4+ OT-II T cells with me-Ant-K-treated, OVA323-339 peptide (OVAp)-pulsed DC for 72 h, following which T-cell proliferation was determined by [3H]thymidine incorporation. Consistent with upregulation of co-stimulatory molecules and induction of cytokine production, me-Ant-K-activated DC induced more OT-II T-cell proliferation than control cells in vitro (Fig. 4A). Then, we performed Ag recall assays to evaluate the effect of me-AntK on T-cell priming in vivo. C57BL/6 mice were immunized with incomplete Freund's adjuvant (IFA) plus OVAp with DMSO (solvent for me-Ant K) or me-AntK. Draining lymph node cells were isolated after 10 days and cultured in the presence of OVAp for 3 days. Consistently, cells isolated from IFA/OVAp/me-Ant-K-immunized mice showed more proliferation than cells from control mice in response to OVAp along (Fig. 4B). These data reveal that me-Ant-K-treated DC induce Ag-specific T-cell proliferation both in vitro and in vivo, and also support the conclusion that me-AntK causes a relative enhancement in DC maturation.

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Figure 4. Me-Ant-K-treated DC induce Ag-specific T-cell activation. (A) CD4+ T cells were isolated from OT-II TCR transgenic mice and co-cultured with me-AntK (50 μM)-activated DC pulsed with OVAp (2 μg/mL) at indicated ratio of DC: T cell for 72 h. T-cell proliferation was determined by [3H]thymidine incorporation. (B) C57BL/6 mice were immunized with IFA+OVAp+DMSO (control) or IFA+OVAp+me-AntK (50 μg). Cells were isolated from draining lymph nodes after 10 days, and then incubated with OVAp along at indicated concentrations. T-cell proliferation was determined by [3H]thymidine incorporation. Error bars indicated mean+SD of triplicate samples. The significances NSp>0.05, *p<0.05, **p<0.01 (Student's t-test) were obtained as compared me-Ant-K-treated to DMSO-treated DC. Data shown are representative of three independent experiments.

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Facilitation of Th2 differentiation by me-Ant-K-treated DC

After antigenic stimulation, we determined what type of Th differentiation is induced by me-Ant-K-activated DC. RNA was extracted from OT-II T cells co-cultured with non-treated or treated DC for 3 days. Then, we performed real-time PCR to quantify the transcription factors and cytokines that were expressed in Th cells. As expected, DC stimulated with LPS induced Th1 cell polarization, as evidenced by upregulation of IFN-γ and T-bet expression in the responding T cells. Surprisingly, me-Ant-K-treated DC induced polarization of T cells to the Th2 phenotype, as shown by the increased mRNA expression of IL-4, IL-5, IL-13, and GATA-3 (Fig. 5A). To confirm these results, we measured cytokine production from the T cells stimulated by differentially treated DC. As seen at the RNA level, LPS-treated DC induced production of Th1-related IFN-γ from the OT-II cells, while me-Ant-K-stimulated DC polarized T cells to the Th2 phenotype (Fig. 5B). Furthermore, we determined the OVA-specific IgG isotype spectrum in mice immunized with OVA and me-AntK. Consistent with in vitro results, me-Ant-K-immunized mice showed a higher level of Th2-related IgG1 Ab when compared with the DMSO control (Fig. 5C). We conclude that me-AntK instructs DC to induce Th2 cell differentiation both in vitro and in vivo.

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Figure 5. Me-Ant-K-treated DC facilitate Th2 differentiation. (A) OT-II T cells were co-cultured with DMSO-, LPS (100 ng/mL)-, or me-AntK (50 μM)-activated DC pulsed with OVAp (2 μg/mL) at indicated ratio of DC:T cell. Total RNA was isolated from DC:T cell=1:4 cultures and cDNA was synthesized from total RNA. The mRNA expression levels of IL-4, IL-5, IL-13, IFN-γ, GATA-3, and T-bet were quantified by real-time PCR and normalized to the levels of β-actin mRNA. (B) Supernatants were collected from DC/OT-II T-cell co-cultures after 4 days and IL-4, IL-5, IL-13, and IFN-γ were measured by ELISA. (C) OVA-specific Ab were determined by ELISA assays of serial dilutions of serum obtained from mice on day 30 after subcutaneous immunization with a mixture of OVA (100 μg)+DMSO (control), me-AntK (50 μg), or LPS (10 μg) and boosting on day 14. Upper panel: dilution curves of pre- and post immune sera for IgG1 (left) and IgG2a (right). Lower panel: absolute titers of IgG1 (left) and IgG2a (right) in ng/mL. Error bars indicated mean+SD of (A) triplicate samples, (B) two to four independent experiments, and (C) triplicate samples. The significances NSp>0.05, *p<0.05, **p<0.01 (Student's t-test) were obtained as compared me-Ant-K- or LPS-treated to DMSO-treated DC. Data shown are representative of two to four separate experiments.

