Lipoproteins are key immunostimulatory components of Bacillus species for dendritic cell maturation and activation.

Dendritic cells (DCs) play an important role in immunity by sensing and responding to invasive microbes. Bacillus species are rod-shaped sporulating bacteria that include the pathogenic Bacillus cereus and commensal Bacillus subtilis. Although the interaction between DC and these two Bacillus species has been studied, their key structural component that prompts DC activation is poorly understood. Here, we investigated the two Bacillus species in DC activation by whole cells and their representative microbe-associated molecular patterns (MAMPs). MAMPs including lipoteichoic acid (LTA), lipoprotein (LPP), and peptidoglycan (PGN) were purified from the two Bacillus species. Among the MAMPs, LPP from both species most potently induced the maturation and activation of DCs while PGN, but not LTA, moderately stimulated DCs. LPPs from both Bacillus species enhanced the expression of DC maturation markers including CCR7, CD40, CD80, CD83, CD86, CD205, MHC-I, and MHC-II. Among the MAMPs from B. cereus, PGN most considerably lowered the endocytic capacity of DCs implying DC maturation whereas PGN from B. subtilis lowered it to a similar degree to its LPP. Furthermore, DCs sensitized with LPPs from both Bacillus species and PGN from B. subtilis moderately induced TNF-α and IL-6 production. Notably, a combination of MAMPs did not show any synergistic effect on DC activation. Taken together, our results demonstrate that LPP is the key structural component in B. cereus and B. subtilis that leads to DC activation.

Short-chain fatty acids inhibit the biofilm formation of Streptococcus gordonii through negative regulation of competence-stimulating peptide signaling pathway.

Streptococcus gordonii, a Gram-positive commensal bacterium, is an opportunistic pathogen closely related to initiation and progression of various oral diseases, such as periodontitis and dental caries. Its biofilm formation is linked with the development of such diseases by enhanced resistance against antimicrobial treatment or host immunity. In the present study, we investigated the effect of short-chain fatty acids (SCFAs) on the biofilm formation of S. gordonii. SCFAs, including sodium acetate (NaA), sodium propionate (NaP), and sodium butyrate (NaB), showed an effective inhibitory activity on the biofilm formation of S. gordonii without reduction in bacterial growth. SCFAs suppressed S. gordonii biofilm formation at early time points whereas SCFAs did not affect its preformed biofilm. A quorum-sensing system mediated by competence-stimulating peptide (CSP) is known to regulate biofilm formation of streptococci. Interestingly, SCFAs substantially decreased mRNA expression of comD and comE, which are CSP-sensor and its response regulator responsible for CSP pathway, respectively. Although S. gordonii biofilm formation was enhanced by exogenous synthetic CSP treatment, such effect was not observed in the presence of SCFAs. Collectively, these results suggest that SCFAs have an anti-biofilm activity on S. gordonii through inhibiting comD and comE expression which results in negative regulation of CSP quorum-sensing system. SCFAs could be an effective anti-biofilm agent against S. gordonii for the prevention of oral diseases.

Bacterial Lipoproteins Induce BAFF Production via TLR2/MyD88/JNK Signaling Pathways in Dendritic Cells.

