Lactoferrin-derived chimeric peptide (LFch) strongly boosts TGFß1-mediated inducible Treg differentiation possibly through downregulating TCR/CD28 signalling.

We recently reported that lactoferrin (LF) induces Foxp3+ Treg differentiation through binding to TGFß receptor III (TßRIII), and this activity was further enhanced by TGFß1. Generally, a low T-cell receptor (TCR) signal strength is favourable for Foxp3+ Treg differentiation. In the present study, we explored the effect of lactoferrin chimera (LFch, containing lactoferricin [aa 17-30] and lactoferrampin [aa 265-284]), along with TGFß1 on Foxp3+ Treg differentiation. LFch alone did not induce Foxp3 expression, yet LFch dramatically enhanced TGFß1-induced Foxp3 expression. LFch had little effect on the phosphorylation of Smad3, a canonical transcriptional factor of TGFß1. Instead, LFch attenuated the phosphorylation of S6 (a target of mTOR), IκB and PI3K. These activities of LFch were completely abrogated by pretreatment of LFch with soluble TGFß1 receptor III (sTßRIII). Consistent with this, the activity of LFch on TGFß1-induced Foxp3 expression was also abrogated by treatment with sTßRIII. Finally, the TGFß1/LFch-induced T cell population substantially suppressed the proliferation of responder CD4+ T cells. These results indicate that LFch robustly enhances TGFß1-induced Foxp3+ Treg differentiation by diminishing TCR/CD28 signal intensity.

GSK3 Restrains Germinal Center B Cells to Form Plasma Cells.

B cells in the germinal center (GC) are programmed to form plasma cells (PCs) or memory B cells according to signals received by receptors that are translated to carry out appropriate activities of transcription factors. However, the precise mechanism underlying this process to complete the GC reaction is unclear. In this study, we show that both genetic ablation and pharmacological inhibition of glycogen synthase kinase 3 (GSK3) in GC B cells of mice facilitate the cell fate decision toward PC formation, accompanied by acquisition of dark zone B cell properties. Mechanistically, under stimulation with CD40L and IL-21, GSK3 inactivation synergistically induced the transcription factors Foxo1 and c-Myc, leading to increased levels of key transcription factors required for PC differentiation, including IRF4. This GSK3-mediated alteration of transcriptional factors in turn facilitated the dark zone transition and consequent PC fate commitment. Our study thus reveals the upstream master regulator responsible for interpreting external cues in GC B cells to form PCs mediated by key transcription factors.

Lactoferrin Potentiates Inducible Regulatory T Cell Differentiation through TGF-ß Receptor III Binding and Activation of Membrane-Bound TGF-ß.

Lactoferrin (LF) is known to possess anti-inflammatory activity, although its mechanisms of action are not well-understood. The present study asked whether LF affects the commitment of inducible regulatory T cells (Tregs). LF substantially promoted Foxp3 expression by mouse activated CD4+T cells, and this activity was further enhanced by TGF-ß1. Interestingly, blocking TGF-ß with anti-TGF-ß Ab completely abolished LF-induced Foxp3 expression. However, no significant amount of soluble TGF-ß was released by LF-stimulated T cells, suggesting that membrane TGF-ß (mTGF-ß) is associated. Subsequently, it was found that LF binds to TGF-ß receptor III, which induces reactive oxygen species production and diminishes the expression of mTGF-ß-bound latency-associated peptide, leading to the activation of mTGF-ß. It was followed by phosphorylation of Smad3 and enhanced Foxp3 expression. These results suggest that LF induces Foxp3+ Tregs through TGF-ß receptor III/reactive oxygen species-mediated mTGF-ß activation, triggering canonical Smad3-dependent signaling. Finally, we found that the suppressive activity of LF-induced Tregs is facilitated mainly by CD39/CD73-induced adenosine generation and that this suppressor activity alleviates inflammatory bowel disease.

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92.

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.

The TLR7 agonist imiquimod selectively inhibits IL-4-induced IgE production by suppressing IgG1/IgE class switching and germline ε transcription through the induction of BCL6 expression in B cells.