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Activation of JNK and ERK after me-AntK stimulation in DC

To explore the molecular mechanisms underlying DC activation by me-AntK we examined activation of MAPK isoforms and NF-κB in treated cells. A natural triterpenoid ursolic acid has been reported to enhance the production of several cytokines in macrophages via p38 MAPK, ERK, and NF-κB activation 40, 41. DC were treated with me-Ant K and both the inactive and active forms of JNK, ERK, and p38 MAPK were analyzed by Western blotting. Significantly, me-AntK induced JNK and ERK activation, whereas phosphorylation of p38 MAPK was not observed (Fig. 6). In contrast, me-AntK failed to induce degradation of IκB, suggesting that this compound does not activate the NF-κB pathway. These results suggest that me-AntK promotes DC maturation and function by activating JNK and ERK pathways.

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Figure 6. Me-AntK induces JNK and ERK activation in DC. DC were harvested, starved, and treated with me-AntK (100 μM) or LPS (100 ng/mL), and then lysed at indicated time points. Samples were separated on SDS-PAGE gels, transferred to nitrocellulose membrane, and then analyzed by Western blotting. The JNK, ERK, and p38 MAPK proteins with and without phosphorylation were detected by anti-phosphospecific and anti-protein Ab, respectively. IκB degradation was determined by anti-IκB Ab. Data shown are representative of three independent experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, we have shown that me-AntK promotes DC activation and maturation to enhance both innate and adaptive immune responses. Surprisingly, me-AntK treatment of DC facilitates their ability to induce T cells to differentiate toward a Th2 phenotype. The Th1 promotion and inhibitory effect of ginsenosides, triterpenoids isolated from the medicinal herb ginseng, have been reported on DC 31, 32. Thus, this is the first example of a natural triterpenoid can activate DC to induce Th2 responses. A number of researchers have focused on Ac as a source of biomodulatory compounds. Our report demonstrates the effect of an Ac compound on DC and supports the conclusion that Ac can be a rich source for pharmacological and therapeutic agents.

Although me-AntK enhanced the expression of maturation markers on DC, the upregulation of MHC class II and CD86 was less than that induced by LPS (Fig. 1B) while no effect was seen on CD40 and CD80 expression (Fig. 2A). In addition, me-Ant-K-stimulated DC failed to produce a number of cytokines and chemokines when compared with LPS-treated DC 42, 43. Thus, we conclude that me-AntK triggers a different activation pathway compared with TLR ligands in DC. It makes evolutionary sense that triterpenoids, the natural compounds derived from metabolic intermediates in fungi or plants, are less potent activators of DC compared with PAMP present on true pathogens. Nevertheless, our study points out that triterpenoid me-AntK still plays a modulatory role in DC function and immune responses by promoting DC to produce a number of cytokines/chemokines (Fig. 3) and inducing them to prime Th2 T-cell differentiation (Figs. 4 and 5). In vivo immunization in the presence of me-Ant K resulted in the high production of Th2-associated IgG1 isotype Ab (Fig. 5C). The 103-fold difference between serum dilutions of anti-OVA IgG1versus IgG2a has been reported by other groups 44, 45. However, the difference in absolute titers of anti-OVA IgG1versus IgG2a appeared less dramatic, suggesting that anti-IgG1 and -IgG2a Ab have different sensitivities. Several stimuli have been shown in literature to direct Th2 differentiation by DC, for example, cholera toxin and thymic stromal lymphopoietin 46. The upregulation of OX-40 ligand expression was proposed to involve in the Th2 development 47, 48. However, OX-40 ligand expression was not increased by me-AntK treatment in DC (data not shown). It will be interesting to reveal the mechanism for Th2 differentiation instructed by me-Ant-K-activated DC.

How does me-AntK activate DC? It has been shown that some triterpenoids interact with CD36, insulin receptor, and peroxisome proliferator-activated receptor γ PPAR-γ to activate p38 MAPK, ERK, Akt, and AMP-activated protein kinase AMPκ 41, 49–52. In addition, a natural triterpenoid ursolic acid enhances the cytokine production in macrophages via NF-κB, p38 MAPK, and ERK activation 40, 41. We found that me-AntK induced both ERK and JNK activation, but not p38 MAPK and NF-κB activation, in DC (Fig. 6). We do not know yet whether a receptor is required for me-Ant-K-induced signaling or not. Although a synthetic triterpenoid also activates JNK pathway in human breast cancer cells, it actually induces apoptosis but not cell activation 53. Thus, we conclude that me-AntK uniquely triggers a signaling pathway that activates JNK leading to DC maturation and function.

