Strategies for Overcoming Immune Evasion in Bladder Cancer.

Tumors intricately shape a highly immunosuppressive microenvironment, hampering effective antitumor immune responses through diverse mechanisms. Consequently, achieving optimal efficacy in cancer immunotherapy necessitates the reorganization of the tumor microenvironment and restoration of immune responses. Bladder cancer, ranking as the second most prevalent malignant tumor of the urinary tract, presents a formidable challenge. Immunotherapeutic interventions including intravesical BCG and immune checkpoint inhibitors such as atezolizumab, avelumab, and pembrolizumab have been implemented. However, a substantial unmet need persists as a majority of bladder cancer patients across all stages do not respond adequately to immunotherapy. Bladder cancer establishes a microenvironment that can actively hinder an efficient anti-tumor immune response. A deeper understanding of immune evasion mechanisms in bladder cancer will aid in suppressing recurrence and identifying viable therapeutic targets. This review seeks to elucidate mechanisms of immune evasion specific to bladder cancer and explore novel pathways and molecular targets that might circumvent resistance to immunotherapy.

Hexosamine Biosynthetic Pathway-Derived O-GlcNAcylation Is Critical for RANKL-Mediated Osteoclast Differentiation.

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) performed by O-GlcNAc transferase (OGT) is a nutrient-responsive post-translational modification (PTM) via the hexosamine biosynthetic pathway (HBP). Various transcription factors (TFs) are O-GlcNAcylated, affecting their activities and significantly contributing to cellular processes ranging from survival to cellular differentiation. Given the pleiotropic functions of O-GlcNAc modification, it has been studied in various fields; however, the role of O-GlcNAcylation during osteoclast differentiation remains to be explored. Kinetic transcriptome analysis during receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that the nexus of major nutrient metabolism, HBP was critical for this process. We observed that the critical genes related to HBP activation, including Nagk, Gfpt1, and Ogt, were upregulated, while the global O-GlcNAcylation was increased concomitantly during osteoclast differentiation. The O-GlcNAcylation inhibition by the small-molecule inhibitor OSMI-1 reduced osteoclast differentiation in vitro and in vivo by disrupting the translocation of NF-κB p65 and nuclear factor of activated T cells c1 (NFATc1) into the nucleus by controlling their PTM O-GlcNAcylation. Furthermore, OSMI-1 had a synergistic effect with bone target therapy on osteoclastogenesis. Lastly, knocking down Ogt with shRNA (shOgt) mimicked OSMI-1's effect on osteoclastogenesis. Targeting O-GlcNAcylation during osteoclast differentiation may be a valuable therapeutic approach for osteoclast-activated bone diseases.

Fetuin-B regulates vascular plaque rupture via TGF-ß receptor-mediated Smad pathway in vascular smooth muscle cells.

Fetuin-B is a serum protein linked to the regulation of physiological or pathophysiological events such as fertility, energy metabolism, and liver disease. Recently, fetuin-B has been reported to be involved in the modulation of the rupture of atherosclerotic plaques associated with acute myocardial infarction. However, the exact mechanism involved in the modulation of atherosclerotic plaque rupture event by fetuin-B is not fully elucidated yet. In the present study, we investigated whether fetuin-B could influence atherosclerotic plaque rupture through vascular smooth muscle cells (VSMCs). Immunoprecipitation assay using membrane proteins from VSMCs revealed that fetuin-B tightly bound to transforming growth factor-ß receptor (TGF-ßR). Fetuin-B treatment elevated TGF-ßR signals (e.g., phosphorylation of Smad2 and Smad3) in VSMCs. Fetuin-B also stimulated nuclear translocation of phosphorylated Smads. Phosphorylation of Smad and its nuclear translocation by treatment with fetuin-B were inhibited in VSMCs by treatment with SB431542, a selective inhibitor of TGF-ßR. Fetuin-B enhanced expression levels of plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase-2 (MMP-2) in VSMCs through its epigenetic modification including recruitments of both histone deacetylase 1 and RNA polymerase II. These epigenetic alterations in VSMCs were also inhibited by treatment with SB431542. In vivo administration of fetuin-B protein increased expression levels of PAI-1 and MMP-2 in the vascular plaque. However, these increases in expression were inhibited by the administration of SB43154. These results indicate that fetuin-B may modulate vascular plaque rupture by promoting expression of PAI-1 and MMP-2 in VSMCs via TGF-ßR-mediated Smad pathway.