Imiquimod (IMQ) is a selective toll-like receptor 7 (TLR7) agonist. TLR7 activation leads to the production of IFN-γ and pro-inflammatory cytokines by innate immune cells. However, the role of TLR7 in B cells is not fully understood. In this study, we investigated the direct effect of in vitro stimulation with IMQ on Ab production and isotype switching in B cells. IMQ selectively diminished IL-4-induced IgE and IgG1 production in anti-CD40-activated mouse B cells. IMQ also inhibited germline ε transcripts (GLTε)/GLTγ1 and post-switch ε transcripts (PSTε)/PSTγ1 expression, while enhancing GLTγ2c and PSTγ2c expression in anti-CD40/IL-4-stimulated B cells. Interestingly, IMQ abrogated IL-4-induced circle transcripts ε-γ1 (CTε-γ1) expression, indicative of sequential switching from IgG1 to IgE. Furthermore, IMQ repressed IL-4-induced surface IgE/IgG1 expression while increasing surface IgG2c expression. The selective inhibition of IgE synthesis was not due to IMQ-induced production of IFN-γ or IL-12 in the same culture. IMQ also enhanced BCL6 expression, a transcriptional repressor for the GLTε promoter, in anti-CD40/IL-4-stimulated B cells. In addition, BCL6 siRNA restored IMQ-mediated suppression of GLTε transcription. Therefore, these results indicate that TLR7 engagement by IMQ inhibits IL-4-induced GLTε transcription by enhancing BCL6 expression and inhibits IL-4-induced sequential switching from IgM to IgE via IgG1, thus resulting in the downregulation of IgE production by B cells.

Wnt3A Induces GSK-3ß Phosphorylation and ß-Catenin Accumulation Through RhoA/ROCK.

In canonical pathway, Wnt3A has been known to stabilize ß-catenin through the dissociation between ß-catenin and glycogen synthase kinase-3ß (GSK-3ß) that suppresses the phosphorylation and degradation of ß-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling; however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3ß and accumulation of ß-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3ß and accumulation of ß-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3ß phosphorylation and ß-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3ß in vitro. In addition, Wnt3A-induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3ß phosphorylation and ß-catenin accumulation through RhoA and ROCK activation. J. Cell. Physiol. 232: 1104-1113, 2017. © 2016 Wiley Periodicals, Inc.

Tetrameric structure of the flagellar cap protein FliD from Serratia marcescens.

Bacterial motility is provided by the flagellum. FliD is located at the distal end of the flagellum and plays a key role in the insertion of each flagellin protein at the growing tip of the flagellar filament. Because FliD functions as an oligomer, the determination of the oligomeric state of FliD is critical to understanding the molecular mechanism of FliD-mediated flagellar growth. FliD has been shown to adopt a pentameric or a hexameric structure depending on the bacterial species. Here, we report another distinct oligomeric form of FliD based on structural and biochemical studies. The crystal structures of the D2 and D3 domains of Serratia marcescens FliD (smFliD) were determined in two crystal forms and together revealed that smFliD assembles into a tetrameric architecture that resembles a four-pointed star plate. smFliD tetramerization was also confirmed in solution by cross-linking experiments. Although smFliD oligomerizes in a head-to-tail orientation using a common primary binding interface between the D2 and D3' domains (the prime denotes the second subunit in the oligomer) similarly to other FliD orthologs, the smFliD tetramer diverges to present a unique secondary D2-D2' binding interface. Our structure-based comparative analysis of FliD suggests that bacteria have developed diverse species-specific oligomeric forms of FliD that range from tetramers to hexamers for flagellar growth.

Mechanism underlying the suppressor activity of retinoic acid on IL4-induced IgE synthesis and its physiological implication.