B-cell activating factor (BAFF) plays a crucial role in survival, differentiation, and antibody secretion of B cells. Microbial products with B-cell mitogenic properties can indirectly promote expansion and activation of B cells by stimulating accessory cells, such as dendritic cells (DCs), to induce BAFF. Although bacterial lipoproteins are potent B-cell mitogen like lipopolysaccharides (LPSs), it is uncertain whether they can stimulate DCs to induce BAFF expression. Here, we evaluated the effect of bacterial lipoproteins on BAFF expression in mouse bone marrow-derived DCs. Lipoprotein-deficient Staphylococcus aureus mutant induced relatively low expression level of membrane-bound BAFF (mBAFF) and the mRNA compared with its wild-type strain, implying that bacterial lipoproteins can positively regulate BAFF induction. The synthetic lipopeptides Pam2CSK4 and Pam3CSK4, which mimic bacterial lipoproteins, dose-dependently induced BAFF expression, and their BAFF-inducing capacities were comparable to those of LPS in DCs. Induction of BAFF by the lipopeptide was higher than the induction by other microbe-associated molecular patterns, including peptidoglycan, flagellin, zymosan, lipoteichoic acid, and poly(I:C). Pam3CSK4 induced both mBAFF and soluble BAFF expression in a dose- and time-dependent manner. BAFF expression by Pam3CSK4 was completely absent in DCs from TLR2- or MyD88-deficient mice. Among various MAP kinase inhibitors, only JNK inhibitors blocked Pam3CSK4-induced BAFF mRNA expression, while inhibitors blocking ERK or p38 kinase had no such effect. Furthermore, Pam3CSK4 increased the DNA-binding activities of NF-κB and Sp1, but not that of C/EBP. Pam3CSK4-induced BAFF promoter activity via TLR2/1 was blocked by NF-κB or Sp1 inhibitor. Collectively, these results suggest that bacterial lipoproteins induce expression of BAFF through TLR2/MyD88/JNK signaling pathways leading to NF-κB and Sp1 activation in DCs, and BAFF derived from bacterial lipoprotein-stimulated DCs induces B-cell proliferation.

Fibroblasts from patients with idiopathic pulmonary fibrosis are resistant to cisplatin-induced cell death via enhanced CK2-dependent XRCC1 activity.

Idiopathic pulmonary fibrosis (IPF) is a deadly and progressive fibrotic lung disease, but the precise etiology remains elusive. IPF is characterized by the presence of apoptosis-resistant (myo)fibroblasts that relentlessly produce a collagen-rich extracellular matrix (ECM). Recent studies showed that an anti-cancer chemotherapy drug cisplatin is implicated in the development of pulmonary fibrosis, suggesting that the treatment of cancer patients with cisplatin may alter fibroblast viability. To address this possibility, we investigated the cisplatin-induced cell death mechanism in lung fibroblasts derived from IPF and non-IPF patients in response to a collagen matrix. IPF fibroblasts showed enhanced resistance to cisplatin-induced cell death compared to non-IPF fibroblasts in a time- and dose-dependent manner. Molecular study showed that the expression of γH2AX, PUMA and caspase-3/7 activity was abnormally reduced in IPF fibroblasts, suggesting that DNA damage-induced apoptosis caused by cisplatin was suppressed in IPF fibroblasts. Our study further revealed that DNA repair protein XRCC1 activity was aberrantly increased as a result of CK2 hyper-activation in cisplatin-treated IPF fibroblasts, and this alteration protected IPF fibroblasts from cisplatin-induced cell death. Our results showed that IPF fibroblasts residing in a collagen rich matrix are resistance to cisplatin-induced cell death due to the aberrantly high CK2/XRCC1-dependent DNA repair activity. This finding suggests that pulmonary fibrosis may develop and worsen due to the presence of apoptosis-resistant lung fibroblasts in cisplatin-treated cancer patients.

Hyaluronan-CD44/RHAMM interaction-dependent cell proliferation and survival in lung cancer cells.