Therapeutic value of steroidal alkaloids in cancer: Current trends and future perspectives.

Discovery and development of new potentially selective anticancer agents are necessary to prevent a global cancer health crisis. Currently, alternative medicinal agents derived from plants have been extensively investigated to develop anticancer drugs with fewer adverse effects. Among them, steroidal alkaloids are conventional secondary metabolites that comprise an important class of natural products found in plants, marine organisms and invertebrates, and constitute a judicious choice as potential anti-cancer leads. Traditional medicine and modern science have shown that representatives from this compound group possess potential antimicrobial, analgesic, anticancer and anti-inflammatory effects. Therefore, systematic and recapitulated information about the bioactivity of these compounds, with special emphasis on the molecular or cellular mechanisms, is of high interest. In this review, we methodically discuss the in vitro and in vivo potential of the anticancer activity of natural steroidal alkaloids and their synthetic and semi-synthetic derivatives. This review focuses on cumulative and comprehensive molecular mechanisms, which will help researchers understand the molecular pathways involving steroid alkaloids to generate a selective and safe new lead compound with improved therapeutic applications for cancer prevention and therapy. In vitro and in vivo studies provide evidence about the promising therapeutic potential of steroidal alkaloids in various cancer cell lines, but advanced pharmacokinetic and clinical experiments are required to develop more selective and safe drugs for cancer treatment.

WZ3146 inhibits mast cell Lyn and Fyn to reduce IgE-mediated allergic responses in vitro and in vivo.

Mast cells (MCs) play an important role as effector cells that cause allergic responses in allergic diseases. For these reasons, MC is considered an attractive therapeutic target for allergic disease treatment. In this study, we investigated the inhibitory effect of WZ3146, N-[3-[5-chloro-2-[4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]oxyphenyl]prop-2-enamide, and the mechanisms of its actions on the MC activation and IgE-mediated allergic response by using three types of MCs such as rat basophilic leukemia (RBL)-2H3 cells, mouse bone marrow mast cells (BMMCs), and human Laboratory of Allergic Diseases 2 (LAD2) cells. WZ3146 inhibited antigen-stimulated degranulation in a dose-dependent manner (IC50, ~ 0.35 µM for RBL-2H3 cells; ~ 0.39 µM for BMMCs; ~ 0.41 for LAD2 cells). WZ3146 also suppressed the production of histamine, tumor necrosis factor (TNF)-α and interleukin (IL)-6, which mediate various allergic responses, in a dose-dependent manner. As the mechanism of WZ3146 to inhibit MCs, it inhibited the activation of spleen tyrosine kinase (Syk) and the downstream signaling proteins of Syk such as linker for activation of T cell (LAT) and phospholipase (PL) Cγ1 in the signaling pathway of FcεRI. In addition, WZ3146 inhibited the activation of Akt, extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). However, WZ3146 did not inhibit degranulation of MCs by thapsigargin or ionomycin, which increase calcium concentration in cytosol. Notably, WZ3146 inhibited the activity of Lyn and Fyn, but not Syk. In an following animal experiment, WZ3146 inhibited IgE-dependent passive cutaneous anaphylaxis (PCA) in a dose-dependent manner (ED50, ~ 20 mg/kg). Taken together, in this study we show that the pyrimidine derivative, WZ3146, inhibits the IgE-mediated allergic response by inhibiting Lyn and Fyn Src-family kinases, which are initially activated by antigen stimulation in MCs. Therefore, we propose that WZ3146 could be used as a new therapeutic agent for the treatment of allergic diseases.

Fibrinogen cleavage products and Toll-like receptor 4 promote the generation of programmed cell death 1 ligand 2-positive dendritic cells in allergic asthma.