The present study extends an earlier report that retinoic acid (RA) down-regulates IgE Ab synthesis in vitro. Here, we show the suppressive activity of RA on IgE production in vivo and its underlying mechanisms. We found that RA down-regulated IgE class switching recombination (CSR) mainly through RA receptor α (RARα). Additionally, RA inhibited histone acetylation of germ-line ε (GL ε) promoter, leading to suppression of IgE CSR. Consistently, serum IgE levels were substantially elevated in vitamin A-deficient (VAD) mice and this was more dramatic in VAD-lecithin:retinol acyltransferase deficient (LRAT-/-) mice. Further, serum mouse mast cell protease-1 (mMCP-1) level was elevated while frequency of intestinal regulatory T cells (Tregs) were diminished in VAD LRAT-/- mice, reflecting that deprivation of RA leads to allergic immune response. Taken together, our results reveal that RA has an IgE-repressive activity in vivo, which may ameliorate IgE-mediated allergic disease.

Structural and biochemical characterization of the Bacillus cereus 3-hydroxyisobutyrate dehydrogenase.

The 3-hydroxyisobutyrate dehydrogenase (HIBADH) family catalyzes the NAD(+)- or NADP(+)-dependent oxidation of various ß-hydroxyacid substrates into their cognate semialdehydes for diverse metabolic pathways. Because HIBADH group members exhibit different substrate specificities, the substrate-recognition mode of each enzyme should be individually characterized. In the current study, we report the biochemical and structural analysis of a HIBADH group enzyme from Bacillus cereus (bcHIBADH). bcHIBADH mediates a dehydrogenation reaction on S-3-hydroxyisobutyrate substrate with high catalytic efficiency in an NAD(+)-dependent manner; it also oxidizes l-serine and 3-hydroxypropionate with lower activity. bcHIBADH consists of two domains and is further assembled into a functional dimer rather than a tetramer that has been commonly observed in other prokaryotic HIBADH group members. In the bcHIBADH structure, the interdomain cleft forms a putative active site and simultaneously accommodates both an NAD(+) cofactor and a substrate mimic. Our structure-based comparative analysis highlights structural motifs that are important in the cofactor and substrate recognition of the HIBADH group.

Small GTPase Rap1 regulates cell migration through regulation of small GTPase RhoA activity in response to transforming growth factor-ß1.

Transforming growth factor (TGF)-ß1 regulates diverse cellular functions. Particularly, TGF-ß1 induces monocyte migration to sites of injury or inflammation in early period, whereas TGF-ß1 inhibits cell migration in late phase. In this study, we attempted to understand how TGF-ß1 suppresses cell migration in late phase. We found that TGF-ß1 of short exposure induces the production of chemokines, such as macrophage inflammatory protein (MIP)-1α, by Raw 264.7 cells. However, knock-down of small GTPase RhoA by sh-RhoA inhibited the production of MIP-1α and macrophage migration, suggesting that RhoA is essential for expression of this chemokine. An activator of Epac (exchange proteins directly activated by cAMP; a guanine nucleotide exchange factor of Rap1), 8CPT-2Me-cAMP which leads to Rap1 activation abrogated MIP-1α expression and macrophage migration. Indeed, GTP-RhoA and GTP-Rap1 levels were reciprocally regulated in a time-dependent manner following TGF-ß1 stimulation. 8CPT-2Me-cAMP suppressed GTP-RhoA levels, whereas si-Rap1 augmented GTP-RhoA levels and cell migration. TGF-ß1 produced cAMP in late period and si-RNAs of Epac1 and Epac2 reduced GTP-Rap1 levels leading to promotion of GTP-RhoA levels. Furthermore, si-RNA of ARAP3 (Rap-dependent RhoGAP) increased GTP-RhoA level and cell migration. Therefore, we propose the mechanism that prolonged TGF-ß1 treatment produce cAMP, which activates sequentially Epac, Rap1 and ARAP3, resulting in suppression of RhoA, chemokine expression, and macrophage migration. Contrary to the general concept that Rap1 stimulates cell migration, we demonstrated in this study that Rap1 inhibits cell migration by suppression of RhoA activity in response to TGF-ß1.

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.

Combined Treatment With TGF-ß1, Retinoic Acid, and Lactoferrin Robustly Generate Inducible Tregs (iTregs) Against High Affinity Ligand.