Although members of the hyaluronan (HA)-CD44/HA-mediated motility receptor (RHAMM) signaling pathway have been shown to be overexpressed in lung cancer, their role in lung tumorigenesis is unclear. In the present study, we first determined levels of HA and its receptors CD44 and RHAMM in human non-small cell lung cancer (NSCLC) cells and stromal cells as well as mouse lung tumors. Subsequently, we examined the role of HA-CD44/RHAMM signaling pathway in mediating the proliferation and survival of NSCLC cells and the cross-talk between NSCLC cells and normal human lung fibroblasts (NHLFs)/lung cancer-associated fibroblasts (LCAFs). The highest levels of HA and CD44 were observed in NHLFs/LCAFs followed by NSCLC cells, whereas THP-1 monocytes/macrophages showed negligible levels of both HA and CD44. Simultaneous silencing of HA synthase 2 (HAS2) and HAS3 or CD44 and RHAMM suppressed cell proliferation and survival as well as the EGFR/AKT/ERK signaling pathway. Exogenous HA partially rescued the defect in cell proliferation and survival. Moreover, conditioned media (CM) generated by NHLFs/LCAFs enhanced the proliferation of NSCLC cells in a HA-dependent manner as treatment of NHLFs and LCAFs with HAS2 siRNA, 4-methylumbelliferone, an inhibitor of HASs, LY2228820, an inhibitor of p38MAPK, or treatment of A549 cells with CD44 blocking antibody suppressed the effects of the CM. Upon incubation in CM generated by A549 cells or THP-1 macrophages, NHLFs/LCAFs secreted higher concentrations of HA. Overall, our findings indicate that targeting the HA-CD44/RHAMM signaling pathway could be a promising approach for the prevention and therapy of lung cancer.

FoxM1-dependent RAD51 and BRCA2 signaling protects idiopathic pulmonary fibrosis fibroblasts from radiation-induced cell death.

Radiation therapy is critical for the control of many tumors and lung is an important dose-limiting organ that impacts radiation dose prescribed to avoid irreversible pulmonary fibrosis in cancer survivors. Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible lung disease caused by aberrantly activated lung (myo)fibroblasts. The presence of pro-fibrotic, apoptosis-resistant fibroblasts in IPF promotes progressive fibrosis and may have a role in other diseases, if these resistant cells are selected for as a consequence of treatment. However, the pathological response of IPF fibroblasts to radiation compared to non-IPF lung fibroblasts is not known. To address this, we examined fibroblast viability following radiation in lung fibroblasts from IPF and non-IPF patients and the underlying mechanism that protects IPF fibroblasts from radiation-induced death. IPF fibroblasts are significantly more resistant to apoptosis compared to non-IPF lung fibroblasts, suggesting that resistance to radiation-induced cell death is a predominant mechanism leading to lung fibrosis. Analysis of γH2AX induction demonstrated that radiation-induced DNA damage is reduced in IPF fibroblasts and correlates to the activation of the transcription factor forkhead box M1 (FoxM1) and subsequent upregulation of DNA repair proteins RAD51 and BRCA2. FoxM1 activation occurs secondary to FoxO3a suppression in IPF fibroblasts while restoration of FoxO3a function sensitizes IPF fibroblasts to radiation-induced cell death and downregulates FoxM1, RAD51, and BRCA2. Our findings support that increased FoxO3a/FoxM1-dependent DNA repair may be integral to the preservation of death-resistant fibrotic fibroblasts after radiation and that selective targeting of radioresistant fibroblasts may mitigate fibrosis.

The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts.

Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6-78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts.

Desensitization of idiopathic pulmonary fibrosis fibroblasts to Alternaria alternata extract-mediated necrotic cell death.

Alternaria alternata is an allergenic fungus and known to cause an upper respiratory tract infection and asthma in humans with compromised immunity. Although A. alternata's effect on airway epithelial cells has previously been examined, the potential role of A. alternata on lung fibroblast viability is not understood. Since lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF) display a distinct phenotype that is resistant to stress and cell death inducing conditions, the investigation of the role of Alternaria on pathological IPF fibroblasts provides a better understanding of the fibrotic process induced by an allergenic fungus. Therefore, we examined cell viability of control and IPF fibroblasts (n = 8 each) in response to A. alternata extract. Control fibroblast cell death was increased while IPF fibroblasts were resistant when exposed to 50-100 µg/mL of A. alternata extract. However, there was no significant difference in kinetics or magnitude of Ca2+ responses from control lung and IPF fibroblasts. In contrast, unlike control fibroblasts, intracellular reactive oxygen species (ROS) levels remained low when IPF cells were treated with A. alternata extracts as a function of time. Caspase 3/7 and TUNEL assay revealed that enhanced cell death caused by A. alternata extract was likely due to necrosis, and 7-AAD assay and the use of sodium pyruvate for ATP generation further supported our findings that IPF fibroblasts become resistant to A. alternata extract-induced necrotic cell death. Our results suggest that exposure to A. alternata potentially worsens the fibrotic process by promoting normal lung fibroblast cell death in patients with IPF.