BACKGROUND: Inhaled protease allergens preferentially trigger TH2-mediated inflammation in allergic asthma. The role of dendritic cells (DCs) on induction of TH2 cell responses in allergic asthma has been well documented; however, the mechanism by which protease allergens induce TH2-favorable DCs in the airway remains unclear. OBJECTIVE: We sought to determine a subset of DCs responsible for TH2 cell responses in allergic asthma and the mechanism by which protease allergens induce the DC subset in the airway. METHODS: Mice were challenged intranasally with protease allergens or fibrinogen cleavage products (FCPs) to induce allergic airway inflammation. DCs isolated from mediastinal lymph nodes were analyzed for surface phenotype and T-cell stimulatory function. Anti-Thy1.2 and Mas-TRECK mice were used to deplete innate lymphoid cells and mast cells, respectively. Adoptive cell transfer, bone marrow DC culture, anti-IL-13, and Toll-like receptor (TLR) 4-deficient mice were used for further mechanistic studies. RESULTS: Protease allergens induced a remarkable accumulation of TH2-favorable programmed cell death 1 ligand 2 (PD-L2)+ DCs in mediastinal lymph nodes, which was significantly abolished in mice depleted of mast cells and, to a lesser extent, innate lymphoid cells. Mechanistically, FCPs generated by protease allergens triggered IL-13 production from wild-type mast cells but not from TLR4-deficient mast cells, which resulted in an increase in the number of PD-L2+ DCs. Intranasal administration of FCPs induced an increase in numbers of PD-L2+ DCs in the airway, which was significantly abolished in TLR4- and mast cell-deficient mice. Injection of IL-13 restored the PD-L2+ DC population in mice lacking mast cells. CONCLUSION: Our findings unveil the "protease-FCP-TLR4-mast cell-IL-13" axis as a molecular mechanism for generation of TH2-favorable PD-L2+ DCs in allergic asthma and suggest that targeting the PD-L2+ DC pathway might be effective in suppressing allergic T-cell responses in the airway.

DJ-1 is involved in epigenetic control of sphingosine-1-phosphate receptor expression in vascular neointima formation.

DJ-1 and sphingosine-1-phosphate (S1P) receptors (S1PRs) are implicated in the control of physiology and pathophysiology of cardiovascular systems such as blood pressure, atherosclerosis, and restenosis. Here, we investigated whether DJ-1 with antioxidant function participates in the regulation of S1PR1 and S1PR2 expression in vascular smooth muscle cells (VSMCs) and whether this response is related to vascular neointima formation. In vitro studies used cellular migration assay, western blot, reverse transcriptase and real-time PCR analysis, and immunocytochemistry. In vivo studies were performed using the carotid artery ligation model together with immunohistochemistry in DJ-1 knockout (DJKO) and corresponding wild-type (DJWT) mice. S1P stimulated migration of VSMCs from DJKO and DJWT mice. VSMC migration was suppressed by S1PR1 inhibitor but was elevated by S1PR2 inhibitor. Compared with DJWT mice, S1PR1 expression was higher in VSMCs and neointimal plaque from DJKO mice, but S1PR2 expression was lower. Overexpression of DJ-1 in DJKO VSMCs reduced S1PR1 expression and elevated S1PR2 expression. Compared with DJWT mice, histone deacetylase-1 recruitment and histone H3 acetylation at the S1PR1 promoter region were lower and higher, respectively, but this pattern was reversed at the S1PR2 promoter region in DJKO VSMCs. S1PR expressions and epigenetic changes at S1PR promoter regions in DJWT VSMCs treated with H2O2 showed similar patterns to those in DJKO VSMCs. Our findings suggest that DJ-1 may be involved in the regulation of S1PR1 and S1PR2 expression via H2O2-mediated histone modification in VSMCs. Consequently, this modification may affect S1P-induced VSMC migration and be related to vascular neointima formation.

Angiotensin II facilitates neointimal formation by increasing vascular smooth muscle cell migration: Involvement of APE/Ref-1-mediated overexpression of sphingosine-1-phosphate receptor 1.

