Seok-Rae Park

Retinoic acid, acting as a highly specific IgA isotype switch factor, cooperates with TGF‐β1 to enhance the overall IgA response

The present study demonstrates that RA has activity of an IgA switch factor and is more specific than TGF‐β1. RA independently caused only IgA switching, whereas TGF‐β1 caused IgA and IgG2b switching. We found that RA increased IgA production and that this was a result of its ability to increase the frequency of IgA‐secreting B cell clones. Increased IgA production was accompanied by an increase of GLTα. RA activity was abrogated by an antagonist of the RAR. Additionally, RA affected intestinal IgA production in mice. Surprisingly, RA, in combination with TGF‐β1, notably enhanced not only IgA production and GLTα expression but also CCR9 and α4β7 expression on B cells. These results suggest that RA selectively induces IgA isotype switching through RAR and that RA and TGF‐β have important effects on the overall gut IgA antibody response.

Activation-induced Cytidine Deaminase in B Cell Immunity and Cancers

Activation-induced cytidine deaminase (AID) is an enzyme that is predominantly expressed in germinal center B cells and plays a pivotal role in immunoglobulin class switch recombination and somatic hypermutation for antibody (Ab) maturation. These two genetic processes endow Abs with protective functions against a multitude of antigens (pathogens) during humoral immune responses. In B cells, AID expression is regulated at the level of either transcriptional activation on AID gene loci or post-transcriptional suppression of AID mRNA. Furthermore, AID stabilization and targeting are determined by post-translational modifications and interactions with other cellular/nuclear factors. On the other hand, aberrant expression of AID causes B cell leukemias and lymphomas, including Burkitts lymphoma caused by c-myc/IgH translocation. AID is also ectopically expressed in T cells and non-immune cells, and triggers point mutations in relevant DNA loci, resulting in tumorigenesis. Here, I review the recent literatures on the function of AID, regulation of AID expression, stability and targeting in B cells, and AID-related tumor formation.

Anti-hyperlipidemic Effect of Polyphenol Extract (Seapolynol™) and Dieckol Isolated from Ecklonia cava in in vivo and in vitro Models

The inhibitory effect of polyphenol extracts (Seapolynol™, SPN) of the marine brown algae Ecklonia cava and dieckol, a major component of SPN, on hyperlipidemia was investigated in ICR mice fed a high-fat diet (HFD) for five weeks. For analysis of the anti-hyperlipidemic effects of SPN and dieckol, these two agents were given orally on a daily basis to HFD-fed mice for four weeks, starting one week after the beginning of HFD feeding. Groups administered with SPN as well as dieckol showed lower body weight gains than the HFD only group. Administration of SPN and dieckol also resulted in a significant reduction of the level of total cholesterol (TCHO), triglyceride (TG), and low-density lipoprotein (LDL) cholesterol in the serum of HFD-fed mice. In Oil Red O staining using 3T3-L1 preadipocytes, it was shown that both SPN and dieckol markedly inhibited lipid accumulation of 3T3-L1 cells. Furthermore, SPN and dieckol (50 μg/mL) significantly inhibited 3-hydroxyl-methyl glutaryl coenzyme A (HMGCoA) reductase activity in vitro. Taken together, these results suggest that polyphenols of Ecklonia cava (SPN) and dieckol reduce body weight gain and fat accumulation in HFD-induced obese mice, and that their hypolipidemic effect is related to the inhibition of adipogenesis of adipocytes and HMGCoA reductase activity.

Retinoic acid acts as a selective human IgA switch factor

Retinoic acid (RA) is known to have several functions that lead to a potent mucosal IgA response. Nevertheless, its exact role in human IgA synthesis has yet to be elucidated. Thus, we investigated the role of RA in promoting IgA isotype switching in human B cells. We found that RA increased IgA production and the expression of germ-line IgA1 and IgA2 transcripts (GLTα1 and GLTα2). This induction occurred alongside an increase in the frequency of IgA1-secreting B cell clones, as assessed by limiting dilution analysis. Under the same conditions, RA did not increase IgM and IgG production. Am80, an agonist of RA receptor α (RARα), increased IgA production. In addition, RA activity was abrogated by LE540, an antagonist of RAR, suggesting that the RAR pathway is involved in RA-induced IgA production. Taken together, these results indicate that RA induces IgA isotype switching mainly through RARα in human B cells.