In this study we also tested two newly identified triterpenoids, antcin N and me-AntN, and found that only me-AntN can weakly activate DC (Fig. 1B). The primary molecular difference between non-activating compounds and me-AntK is the location of an OH group (4β in antcin K/me-AntK versus 12α in antcin N/me-AntN) and methylation (antcin K/N versus me-Ant K/N) (Fig. 1A). Accordingly, methylation is probably more important than the position of OH group for DC activation in the structure of these compounds. The information about this structural and functional relationship might be useful for developing various triterpenoid derivatives with immunomodulatory activity 54.

In summary, our study clearly demonstrates that me-AntK promotes DC activation, maturation, and facilitates the Th2 responses. The data provide not only a valuable evidence for the medical application of Ac but also important information for developing an immunomodulatory agent in pharmacological therapy. It has been suggested that skewing to Th2 differentiation may be a strategy for treating inflammation and autoimmune diseases 55, 56. Thus, me-Ant K can potentially have applications for the control of these types of immune dysfunctions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Mice and DC cultures

BMDC were generated from C57BL/6 mice (National Laboratory Animal Center, Taipei, Taiwan) as previously described 57. In brief, bone marrow cells were isolated and seeded on 24-well culture plates (Costar Corning) in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Maverick) and 10 ng/mL mouse GM-CSF (Peprotech) for 7 days. OT-II TCR transgenic mice were provided by Dr. Clifford Lowell (UCSF, CA). All mice were housed in the barrier facility at NHRI (Taiwan) under an Institutional Animal Care and Use Committee-approved protocol.

Preparation of triterpenoids from Ac and As

All triterpenoids were isolated from fruiting bodies of Ac or As by a series of chromatographic cycles and HPLC, and the structures were determined by various spectrometers and NMR 23, 35–38. Briefly, the chipped fruiting bodies were extracted with water, and then the residue of the fruiting bodies was refluxed with ethanol. The concentrate from the ethanol extracts was partitioned between dichloromethane and water, and then the organic fraction was dissolved in methanol. The methanol-soluble portion was chromatographed on a Sephadex LH-20 column. Then, the fractions were collected and pooled according to their TLC profiles to give the stock fractions. Each stock fraction was re-treated with methanol and chromatographed on silica gel MPLC to generate sub-fractions. Each sub-fraction was further purified by chromatography and HPLC to obtain all compounds used here. All compounds were >90–95% purity as measured by HPLC and dissolved in DMSO for use. Supporting Information Fig. 1 shows the HPLC data for the purity of me-AntK.

DC maturation

For DC maturation, BMDC were cultured in 24-well plates for 6 days and treated with compounds or LPS at the indicated concentrations. DMSO is less than 0.5% v/v in all tests and used as control. After 16 h, the cells were blocked with anti-CD16/CD32 mAb 2.4G2 (BD Pharmingen), stained with mAb against CD11c, CD40, CD54, CD80, CD86, CD119, OX-40L, and I-Ab (Biolegend), and analyzed by flow cytometry. For endocytosis assay, treated or non-treated DC were incubated with 200 μg/mL Dextran-FITC (MW ∼77 kD, Sigma-Aldrich) for 1 h at 4 or 37°C. Cells were washed with cold PBS, stained with anti-CD11c mAb, and then analyzed by flow cytometry.

Cytokine and chemokine production

For intracellular cytokine production, 7-day-cultured DC were treated with me-Ant K for 6 h, and 10 μg/mL Brefeldin A (Biolegend) was included in the last 4 h. Cells were then blocked with 2.4G2 mAb, stained with anti-CD11c, fixed, permeabilized, and then stained with mAb against TNFα, IL-6, and IL-12 p40 (Biolegend), and analyzed by flow cytometry. For ELISA, supernatants were collected from 1×106 DC/mL incubated with or without me-Ant K after 24 h (or 6 h for TNF-α), and the production of TNF-α, IL-1α IL-1β, IL-2, IL-6, IL-10, IL-12 p70, IFN-γ and chemokines MCP-1, MIP-1α, MIP-1β, and RANTES were assayed using ELISA kits (R&D system and e-Bioscience).