Angiotensin II (Ang II) is implicated in the development of cardiovascular disorders including hypertension and atherosclerosis. However, the role of Ang II in the interaction between apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) and sphingosine-1-phosphate (S1P) signals in relation to vascular disorders remains to be clarified. This study aimed to determine whether APE/Ref-1 plays a role in epigenetic regulation of the S1P receptor (S1PR) in response to Ang II in vascular smooth muscle cell (VSMC) migration and vascular neointima formation. Ang II augmented the expression of S1PR1 in aortic smooth muscle cells of Sprague Dawley rats (RASMCs), which was attenuated by Ang II receptor (AT) 1 inhibitors, antioxidants, and APE/Ref-1 knockdown with small interference RNA. Ang II stimulation produced H2O2, and exogenous H2O2 elevated S1PR1 expression in RASMCs. Moreover, Ang II caused translocation of cytoplasmic APE/Ref-1 into the nucleus in RASMCs. H3 histone acetylation and APE/Ref-1 binding at the S1PR1 promoter were increased in RASMCs treated with Ang II. In addition, Ang II induced migration in RASMCs, which was suppressed by AT1 and S1PR1 inhibitors. The expression of S1PR1, and colocalization of APE/Ref-1 and acetylated histone H3 in vascular neointima, were greater in Ang II-infused rats compared with a control group. These findings demonstrate that Ang II stimulates the epigenetic regulation of S1PR1 expression via H2O2-mediated APE/Ref-1 translocation, which may consequently be involved in Ang II-induced VSMC migration and vascular neointima formation. Therefore, APE/Ref-1-mediated overexpression of S1PR1 may be implicated in the vascular dysfunction evoked by Ang II.

Suppression of IgE-mediated mast cell activation and mouse anaphylaxis via inhibition of Syk activation by 8-formyl-7-hydroxy-4-methylcoumarin, 4µ8C.

Mast cells trigger IgE-mediated allergic reactions by releasing various allergic mediators. 8-Formyl-7-hydroxy-4-methylcoumarin, also called 4µ8C, was originally known as an inositol-requiring enzyme 1 (IRE1) suppressant, but no study has examined its relationship with mast cells and allergic diseases. Therefore, the purpose of this study was to determine whether 4µ8C is effective in suppressing allergic reactions in mast cells and in IgE-mediated allergic animal model. 4µ8C suppressed the degranulation of IgE-mediated mast cells (IC50=3.2µM) and the production of cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) in a dose-dependent manner. 4µ8C also suppressed passive cutaneous anaphylaxis (PCA) in mice (ED50=25.1mg/kg). In an experiment on mast cell signaling pathways stimulated by antigen, the phosphorylation and activation of Syk was decreased by 4µ8C, and phosphorylation of downstream signaling molecules, such as linker for activated T cells (LAT), Akt, and the three MAP kinases, ERK, p38, and JNK, were suppressed. Mechanistic studies showed that 4µ8C inhibited the activity of Lyn and Fyn in vitro. Based on the results of those experiments, the suppressor mechanism of allergic reaction by 4µ8C involved reduced activity of Lyn and Fyn, which is pivotal in an IgE-mediated signaling pathway. In summary, for the first time, this study shows that 4µ8C inhibits Lyn and Fyn, thus suppressing allergic reaction by reducing the degranulation and the production of inflammatory cytokines. This suggests that 4µ8C can be used as a new medicinal candidate to control allergic diseases such as seasonal allergies and atopic dermatitis.

DJ-1 regulates the expression of renal (pro)renin receptor via reactive oxygen species-mediated epigenetic modification.