APRIL stimulates NF-κB-mediated HoxC4 induction for AID expression in mouse B cells.

Activation-induced cytidine deaminase (AID) plays a key role in B cell immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM). We have previously reported that the highly conserved homeodomain HoxC4 transcription factor binds to the Aicda (AID gene) promoter to induce AID expression. Here, we investigated the regulation of HoxC4 transcription by a proliferation-inducing ligand (APRIL) and B cell-activating factor belonging to the TNF family (BAFF) in mouse B cells. APRIL substantially increased both HoxC4 and AID expression, whereas BAFF induced the expression of AID but not HoxC4. To elucidate the underlying mechanisms, we constructed a HoxC4 gene promoter reporter vector and analyzed the promoter induction after APRIL stimulation. APRIL enhanced the HoxC4 promoter activity by 2.3-fold, and this increase disappeared when the second putative NF-κB-binding promoter element (NBE2) was mutated. Based on ChIP assays, we found that NF-κB bound to the HoxC4 promoter NBE2 region. Furthermore, the overexpression of NF-κB augmented the APRIL-induced HoxC4 promoter activity, while the expression of dominant negative-IκBα suppressed it. Taken together, our findings suggest that NF-κB mediates APRIL-induced HoxC4 transcription.

Retinoic acid induces expression of Ig germ line α transcript, an IgA isotype switching indicative, through retinoic acid receptor

Retinoic acid (RA) is considered to possess an activity of IgA isotype switching. Thus far, TGF-β1 is known to be the most powerful IgA isotype switch factor. To elucidate the molecular mechanisms underlying the Ig germ line (GL) α transcriptional regulation by RA, we constructed three different sizes of mouse GLα promoter reporters; short-GLα(−130/+14), middle-GLα(−448/+72) and long-GLα(−3028/+72). Based on luciferase assay, RA increased the activity of all three GLα promoter reporters by approximately 2-fold and the effect was further enhanced by TGF-β1. Overexpression of Smad3/4 increased TGF-β1-induced GLα promoter activities but had no effect on RA-induced GLα promoter activities. In order to analyze the characteristics of the RA-inducible GLα promoter region, we also constructed two mutant reporters: Smad3 binding elements (SBEs)-substituted short-GLα (short-GLα mSBE) and Runx3 binding elements (RBEs)-substituted short-GLα (short-GLα mRBE) promoter reporters. Promoter activities of the two mutant reporters to RA were comparable to that of wild type reporter, while those of the two mutant reporters to TGF-β1 were markedly diminished as compared to that of WT short-GLα. Finally, RA-induced GLα transcription was virtually disappeared by LE540, an antagonist of RA receptor (RAR). Taken together, these results suggest that RA induces GLα transcription mainly through RAR pathway, where neither Smad3/4 nor Runx3 is involved.

Lactoferrin Combined with Retinoic Acid Stimulates B1 Cells to Express IgA Isotype and Gut-homing Molecules.

It is well established that TGF-β1 and retinoic acid (RA) cause IgA isotype switching in mice. We recently found that lactoferrin (LF) also has an activity of IgA isotype switching in spleen B cells. The present study explored the effect of LF on the Ig production by mouse peritoneal B cells. LF, like TGF-β1, substantially increased IgA production in peritoneal B1 cells but little in peritoneal B2 cells. In contrast, LF increased IgG2b production in peritoneal B2 cells much more strongly than in peritoneal B1 cells. LF in combination with RA further enhanced the IgA production and, interestingly, this enhancement was restricted to IgA isotype and B1 cells. Similarly, the combination of the two molecules also led to expression of gut homing molecules α4β7 and CCR9 on peritoneal B1 cells, but not on peritoneal B2 cells. Thus, these results indicate that LF and RA can contribute to gut IgA response through stimulating IgA isotype switching and expression of gut-homing molecules in peritoneal B1 cells.