To determine Th differentiation, supernatants were collected from DC/OT-II T-cell cultures after 4 days as described in Assay for T-cell activation. The production of IL-4, IL-5, IL-13, and IFN-γ was determined by ELISA kits (eBioscience).

Assay for T-cell activation

Ag presentation by DC was determined by using an OVA-specific T-cell proliferation assay, as described previously 58. Briefly, DC were purified by using EasySep Positive Selection Kit (Stem Cell Technology), seeded in 96-well flat-bottom plates (Costar Corning) at 2.5×104 cells/well with or without me-AntK (50 μM) and OVAp (2 μg/mL), and incubated for 3 h. CD4+T cells were isolated from OT-II TCR transgenic mice with EasySep Positive Selection Kit and added to DC cultures at various DC:T cell ratio as indicated. Cells were incubated for 72 h, [3H]thymidine (1 μCi/well) was added during the last 16 h of culture, and the [3H]thymidine incorporation was measured by scintillation counting.

For recall assays, C57BL/6 mice were immunized with 10 μg OVAp mixed with IFA (Sigma-Aldrich)+DMSO (control) or IFA+me-AntK (50 μg) via footpad injection. Total draining lymph node cells were isolated from immunized mice after 10 days and cultured in 96-well plates at 5×105 cells/well with indicated concentration of OVAp for 3 days. T-cell proliferation was determined by [3H]thymidine incorporation.

Real-time PCR analysis

Total RNA was isolated from OT-II T cells co-cultured with non-treated or LPS- and me-Ant-K-treated DC using Trizol reagent (Invitrogen). The cDNA was synthesized from total RNA with ProtoScript First Strand cDNA Synethesis kit (New English Biolab) using oligo(dT) primers. The mRNA expression levels were quantified by real-time PCR using a Mastercycler ep realplex instrument (Eppendorf) with the SYBR Green PCR Master Mix (Roche Applied Science) in a one-step reaction according to the manufacturer's instructions and normalized to the levels of β-actin mRNA. The expression levels of all mRNAs from untreated DC/T-cell culture represented one fold. Primer pairs used for IL-4, IL-5, IL-13, IFN-γ, GATA-3, and T-bet were described previously 59.

IgG isotype spectrum analysis

C57BL/6 mice were immunized with 100 μg OVA (Sigma-Aldrich)+DMSO (control), me-Ant K (50 μg), or LPS (10 μg) via subcutaneous injection and boosted with the same mixtures 14 days later. Mouse sera were collected after 30 days, and OVA-specific IgG isotypes were determined by direct ELISA 60. The 96-well plates (Corning) were coated with 10 μg/mL OVA for detection or purified mouse IgG1 and IgG2a (Biolegend) for the generation of standard curves. Mouse sera were diluted (101–7and 101–4-fold for IgG1 and IgG2a, respectively) into PBS containing 1% BSA and analyzed for Ig isotypes by using HRP-rabbit anti-mouse IgG1 and IgG2a (Zymed). Plates were developed with TMB solution (eBioscience) and absorbance was read at 450 nm. Titers were calculated by plotting ODs on the standard curves and multiplying with the dilution factors.

Western blot assay

BMDC were harvested, starved in PBS for 3 h, and then treated with me-AntK (100 μM). Cells were lysed at indicated time points, boiled in sample buffer, and then separated on SDS-PAGE gels and transferred to Immunobilon NC membrane (Millipore). After blocking with 2% BSA in TBST, blots were incubated overnight at 4°C with anti-phosphospecific Ab directed against p38 MAPK (Thr180/Tyr182), ERK (Thr202/Tyr204), and JNK (Thr183/Tyr185) (Cell Signaling Technology) or Ab directed against p38 MAPK (Millipore), ERK (BD Transduction Laboratories), JNK, and IκB (Santa Cruz Biotechnology), followed by HRP-conjugated goat anti-mouse or rabbit Ab (Chemicon). Immunoreactivity was detected using the ECL detection reagent (Pierce).

Data analysis

Significance of T-cell proliferation, cytokine and chemokine production, and Th responses of me-AntK or LPS treatment in comparison with control was determined using a Student's t-test with two-sample equal variance with a two-tailed distribution.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Dr. Ing-Kang Ho (NHRI, Taiwan) and Dr. Clifford Lowell (UCSF, CA) for critically reading the manuscript, and Dr. Ming-Tao Hsu for constructive suggestion in initiating this project. This work was supported by NHRI grant VC096PP04, IM097PP03, and NSC grant NSC962320B400003 (in part) of Taiwan.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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