BACKGROUND: DJ-1 protein plays multifunctional roles including transcriptional regulation and scavenging oxidative stress; thus, it may be associated with the development of renal disorders. We investigated whether DJ-1 protein regulates the expression of (pro)renin receptor (PRR), a newly identified member of renin-angiotensin system. METHODS: The levels of mRNA and protein were determined by real-time PCR and western blot, respectively. H2O2 production was tested by using fluorescence probe. Histone modification was determined by chromatin immunoprecipitation. RESULTS: The expression of PRR was significantly higher in the kidney from DJ-1 knockout mice (DJ-1-/-) compared with wild-type mice (DJ-1+/+). Histone deacetylase 1 recruitment at the PRR promoter was lower, and histone H3 acetylation and RNA polymerase II recruitment were higher in DJ-1-/- than in DJ-1+/+. Knockdown or inhibition of histone deacetylase 1 restored PRR expression in mesangial cells from DJ-1+/+. H2O2 production was greater in DJ-1-/- cells compared with DJ-1+/+ cells. These changes in PRR expression and epigenetic modification in DJ-1-/- cells were induced by H2O2 treatment and reversed completely by addition of an antioxidant reagent. Prorenin-stimulated ERK1/2 phosphorylation was greater in DJ-1-/- than in DJ-1+/+ cells and this was inhibited by a PRR-inhibitory peptide, and by AT1 and AT2 receptor inhibitors. The expression of renal fibrotic genes was higher in DJ-1-/- than in DJ-1+/+ cells and decreased in PRR-knockdown DJ-1-/- cells. CONCLUSIONS: We conclude that DJ-1 protein regulates the expression of renal PRR through H2O2-mediated epigenetic modification. GENERAL SIGNIFICANCE: We suggest that renal DJ-1 protein may be an important molecule in the acceleration of renal pathogenesis through PRR regulation.

Psammaplin A induces Sirtuin 1-dependent autophagic cell death in doxorubicin-resistant MCF-7/adr human breast cancer cells and xenografts.

BACKGROUND: Psammaplin A (PsA) is a natural product isolated from marine sponges, which has been demonstrated to have anticancer activity against several human cancer cell lines via the induction of cell cycle arrest and apoptosis. New drugs that are less toxic and more effective against multidrug-resistant cancers are urgently needed. METHODS: We tested cell proliferation, cell cycle progression and autophagic cell death pathway in doxorubicin-resistant MCF-7 (MCF-7/adr) human breast cancer cells. The potency of PsA was further determined using an in vivo xenograft model. RESULTS AND CONCLUSION: PsA significantly inhibited MCF-7/adr cells proliferation in a concentration-dependent manner, with accumulation of cells in G2/M phase of the cell cycle. PsA significantly decreased SIRT1 enzyme activity and reduced expression of SIRT1 protein in the cultured cells with greater potency than sirtinol or salermide. Acetylation of p53, a putative target of SIRT1, increased significantly following PsA treatment. In addition, PsA markedly increased the expression levels of autophagy-related proteins. In support of this, it was found that PsA significantly increased the expression of damage-regulated autophagy modulator (DRAM), a p53-induced protein. GENERAL SIGNIFICANCE: The results of this study suggest that PsA is sufficient to overcome multidrug-resistant cancer via SIRT1-mediated autophagy in MCF-7/adr breast cancer cells, indicating that PsA has therapeutic potential for clinical use.

A novel role for flotillin-1 in H-Ras-regulated breast cancer aggressiveness.

Elevated expression and aberrant activation of Ras have been implicated in breast cancer aggressiveness. H-Ras, but not N-Ras, induces breast cell invasion. A crucial link between lipid rafts and H-Ras function has been suggested. This study sought to identify the lipid raft protein(s) responsible for H-Ras-induced tumorigenicity and invasiveness of breast cancer. We conducted a comparative proteomic analysis of lipid raft proteins from invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein flotillin-1 as an important regulator of H-Ras activation and breast cell invasion. Flotillin-1 was required for epidermal growth factor-induced activation of H-Ras, but not that of N-Ras, in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Flotillin-1 knockdown inhibited the invasiveness of MDA-MB-231 and Hs578T TNBC cells in vitro and in vivo. In xenograft mouse tumor models of these TNBC cell lines, we showed that flotillin-1 played a critical role in tumor growth. Using human breast cancer samples, we provided clinical evidence for the metastatic potential of flotillin-1. Membrane staining of flotillin-1 was positively correlated with metastatic spread (p = 0.013) and inversely correlated with patient disease-free survival rates (p = 0.005). Expression of flotillin-1 was associated with H-Ras in breast cancer, especially in TNBC (p < 0.001). Our findings provide insight into the molecular basis of Ras isoform-specific interplay with flotillin-1, leading to tumorigenicity and aggressiveness of breast cancer.