Dectin-1 Stimulation Selectively Reinforces LPS-driven IgG1 Production by Mouse B Cells

Dectin-1, which specifically recognizes β-glucan of fungal cell walls, is a non-Toll-like receptor (TLR) pattern recognition receptor and a representative of C-type lectin receptors (CLRs). The importance of Dectin-1 in innate immune cells, such as dendritic cells and macrophages, has previously been well studied. However, the function of Dectin-1 in B cells is very poorly understood. To determine the role of Dectin-1 in B cell activation, we first investigated whether mouse B cells express Dectin-1 and then assessed the effect of Dectin-1 stimulation on B cell proliferation and antibody production. Mouse B cells express mRNAs encoding CLRs, including Dectin-1, and surface Dectin-1 was expressed in B cells of C57BL/6 rather than BALB/c strain. Dectin-1 agonists, heat-killed Candida albicans (HKCA) and heat-killed Saccharomyces cerevisiae (HKSC), alone induced B cell proliferation but not antibody production. Interestingly, HKSC, HKCA, and depleted zymosan (a selective Dectin-1 agonist) selectively enhanced LPS-driven IgG1 production. Taken together, these results suggest that, during fungal infection, β-glucan-stimulated Dectin-1 may cooperate with TLR4 to specifically enhance IgG1 production by mouse B cells.

Kinetic Analysis of CpG-Induced Mouse B Cell Growth and Ig Production

Immune cells express toll-like receptors (TLRs) and respond to molecular patterns of various pathogens. CpG motif in bacterial DNA activates innate and acquired immune systems through binding to TLR9 of immune cells. Several studies reported that CpG can directly regulate B cell activation, differentiation, and Ig production. However, the role of CpG in B cell growth and Ig production is not fully understood. In this study, we analyzed the effect of CpG on the kinetics of mouse B cell viability, proliferation, and Igs production. Overall, CpG enhanced mouse B cell growth and production of Igs in a dose-dependent manner. Unlike LPS, 100 nM CpG (high dose) did not support TGF-β1-induced IgA and IgG2b production. Moreover, 100 nM CpG treatment abrogated either LPS-induced IgM or LPS/TGF-β1-induced IgA and IgG2b production, although B cell growth was enhanced by CpG under the same culture conditions. We subsequently found that 10 nM CpG (low dose) is sufficient for B cell growth. Again, 10 nM CpG did not support TGF-β1-induced IgA production but, interestingly enough, supported RA-induced IgA production. Further, 10 nM CpG, unlike 100 nM, neither abrogated the LPS/TGF-β1-nor the LPS/RA-induced IgA production. Taken together, these results suggest that dose of CpG is critical in B cell growth and Igs production and the optimal dose of CpG cooperates with LPS in B cell activation and differentiation toward Igs production.

Anti-Inflammatory Effects of Liriope platyphylla in LPS-Stimulated Macrophages and Endotoxemic Mice

In the present study, the anti-inflammatory and antisepticemic activities of a water extract of Liriope platyphylla (LP) were investigated. We first estimated the scavenging activity of DPPH and the hydroxyl radical and total phenolic contents of LP. Results indicated that LP, a rich source of phenolic compounds, showed a remarkable radical scavenging capacity. A MTT assay showed that LP treatment did not affect the toxicity against the RAW 264.7 macrophage cells, up to the concentration of 500[Formula: see text][Formula: see text]g/mL. Treatment of LP significantly attenuated the production of inflammatory mediators, such as nitric oxide (NO), interleukin-6 (IL-6), tumor-necrosis factor (TNF)-[Formula: see text] and prostaglandin (PG)E2 in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages cells. Moreover, LP contributed to the down-regulation of inducible NO synthase (iNOS) and TNF-[Formula: see text] mRNA expression, as well as cyclooxygenase-2 (COX-2) protein expression. A western blotting assay further showed that LP inhibited activation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-[Formula: see text]B. In an animal experiment using an LPS-induced septicemia model in C57BL/6 mice, oral administration of LP (40[Formula: see text]mg/kg body weight) markedly reduced the level of TNF-[Formula: see text] and IL-6 in serum and protected against LPS-induced lethal shock in mice. Taken together, the results of treatments of LP on inhibited LPS-induced inflammatory responses in both in vitro and in vivo models and indicate it may be a promising neutraceutical or medicinal agent to prevent or cure inflammation-related disease.