Advanced Therapeutic Strategies for Chronic Lung Disease Using Nanoparticle-Based Drug Delivery.

Chronic lung diseases include a variety of obstinate and fatal diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF), and lung cancers. Pharmacotherapy is important for the treatment of chronic lung diseases, and current progress in nanoparticles offers great potential as an advanced strategy for drug delivery. Based on their biophysical properties, nanoparticles have shown improved pharmacokinetics of therapeutics and controlled drug delivery, gaining great attention. Herein, we will review the nanoparticle-based drug delivery system for the treatment of chronic lung diseases. Various types of nanoparticles will be introduced, and recent innovative efforts to utilize the nanoparticles as novel drug carriers for the effective treatment of chronic lung diseases will also be discussed.

Idiopathic pulmonary fibrosis fibroblasts become resistant to Fas ligand-dependent apoptosis via the alteration of decoy receptor 3.

Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiology. IPF patients' lung fibroblasts express inappropriately high Akt activity, protecting them in response to an apoptosis-inducing type I collagen matrix. FasL, a ligand for Fas, is known to be increased in the lung tissues of patients with IPF, implicated with the progression of IPF. Expression of Decoy Receptor3 (DcR3), which binds to FasL, thereby subsequently suppressing the FasL-Fas-dependent apoptotic pathway, is frequently altered in various human disease. However, the role of DcR3 in IPF fibroblasts in regulating their viability has not been examined. We found that enhanced DcR3 expression exists in the majority of IPF fibroblasts on collagen matrices, resulting in the protection of IPF fibroblasts from FasL-induced apoptosis. Abnormally high Akt activity suppresses GSK-3ß function, thereby accumulating the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) in the nucleus, increasing DcR3 expression in IPF fibroblasts. This alteration protects IPF cells from FasL-induced apoptosis on collagen. However, the inhibition of Akt or NFATc1 decreases DcR3 mRNA and protein levels, which sensitizes IPF fibroblasts to FasL-mediated apoptosis. Furthermore, enhanced DcR3 and NFATc1 expression is mainly present in myofibroblasts in the fibroblastic foci of lung tissues derived from IPF patients. Our results showed that when IPF cells interact with collagen matrix, aberrantly activated Akt increases DcR3 expression via GSK-3ß-NFATc1 and protects IPF cells from the FasL-dependent apoptotic pathway. These findings suggest that the inhibition of DcR3 function may be an effective approach for sensitizing IPF fibroblasts in response to FasL, limiting the progression of lung fibrosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Reduced FoxO3a expression causes low autophagy in idiopathic pulmonary fibrosis fibroblasts on collagen matrices.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease, and fibroblasts derived from patients with IPF are resistant to type I collagen matrix-induced cell death. The alteration of the PTEN-Akt axis permits IPF fibroblasts to maintain a pathological phenotype on collagen by suppressing autophagy. However, the precise underlying mechanism by which the Akt downstream molecule suppresses autophagic activity remains elusive. FoxO3a is a direct target of Akt and is implicated with the transcriptional activation of autophagy. Therefore, we investigated whether reduced FoxO3a expression causes abnormally low autophagy in IPF fibroblasts on collagen. We found that FoxO3a mRNA and protein levels are low in IPF fibroblasts, which subsequently suppresses the autophagosomal marker LC3B expression on collagen matrix. In contrast, the majority of control fibroblasts showed an increase in FoxO3a and LC3B expression at both the mRNA and protein levels. The luciferase assay confirmed that FoxO3a binds to the promoter region of LC3B and transcriptionally activates LC3B. The overexpression of wild-type FoxO3a increased LC3B mRNA and protein expression in IPF fibroblasts, whereas the dominant negative FoxO3a decreased the LC3B level in control fibroblasts. The inhibition of autophagic activity sensitized control fibroblasts to collagen matrix-induced cell death. In contrast, enhanced viability was found when autophagic function was inhibited in IPF fibroblasts. Our study showed that aberrantly low FoxO3a expression participates in reducing autophagic activity via transcriptional suppression of LC3B in IPF fibroblasts on collagen. This suggests that low autophagic activity by the alteration of FoxO3a may contribute to IPF progression.