Resveratrol enhances chemosensitivity of doxorubicin in multidrug-resistant human breast cancer cells via increased cellular influx of doxorubicin.

BACKGROUND: Multidrug resistance is a major problem in the treatment of breast cancer, and a number of studies have attempted to find an efficient strategy with which to overcome it. In this study, we investigate the synergistic anticancer effects of resveratrol (RSV) and doxorubicin (Dox) against human breast cancer cell lines. METHODS: The synergistic effects of RSV on chemosensitivity were examined in Dox-resistant breast cancer (MCF-7/adr) and MDA-MB-231 cells. In vivo experiments were performed using a nude mouse xenograft model to investigate the combined sensitization effect of RSV and Dox. RESULTS AND CONCLUSION: RSV markedly enhanced Dox-induced cytotoxicity in MCF-7/adr and MDA-MB-231 cells. Treatment with a combination of RSV and Dox significantly increased the cellular accumulation of Dox by down-regulating the expression levels of ATP-binding cassette (ABC) transporter genes, MDR1, and MRP1. Further in vivo experiments in the xenograft model revealed that treatment with a combination of RSV and Dox significantly inhibited tumor volume by 60%, relative to the control group. GENERAL SIGNIFICANCE: These results suggest that treatment with a combination of RSV and Dox would be a helpful strategy for increasing the efficacy of Dox by promoting an intracellular accumulation of Dox and decreasing multi-drug resistance in human breast cancer cells.

Diminished Lipid Raft SNAP23 Increases Blood Pressure by Inhibiting the Membrane Fluidity of Vascular Smooth-Muscle Cells.

Synaptosomal-associated protein 23 (SNAP23) is involved in microvesicle trafficking and exocytosis in various cell types, but its functional role in blood pressure (BP) regulation has not yet been defined. Here, we found that lipid raft SNAP23 expression was much lower in vascular smooth-muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) than in those from normotensive Wistar-Kyoto (WKY) rats. This led us to investigate the hypothesis that this lower expression may be linked to the spontaneous hypertension found in SHR. The expression level of lipid raft SNAP23 and the fluidity in the plasma membrane of VSMCs were lower in SHR than in WKY rats. Cholesterol content in the VSMC membrane was higher, but the secreted cholesterols found in VSMC-conditioned medium and in the blood serum were lower in SHR than in WKY rats. SNAP23 knockdown in WKY rat VSMCs reduced the membrane fluidity and increased the membrane cholesterol level. Systemic overexpression of SNAP23 in SHR resulted in an increase of cholesterol content in their serum, a decrease in cholesterol in their aorta and the reduction of their BP. Our findings suggest that the low expression of the lipid raft SNAP23 in VSMCs might be a potential cause for the characteristic hypertension of SHR.

An indoxyl compound 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, suppresses activation of Fyn kinase in mast cells and IgE-mediated allergic responses in mice.

Mast cells, constituents of virtually all organs and tissues, are critical cells in IgE-mediated allergic responses. The aim of this study was to investigate the effect and mechanism of an indoxyl chromogenic compound, 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, on IgE-mediated mast cell activation and allergic responses in mice. CAC-0982 reversibly suppressed antigen-stimulated degranulation in murine mast cells (IC50, ~3.8µM) and human mast cells (IC50, ~3.0µM). CAC-0982 also inhibited the expression and secretion of IL-4 and TNF-α in mast cells. Furthermore, CAC-0982 suppressed the mast cell-mediated allergic responses in mice in a dose-dependent manner (ED50 27.9mg/kg). As for the mechanism, CAC-0982 largely suppressed the phosphorylation of Syk and its downstream signaling molecules, including LAT, Akt, Erk1/2, p38, and JNK. Notably, the tyrosine kinase assay of antigen-stimulated mast cells showed that CAC-0982 inhibited Fyn kinase, one of the upstream tyrosine kinases for Syk activation in mast cells. Taken together, these results suggest that CAC-0982 may be used as a new treatment for regulating IgE-mediated allergic diseases through the inhibition of the Fyn/Syk pathway in mast cells.