Forkhead box P3-positive (Foxp3+)-inducible Tregs (iTregs) are readily generated by TGF-ß1 at low TCR signaling intensity. TGF-ß1-mediated Foxp3 expression is further enhanced by retinoic acid (RA) and lactoferrin (LF). However, the intensity of TCR signaling required for induction of Foxp3 expression by TGF-ß1 in combination with RA and LF is unknown. Here, we found that either RA or LF alone decreased TGF-ß1-mediated Foxp3 expression at low TCR signaling intensity. In contrast, at high TCR signaling intensity, the addition of either RA or LF strongly increased TGF-ß1-mediated Foxp3 expression. Moreover, decreased CD28 stimulation was more favorable for TGF-ß1/LF-mediated Foxp3 expression. Lastly, we found that at high signaling intensities of both TCR and CD28, combined treatment with TGF-ß1, RA, and LF induced robust expression of Foxp3, in parallel with powerful suppressive activity against responder T cell proliferation. Our findings that TGFß/RA/LF strongly generate high affinity Ag-specific iTreg population would be useful for the control of unwanted hypersensitive immune reactions such as various autoimmune diseases.

Control of neurite outgrowth by RhoA inactivation.

cAMP induces neurite outgrowth in the rat pheochromocytoma cell line 12 (PC12). In particular, di-butyric cAMP (db-cAMP) induces a greater number of primary processes with shorter length than the number induced by nerve growth factor (NGF). db-cAMP up- and down-regulates GTP-RhoA levels in PC12 cells in a time-dependent manner. Tat-C3 toxin stimulates neurite outgrowth, whereas lysophosphatidic acid (LPA) and constitutively active (CA)-RhoA reduce neurite outgrowth, suggesting that RhoA inactivation is essential for the neurite outgrowth from PC12 cells stimulated by cAMP. In this study, the mechanism by which RhoA is inactivated in response to cAMP was examined. db-cAMP induces phosphorylation of RhoA and augments the binding of RhoA with Rho guanine nucleotide dissociation inhibitor (GDI). Moreover, RhoA (S188D) mimicking phosphorylated RhoA induces greater neurite outgrowth than RhoA (S188A) mimicking dephosphorylated form does. Additionally, db-cAMP increases GTP-Rap1 levels, and dominant negative (DN)-Rap1 and DN-Rap-dependent RhoGAP (ARAP3) block neurite outgrowth induced by db-cAMP. DN-p190RhoGAP and the Src inhibitor PP2 suppress neurite outgrowth, whereas transfection of c-Src and p190RhoGAP cDNAs synergistically stimulate neurite outgrowth. Taken together, RhoA is inactivated by phosphorylation of itself, by p190RhoGAP which is activated by Src, and by ARAP3 which is activated by Rap1 during neurite outgrowth from PC12 cells in response to db-cAMP.

iNOS potentiates mouse Ig isotype switching through AID expression.

The IgA antibody plays an important role in protecting mucosal surfaces against pathogens. It has recently been shown that nitric oxide (NO) plays a critical role in mouse IgA synthesis. In the present study, we further characterized inducible-nitric oxide synthase-deficient (iNOS(-/-)) mice in the context of Ig expression. The amount of IgA in fecal pellets was substantially diminished in iNOS(-/-) mice and was paralleled by a decrease in IgA production by Peyer's patch cells. Interestingly, the amount of all IgG subisotypes, as well as IgA, was substantially diminished in sera and in cultured spleen B cells from iNOS(-/-) mice. Moreover, the synthesis of TGF-ß1-inducible IgA and IgG2b in iNOS(-/-) mice was also lower than that in WT mice. However, levels of Ig germ-line transcripts, and expression of TGF-ß receptor type II (TßRII) and BAFF/APRIL, were comparable between iNOS(-/-) and WT mice. Expression of activation-induced cytidine deaminase (AID) was diminished in iNOS(-/-) B cells, but restored by a NO donor, SNAP. These results indicate that iNOS regulates Ig isotype switching events at the level of AID gene expression.

Analyses of TGF-beta1-inducible Ig germ-line gamma2b promoter activity: involvement of Smads and NF-kappaB.