Bacterial flagellin induces IL-6 expression in human basophils.

Binding of allergen to IgE on basophils positively affects allergic inflammation by releasing inflammatory mediators. Recently, basophils were shown to express pattern-recognition receptors, such as toll-like receptors (TLRs), for recognizing microbe-associated molecular patterns (MAMPs) that are independent of allergen-IgE binding. In this study, we investigated whether MAMP alone can induce IL-6 production in a human basophil cell line, KU812. Stimulation with flagellin in the absence of allergen-IgE association induced IL-6 expression in KU812 cells, while stimulation with lipoteichoic acid, peptidoglycan, or poly I:C did not under the same condition. Flagellin-induced IL-6 expression was also observed in human primary basophils. Flow cytometric analysis showed that KU812 cells expressed flagellin-recognizing TLR5 both on the cell surface and in the cytoplasm while TLR2 and TLR3 were observed only in the cytoplasm. We further demonstrated that although flagellin augmented the phosphorylation of mitogen-activated protein kinases including p38 kinase, ERK, and JNK, flagellin-induced IL-6 production was attenuated by inhibitors for p38 kinase and ERK, but not by JNK inhibitors. In addition, flagellin enhanced phosphorylation of signaling molecules including CREB, PKCδ, and AKT. The inhibitors for PKA and PKC also showed inhibitory effects. Interestingly, flagellin-induced IL-6 production was further enhanced by pretreatment with inhibitors for PI3K, implying that PI3K negatively affects the flagellin-induced IL-6 production. Furthermore, DNA binding activities of NF-κB, AP-1, and CREB, which play pivotal roles in the induction of IL-6 gene expression, were increased by flagellin. These results suggest that flagellin alone is sufficient to induce IL-6 gene expression via TLR5 signaling pathways in human basophils.

MicroRNA-96 inhibits FoxO3a function in IPF fibroblasts on type I collagen matrix.

Idiopathic pulmonary fibrosis (IPF) is a lethal and progressive lung disease characterized by persistent (myo)fibroblasts and the relentless accumulation of collagen matrix. Unlike normal lung fibroblasts, IPF lung fibroblasts have suppressed forkhead box O3a (FoxO3a) activity, which allows them to expand in this diseased environment. microRNA-96 (miR-96) has recently been found to directly bind to the 3'-untranslated region of FoxO3a mRNA, which subsequently inhibits its function. We examined whether aberrantly low FoxO3a expression is in part due to increased miR-96 levels in IPF fibroblasts on polymerized collagen, thereby causing IPF fibroblasts to maintain their pathological properties. miR-96 expression was upregulated in IPF fibroblasts compared with control fibroblasts when cultured on collagen. In contrast, FoxO3a mRNA levels were reduced in most IPF fibroblasts. However, when miR-96 function was inhibited, FoxO3a mRNA and protein expression were increased, suppressing IPF fibroblast proliferation and promoting their cell death in a dose-dependent fashion. Likewise, FoxO3a and its target proteins p21, p27, and Bim expression was also increased in the presence of a miR-96 inhibitor in IPF fibroblasts. However, when control fibroblasts were treated with miR-96 mimic, FoxO3a, p27, p21, and Bim mRNA and protein levels were decreased. In situ hybridization analysis further revealed the presence of enhanced miR-96 expression in cells within the fibroblastic foci of IPF lung tissue. Our results suggest that when IPF fibroblasts interact with collagen-rich matrix, pathologically altered miR-96 expression inhibits FoxO3a function, causing IPF fibroblasts to maintain their pathological phenotype, which may contribute to the progression of IPF.

Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus.

A Gram-positive bacterium, Staphylococcus aureus is known to be one of the major pathogenic bacteria responsible for causing bovine mastitis. Among the various cell wall components of S. aureus, lipoteichoic acid (LTA) and peptidoglycan (PGN) are closely associated with inflammatory responses. However, the role of LTA and PGN derived from S. aureus in bovine mastitis has not been clearly elucidated. In this study, we characterized the gene expression profile of a bovine mammary gland epithelial cell line, MAC-T cells, in the presence of LTA and PGN from S. aureus. LTA plus PGN, but not LTA or PGN alone, activated MAC-T cells. The analysis of transcriptional profiles using an Affymetrix genechip microarray showed that stimulation with LTA plus PGN produced a total of 2019 (fold change >1.2) differentially expressed genes (DEGs), with 801 up-regulated genes and 1218 down-regulated genes. Of the up-regulated genes, 14 inflammatory mediator-related DEGs, 22 intra-cellular signaling molecule-related DEGs, and 15 transcription factor-related DEGs were observed, whereas among the down-regulated DEGs 17 inflammation-related DEGs were found. The microarray results were confirmed using real-time RT-PCR of 18 genes with substantial changes in expression (9 each from the up-regulated and down-regulated DEGs). These results provide a comprehensive analysis of gene-expression profiles elicited by S. aureus LTA and PGN in MAC-T cells, contributing to an understanding of the pathogenesis for S. aureus-induced bovine mastitis.

Induction of BAFF expression by IFN-γ via JAK/STAT signaling pathways in human intestinal epithelial cells.

BAFF plays an important role in the development of B cells. Here, we investigated the effect of IFN-γ on BAFF expression in human intestinal epithelial cells. IFN-γ induced soluble and membrane-bound BAFF production in a dose- and time-dependent manner. IFN-γ-induced BAFF release from polarized intestinal epithelial cells was observed in apical and basolateral compartments. JAK I inhibitor suppressed IFN-γ-induced BAFF expression. Moreover, IFN-γ enhanced STAT1 phosphorylation and expression of IRF-1. Transient transfection and reporter gene assay showed that the BAFF promoter region spanning -750 to -500 bp from the translation initiation site was crucial for IFN-γ-induced BAFF expression. Nucleotide sequence analysis revealed a GAS element in the promoter region. ChIP assay confirmed the enhanced binding of phosphorylated STAT1 to the BAFF promoter region at -800 to -601 bp. Furthermore, IFN-γ enhanced DNA binding to GAS and its transcriptional activation, as determined by the EMSA and reporter gene assay. Collectively, these results suggest that IFN-γ induces BAFF expression in human intestinal epithelial cells through JAK/STAT signaling pathways that might activate the GAS and IRF-1-binding element in the BAFF promoter.

Staphylococcus aureus induces IL-1ß expression through the activation of MAP kinases and AP-1, CRE and NF-κB transcription factors in the bovine mammary gland epithelial cells.