Rhamnus davurica leaf extract inhibits Fyn activation by antigen in mast cells for anti-allergic activity.

BACKGROUND: Complementary and alternative herbal medicines are recently considered as a promising approach for treating various diseases. We screened approximately 100 plant extracts for anti-allergic activity. Rhamnus davurica leaf extract showed the most potent inhibitory effect on the activation of RBL-2H3 mast cells. Although Rhamnus davurica extract has been used to treat pruritus, dysuresia, and constipation as a traditional herbal medicine in some Asian countries, an anti-allergic effect of Rhamnus davurica has not yet been demonstrated. We aimed to investigate the effect and mechanism of the leaf extract of Rhamnus davurica (LERD) on mast cells in vitro and allergic responses in vivo. METHODS: The effects of LERD on the activation of mast cells and mast cell-mediated passive cutaneous anaphylaxis (PCA) were measured in mice and two types of mast cells, mouse bone marrow-derived mast cells (BMMCs) and RBL-2H3 cells in vitro. A mechanistic study of its inhibitory effect was performed by using degranulation assay, reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting analysis. RESULTS: LERD reversibly suppressed antigen-stimulated degranulation in BMMCs and RBL-2H3 cells, and also inhibited mRNA expression and secretion of TNF-α and IL-4 in a dose-dependent manner. In a PCA animal model, LERD significantly inhibited antigen-induced allergic response and degranulation of ear tissue mast cells. As for the mechanism of action, LERD inhibited the activation of Syk, which is the pivotal signaling protein for mast cell activation by antigen. Furthermore, LERD also impeded the activations of well-known downstream proteins such as LAT, Akt and three MAP kinases (Erk, p38 and JNK). In an in vitro kinase assay, LERD suppressed the activation of Fyn in antigen-stimulated mast cells. CONCLUSION: This study demonstrated for the first time that LERD has anti-allergic effects through inhibiting the Fyn/Syk pathway in mast cells. Therefore, this study provides scientific evidence for LERD to be used as an herbal medicine or health food for patients with allergic diseases.

DJ-1 regulates mast cell activation and IgE-mediated allergic responses.

BACKGROUND: DJ-1 is an antioxidant protein known to reduce levels of reactive oxygen species (ROS), but its presence or function in mast cells and allergic diseases is unknown. OBJECTIVES: We sought to determine the role and mechanism of DJ-1 in allergic responses in vitro and in vivo. METHODS: ROS and DJ-1 levels in serum or culture medium were measured with ELISA kits. The role of DJ-1 was evaluated in mast cell cultures and passive cutaneous anaphylaxis in normal or DJ-1 knockout (KO) mice. The mechanism of DJ-1 action was examined by using immunoblotting, immunoprecipitation, RT-PCR, and other molecular biological approaches. RESULTS: Patients with atopic dermatitis had increased levels of ROS and diminished levels of DJ-1. DJ-1 KO mice exhibited enhanced passive cutaneous anaphylaxis and augmented ROS levels in sera and bone marrow-derived mast cells (BMMCs). Furthermore, antigen-induced degranulation and production of TNF-α and IL-4 were significantly amplified in DJ-1 KO and anti-DJ-1 small interfering RNA-transfected BMMCs compared with that seen in wild-type (WT) BMMCs. Studies with these cells and BMMCs transfected with small interfering RNAs against the phosphatases Src homology domain 2-containing protein tyrosine phosphatase (SHP) 1 and SHP-2 revealed that the DJ-1 KO phenotype could be attributed to suppression of SHP-1 activity and enhancement of SHP-2 activity, leading to strengthened signaling through linker for activation of T cells, phospholipase Cγ, and mitogen-activated protein kinases. CONCLUSIONS: A deficiency or constitutive activation of DJ-1 can have implications in mast cell-driven allergic diseases, such as asthma and anaphylaxis.

IL-27-induced PD-L1highSca-1+ innate lymphoid cells suppress contact hypersensitivity in an IL-10-dependent manner.