TGF-beta1 directs class switch recombination to IgG2b as well as IgA. We have shown that Smad3/4, Runx3, and p300 mediate TGF-beta1-induced germ-line (GL) gamma2b transcription and that there is a potential Smad-binding element (SBE, CAGAC, -38/-34) and Runx-binding element (TGTGGGT, +41/+47) in the promoter region. Here, we have characterized more putative transcription factor-binding elements in the promoter. Site-directed mutagenesis revealed that two more putative SBE (GTCTG, -67/-63 and +38/+42) are relevant to TGF-beta1-induced GLgamma2b promoter activity, a finding that was confirmed by EMSA. However, neither overexpression of Ets (i.e. Elf-1, Fli-1, or Pu.1) nor a mutation deleting a putative Ets-binding element (CAGGAA, -4/+2) affected basal or TGF-beta1-induced promoter activity. On the other hand, NF-kappaB repressed promoter activity without direct binding to two putative NF-kappaB-binding elements (GGACTCCCC, -63/-55; GGGCCTTTCC,+237/+246). Instead, NF-kappaB overexpression increased the expression of Smad7 transcripts. Moreover, p300 overexpression failed to rescue the inhibitory effect of NF-kappaB on GLgamma2b promoter activity. These results indicate that there are multiple SBE relevant to GLgamma2b promoter activity and that NF-kappaB acts in cooperation with p300 to downregulate promoter activity through increasing the gene expression of inhibitory Smad7.

Lactoferrin Induces Tolerogenic Bone Marrow-Derived Dendritic Cells.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that initiate both T-cell responses and tolerance. Tolerogenic DCs (tDCs) are regulatory DCs that suppress immune responses through the induction of T-cell anergy and Tregs. Because lactoferrin (LF) was demonstrated to induce functional Tregs and has a protective effect against inflammatory bowel disease, we explored the tolerogenic effects of LF on mouse bone marrow-derived DCs (BMDCs). The expression of CD80/86 and MHC class II was diminished in LF-treated BMDCs (LF-BMDCs). LF facilitated BMDCs to suppress proliferation and elevate Foxp3+ induced Treg (iTreg) differentiation in ovalbumin-specific CD4+ T-cell culture. Foxp3 expression was further increased by blockade of the B7 molecule using CTLA4-Ig but was diminished by additional CD28 stimulation using anti-CD28 Ab. On the other hand, the levels of arginase-1 and indoleamine 2,3-dioxygenase-1 (known as key T-cell suppressive molecules) were increased in LF-BMDCs. Consistently, the suppressive activity of LF-BMDCs was partially restored by inhibitors of these molecules. Collectively, these results suggest that LF effectively causes DCs to be tolerogenic by both the suppression of T-cell proliferation and enhancement of iTreg differentiation. This tolerogenic effect of LF is due to the reduction of costimulatory molecules and enhancement of suppressive molecules.

Cathelicidin-Related Antimicrobial Peptide Regulates CD73 Expression in Mouse Th17 Cells via p38.

The effector function of tumor-infiltrated CD4+ T cells is readily suppressed by many types of immune regulators in the tumor microenvironment, which is one of the major mechanisms of immune tolerance against cancer. Cathelicidin-related antimicrobial peptide (CRAMP), the mouse analog of LL-37 peptide in humans, is a cationic antimicrobial peptide belonging to the cathelicidin family; however, its secretion by cancer cells and role in the tumor microenvironment (TME) remain unclear. In this study, we explored the possibility of an interaction between effector CD4+ T cells and CRAMP using in vitro-generated mouse Th17 cells. We found that CRAMP stimulates Th17 cells to express the ectonucleotidase CD73, while simultaneously inducing cell death. This finding suggested that CD73-expressing Th17 cells may function as immune suppressor cells instead of effector cells. In addition, treatment of pharmacological inhibitors of the transforming growth factor-beta (TGF-ß) signaling pathway showed that induction of CD73 expression is mediated by the p38 signaling pathway. Overall, our findings suggest that tumor-derived LL-37 likely functions as an immune suppressor that induces immune tolerance against tumors through shaping effector Th17 cells into suppressor Th17 cells, suggesting a new intervention target to improve cancer immunotherapy.