Although mastitis caused by Staphylococcus aureus is a problematic inflammatory disease in lactating cows, the innate immunity to S. aureus in the mammary gland is poorly understood. In the present study, we observed that heat-killed S. aureus (HKS) induced IL-1ß expression at both the mRNA and protein levels in the mammary gland epithelial cell-line, MAC-T. IL-1ß production was suppressed by inhibitors of lipid rafts, ERK, JNK, and p38 kinases. Furthermore, HKS augmented the activities of the AP-1, CRE, and NF-κB transcription factors that regulate IL-1ß gene expression. Among staphylococcal cell-wall components with inflammatory potential, Pam2CSK4 (a representative model for diacylated lipoproteins) enhanced IL-1ß mRNA expression, while lipoteichoic acid and peptidoglycan did not. Collectively, we suggest that S. aureus-induced IL-1ß production requires lipid raft formation, activation of MAP kinases, and activation of transcription factors AP-1, CRE, and NF-κB. Lipoprotein seems to be a major cell-wall component for the S. aureus-induced IL-1ß production in bovine mammary gland epithelial cells.

Trp-P-1, a carcinogenic heterocyclic amine, inhibits lipopolysaccharide-induced maturation and activation of human dendritic cells.

Carcinogens frequently provoke immunosuppressive effects thereby allowing cancer cells to persist in the host. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is a carcinogenic heterocyclic amine that is abundantly produced by overcooking meat and fish. Here, we investigated the effect of Trp-P-1 on dendritic cells (DCs), which play a central role in the appropriate activation of the host immune system. When human monocyte-derived DCs were stimulated with lipopolysaccharide (LPS), the DCs became mature with an increase in the expression of co-stimulatory receptors such as CD80, CD86, and MHC molecules and a decrease in phagocytic capacity. Trp-P-1 inhibited all of these phenomena under the same conditions. In addition, Trp-P-1 inhibited production of the cytokines TNF-α and IL-12 in LPS-stimulated DCs. Furthermore, DCs that were pre-exposed to Trp-P-1 were less efficient in inducing activation and proliferation of autologous T cells than control DCs. Trp-P-1 also attenuated the ability of DCs to directly kill T-cell lymphoma Jurkat cells. Mechanism studies showed that Trp-P-1 did not inhibit LPS-binding to Toll-like receptor 4 but interfered with the signaling pathways mediated through p38 kinase. In conclusion, our results suggest that Trp-P-1 is immunosuppressive by inhibiting the functionality of DCs that play an essential role in the appropriate induction of anti-cancer immune responses.

Induction of intercellular adhesion molecule-1 by water-soluble components of Hericium erinaceum in human monocytes.

AIM OF THE STUDY: Hericium erinaceum is a medicinal mushroom that has been traditionally used in Asian countries for the treatment of cancers and infectious diseases. Although the immunomodulating activity of H. erinaceum is considered to be responsible for its medicinal activity, its action mechanisms are poorly understood. In the present study, we investigated the capability of water-extracted H. erinaceum (WEHE) to induce the expression of intercellular adhesion molecule-1 (ICAM-1), which regulates the migration of immune cells. MATERIALS AND METHODS: THP-1, a human monocytic cell-line, or human peripheral blood mononuclear cells (PBMC) were stimulated with WEHE (0-30 µg/mL) and subsequently analyzed using flow cytometry to examine the surface expression of ICAM-1 protein. Steady-state levels of ICAM-1 mRNA were estimated using real-time reverse transcription-polymerase chain reaction analysis. Electrophoretic mobility shift assay was conducted to examine transcription factors involved in ICAM-1 transcription. RESULTS: WEHE induced ICAM-1 expression at both protein and mRNA levels in THP-1 cells in a dose- and time-dependent fashion. A similar pattern of ICAM-1 induction was also observed in CD14(+) monocytes in human PBMC that were stimulated with WEHE. The ICAM-1 expression on THP-1 cells stimulated with WEHE was suppressed by specific inhibitors for extracellular signal-regulated kinases (ERK) and reactive oxygen species (ROS). Additionally, exposure of THP-1 cells to WEHE increased the DNA binding activities of NF-κB, AP-1, SP-1 and STAT-1 transcription factors, all of which are known to be required for ICAM-1 gene expression. CONCLUSIONS: These results suggest that WEHE induces ICAM-1 expression in human monocytes through ERK- and ROS-dependent signaling pathways, resulting in the subsequent activations of NF-κB, AP-1, SP-1, and STAT-1 transcription factors.