Innate lymphoid cells (ILCs) play an important role in maintaining tissue homeostasis and various inflammatory responses. ILCs are typically classified into three subsets, as is the case for T-cells. Recent studies have reported that IL-10-producing type 2 ILCs (ILC210s) have an immunoregulatory function dependent on IL-10. However, the surface markers of ILC210s and the role of ILC210s in contact hypersensitivity (CHS) are largely unknown. Our study revealed that splenic ILC210s are extensively included in PD-L1highSca-1+ ILCs and that IL-27 amplifies the development of PD-L1highSca-1+ ILCs and ILC210s. Adoptive transfer of PD-L1highSca-1+ ILCs suppressed oxazolone-induced CHS in an IL-10-dependent manner Taken together, our results demonstrate that ILC210s are critical for the control of CHS and suggest that ILC210s can be used as target cells for the treatment of CHS.

The Src family kinase Fgr is critical for activation of mast cells and IgE-mediated anaphylaxis in mice.

Mast cells are critical for various allergic disorders. Mast cells express Src family kinases, which relay positive and negative regulatory signals by Ag. Lyn, for example, initiates activating signaling events, but it also induces inhibitory signals. Fyn and Hck are reported to be positive regulators, but little is known about the roles of other Src kinases, including Fgr, in mast cells. In this study, we define the role of Fgr. Endogenous Fgr associates with FcεRI and promotes phosphorylation of Syk, Syk substrates, which include linkers for activation of T cells, SLP76, and Gab2, and downstream targets such as Akt and the MAPKs in Ag-stimulated mast cells. As a consequence, Fgr positively regulates degranulation, production of eicosanoids, and cytokines. Fgr and Fyn appeared to act in concert, as phosphorylation of Syk and degranulation are enhanced by overexpression of Fgr and further augmented by overexpression of Fyn but are suppressed by overexpression of Lyn. Moreover, knockdown of Fgr by small interfering RNAs (siRNAs) further suppressed degranulation in Fyn-deficient bone marrow-derived mast cells. Overexpression of Fyn or Fgr restored phosphorylation of Syk and partially restored degranulation in Fyn-deficient cells. Additionally, knockdown of Fgr by siRNAs inhibited association of Syk with FcεRIγ as well as the tyrosine phosphorylation of FcεRIγ. Of note, the injection of Fgr siRNAs diminished the protein level of Fgr in mice and simultaneously inhibited IgE-mediated anaphylaxis. In conclusion, Fgr positively regulates mast cell through activation of Syk. These findings help clarify the interplay among Src family kinases and identify Fgr as a potential therapeutic target for allergic diseases.

Drug repositioning of anti-microbial agent nifuratel to treat mast cell-mediated allergic responses.

Objectives: Our objective was to assess the effects and mechanisms of nifuratel on IgE-mediated mast cell (MC) degranulation and anaphylaxis in both in vitro and in vivo settings.Methods: The anti-allergic activity of nifuratel was evaluated in mast cell cultures and the passive cutaneous anaphylaxis (PCA) model. The effects of nifuratel on signaling pathways stimulated by antigen in mast cells were measured by immunoblotting, immunoprecipitation, in vitro protein tyrosine kinase assay, and other molecular biological methods.Results: Nifuratel reversibly inhibited antigen-induced degranulation of MCs (IC50, approximately 0.34 µM for RBL-2H3 cells; approximately 0.94 µM for BMMCs) and suppressed the secretion of inflammatory cytokines IL-4 (IC50, approximately 0.74 µM) and TNF-α (IC50, approximately 0.48 µM). Mechanism studies showed that nifuratel inhibited the phosphorylation of Syk by antigen via the inhibition of recruitment of cytosolic Syk to the É£ subunit of FcεRI, and decreased the activation of Syk downstream signaling proteins LAT, Akt, and MAPKs. Finally, nifuratel dose-dependently suppressed the IgE-mediated passive cutaneous anaphylaxis in mice (ED50, approximately 22 mg/kg).Conclusion: Our findings suggest that nifuratel inhibits pathways essential for the activation of mast cells to suppress anaphylaxis, thereby indicating that the anti-microbial drug, nifuratel, could be a potential drug candidate for IgE-mediated allergic disorders.