IL-4 stimulates mouse macrophages to express APRIL through p38MAPK and two different downstream molecules, CREB and Stat6.

APRIL (a proliferation-inducing ligand) is primarily expressed by macrophages and dendritic cells, and has profound effects on B cell physiology. In this study, we investigated the role of IL-4 in APRIL expression by mouse macrophages and the signaling mechanism involved. IL-4 markedly enhanced APRIL expression in mouse macrophages at the transcriptional and protein level. The p38MAPK inhibitor SB203580 completely abolished the IL-4 effect, whereas overexpression of CREB with IL-4 augmented APRIL expression. This increase was abolished by SB203580 treatment, indicating that p38MAPK may activate CREB. Overexpression of Stat6 also augmented IL-4-induced APRIL expression; this effect was partially abolished by SB203580 but not by the Jak inhibitor AG490, indicating that Stat6 mediates IL-4-induced APRIL expression in a Jak-independent manner and that p38MAPK acts as the intermediate. Our results demonstrate that IL-4 up-regulates APRIL expression through two divergent pathways in mouse macrophages, p38MAPK-CREB and p38MAPK-Stat6.

TGF-beta1 and IFN-gamma stimulate mouse macrophages to express BAFF via different signaling pathways.

B cell-activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and dendritic cells and stimulates the proliferation, differentiation, and survival of B cells and their Ig production. In the present study, we examined the pathways by which TGF-beta1 and IFN-gamma induce BAFF expression to see if TGF-beta1 and IFN-gamma regulate B cell differentiation via macrophages. We found that TGF-beta1 stimulated mouse macrophages to express BAFF and that a typical TGF-beta signaling pathway was involved. Thus, Smad3 and Smad4 promoted BAFF promoter activity, and Smad7 inhibited it, and the BAFF promoter was shown to contain three Smad-binding elements. Importantly, TGF-beta1 enhanced the expression of membrane-bound and soluble forms of BAFF. IFN-gamma further augmented TGF-beta1-induced BAFF expression. IFN-gamma caused phosphorylation of CREB, and overexpression of CREB increased IFN-gamma-induced BAFF promoter activity. Furthermore, H89, a protein kinase A (PKA) inhibitor, abrogated the promoter activity. Neither Stat1alpha (a well-known transducing molecule of IFN-gamma) nor AG490 (a JAK inhibitor) affected BAFF expression in response to IFN-gamma. Taken together, these results demonstrate that TGF-beta1 and IFN-gamma up-regulate BAFF expression through independent mechanisms, i.e., mainly Smad3/4 and PKA/CREB, respectively.

Nonsaponin fraction of Korean Red Ginseng attenuates cytokine production via inhibition of TLR4 expression.

BACKGROUND: Ginsenosides of Korean Red Ginseng extracts (RGE) and its saponin components suppress secretion of inflammasome-mediating cytokines, whereas the nonsaponin fraction (NS) of RGE oppositely stimulates cytokine secretion. Although direct exposure of NS to macrophages in mice induces cytokine production, oral administration of NS has not been studied in inflammasome-related disease in animal models. METHODS: Mice were fed RGE or NS for 7 days and then developed peritonitis. Peritoneal cytokines were measured, and peritoneal exudate cells (PECs) were collected to assay expression levels of a set of toll-like receptors (TLRs) and cytokines in response to NS ingestion. In addition, the role of intestinal bacteria in NS-fed mice was assessed. The effect of preexposure to NS in bone marrow-derived macrophages (BMDMs) on cytokine production was further confirmed. RESULTS: NS ingestion attenuated secretion of peritoneal cytokines resulting from peritonitis. In addition, the isolated PECs from NS-fed mice presented lower TLR transcription levels than PECs from control diet-fed mice. BMDMs treated with NS showed downregulation of TLR4 mRNA and protein expression, which was mediated by the TLR4-MyD88-NFκB signal pathway. BMDMs pretreated with NS produced less cytokines in response to TLR4 ligands. CONCLUSION: NS administration directly inhibits TLR4 expression in inflammatory cells such as macrophages, thereby reducing secretion of cytokines during peritonitis.