Induction of ICAM-1 by Armillariella mellea is mediated through generation of reactive oxygen species and JNK activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Armillariella mellea is an edible mushroom that has been traditionally used as an alternative medicine in many countries because of its anti-microbial and anti-cancer effects. AIM OF THE STUDY: In this study, we examined the ability of Armillariella mellea to induce the expression of intercellular adhesion molecule (ICAM)-1, an important cellular adhesion molecule for the recruitment of immune cells to regional inflammatory sites. MATERIALS AND METHODS: A human monocytic cell line, THP-1 or human peripheral blood mononuclear cells (PBMC) were stimulated with Armillariella mellea extract (AME) and subjected to flow cytometry to examine the expression of ICAM-1 protein on the cell surface. Steady-state mRNA level of ICAM-1 was determined by real-time reverse transcription-polymerase chain reaction. The phosphorylation of JNK protein was examined by Western blot analysis using antibodies specific for non-phosphorylated and phosphorylated forms of JNK. For the analysis of transcription factors regulating ICAM-1 transcription, the nuclear fraction was extracted from AME-treated THP-1 cells and subjected to electrophoretic mobility shift assay. RESULTS: AME induced expression of ICAM-1 and its mRNA in THP-1 cells in dose- and time-dependent manners. AME-induced ICAM-1 expression was also observed on CD14-positive monocytes in human PBMC. Interestingly, AME-induced ICAM-1 production was inhibited by the specific inhibitors of reactive oxygen species (ROS) and JNK, whereas no inhibitory effect was observed when inhibitors of ERK, p38 kinase, phosphatidylinositol 3-kinase, or protein kinase C were used. Concomitantly, AME increased phosphorylation of JNK in a time-dependent fashion. DNA binding activities of NF-kappaB, AP-1, SP-1, and STAT-1 were increased by AME treatment. CONCLUSION: These results suggest that AME induces ICAM-1 expression in human monocytic cells through ROS/JNK-dependent signaling pathways leading to the activation of NF-kappaB, AP-1, SP-1, and STAT-1 transcription factors.

Induction of IL-8 expression by Cordyceps militaris grown on germinated soybeans through lipid rafts formation and signaling pathways via ERK and JNK in A549 cells.

AIM OF THE STUDY: In order to elucidate immunoregulatory mechanisms of Cordyceps militaris, a methanol extract of Cordyceps militaris grown on germinated soybeans was prepared and its immunoregulatory effect in the human lung epithelial cells was investigated by examining its ability to induce IL-8 expression. MATERIALS AND METHODS: Cordyceps militaris grown on germinated soybeans was extracted with 80% methanol (GSC4M) and used for stimulation of a human lung epithelial cell-line, A549. An enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction were performed to examine the production of IL-8 protein and its mRNA, respectively. For the analysis of transcription factors regulating IL-8 transcriptional activation, the nuclear fraction was extracted from GSC4M-treated A549 cells and subjected to electrophoretic mobility shift assay. RESULTS: GSC4M induced IL-8 protein secretion and its mRNA expression from A549 cells in a dose- and time-dependent manner. GSC4M-induced IL-8 expression was inhibited by an inhibitor for lipid rafts formation but not by that for clathrin-coated pits. In addition, signaling pathways for GSC4M-induced IL-8 expression were mediated through ERK and JNK but hardly through p38 kinase. Furthermore, GSC4M augmented the DNA-binding activity of the transcription factors AP-1, NF-IL6, and NF-kappaB, all of which are involved in the transcriptional activation of the IL-8 gene. CONCLUSION: Cordyceps militaris grown on germinated soybeans stimulates lung epithelial cells to produce IL-8 through lipid rafts formation and signaling pathways via ERK and JNK.