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Histone deacetylase 6 (HDAC6) has been known to regulate inflammatory diseases. The role of HDAC6 in allergic skin inflammation has not been studied. We studied the role of HDAC6 in atopic dermatitis (AD) and the mechanisms associated with it. The decreased expression or chemical inhibition of HDAC6 suppressed AD by decreasing autophagic flux and cellular features of AD. AD increased expression levels of the Th1 and Th2 cytokines, but decreased expression levels of forkhead box P3 (FoxP3) and interleukin-10 (IL-10) in an HDAC6-dependent manner. CXC chemokine ligand 13 (CXCL13), which was increased in an HDAC6-depenednt manner, mediated AD. MiR-9, negatively regulated by HDAC6, suppressed AD by directly regulating the expression of sirtuin 1 (SIRT1). The downregulation or inhibition of SIRT1 suppressed AD. Experiments employing culture medium and transwell suggested that cellular interactions involving mast cells, keratinocytes, and dermal fibroblast cells could promote AD; HDAC6 and CXCL13 were found to be necessary for these cellular interactions. Mouse recombinant CXCL13 protein increased HDAC6 expression in skin mast cells and dermal fibroblast cells. CXCL13 protein was found to be present in the exosomes of DNCB-treated skin mast cells. Exosomes of DNCB-treated skin mast cells enhanced invasion potentials of keratinocytes and dermal fibroblast cells and increased expression levels of HDAC6, SIRT1 and CXCL13 in keratinocytes and dermal fibroblast cells. These results indicate that HDAC6 and CXCL13 may serve as targets for the developing anti-atopic drugs.

The objective of this study was to investigate mechanisms of allergic inflammation both in vitro and in vivo in details. For this, RNA sequencing was performed. Early growth response 3 gene (Egr3) was one of the most highly upregulated genes in rat basophilic leukemia (RBL2H3) cells stimulated by antigen. The role of Egr3 in allergic inflammation has not been studied extensively. Egr3 was necessary for passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). Egr3 promoter sequences contained potential binding site for NF-κB p65. NF-κB p65 directly regulated Egr3 expression and mediated allergic inflammation in vitro. Histone deacetylases (HDACs) is known to be involved in allergic airway inflammation. HDAC6 promoter sequences contained potential binding site for EGR3. EGR3 showed binding to promoter sequences of HDAC6. EGR3 was necessary for increased expression of histone deacetylase 6 (HDAC6) in antigen-stimulated RBL2H3 cells. HDAC6 mediated allergic inflammation in vitro and PSA. TargetScan analysis predicted that miR-182-5p was a negative regulator of EGR3. Luciferase activity assay confirmed that miR-182-5p was a direct regulator of EGR3. MiR-182-5p mimic inhibited allergic inflammation both in vitro and in vivo. Cytokine array showed that HDAC6 was necessary for increased interleukin-27 (IL-27) expression in BALB/C mouse model of PSA. Antigen stimulation did not affect expression of EBI3, another subunit of IL-27 in RBL2H3 cells or BALB/C mouse model of PCA or PSA. IL-27 receptor alpha was shown to be able to bind to HDAC6. IL-27 p28 mediated allergic inflammation in vitro, PCA, and PSA. Mouse recombinant IL-27 protein promoted features of allergic inflammation in an antigen-independent manner. HDAC6 was necessary for tumorigenic and metastatic potential enhanced by PSA. PSA enhanced the metastatic potential of mouse melanoma B16F1 cells in an IL-27-dependent manner. Experiments employing culture medium and mouse recombinant IL-27 protein showed that IL-27 mediated and promoted cellular interactions involving B16F1 cells, lung macrophages, and mast cells during allergic inflammation. IL-27 was present in exosomes of antigen-stimulated RBL2H3 cells. Exosomes from antigen-stimulated RBL2H3 cells enhanced invasion of B16F1 melanoma cells in an IL-27-dependemt manner. These results present evidence that EGR3-HDAC6-IL-27 axis can regulate allergic inflammation by mediating cellular interactions.

In a previous study, we have demonstrated that p62, a selective receptor of autophagy, can regulate allergic inflammation. In the present study, microRNA array analysis showed that miR-154-5p was increased by antigen (DNP-HSA) in a p62-dependent manner in rat basophilic leukemia cells (RBL2H3). NF-kB directly increased the expression of miR-154-5p. miR-154-5p mediated in vivo allergic reactions, including passive cutaneous anaphylaxis and passive systemic anaphylaxis. Cytokine array analysis showed that antigen stimulation increased the expression of MCP1 in RBL2H3 cells in an miR-154-5p-dependent manner. Reactive oxygen species (ROS)-ERK-NF-kB signaling increased the expression of MCP1 in antigen-stimulated RBL2H3 cells. Recombinant MCP1 protein induced molecular features of allergic reactions both in vitro and in vivo. Anaphylaxis-promoted tumorigenic potential has been known to be accompanied by cellular interactions involving mast cells, and macrophages, and cancer cells. Our experiments employing culture medium, co-cultures, and recombinant MCP1 protein showed that miR-154 and MCP1 mediated these cellular interactions. MiR-154-5p and MCP1 were found to be present in exosomes of RBL2H3 cells. Exosomes from PSA-activated BALB/C mouse induced molecular features of passive cutaneous anaphylaxis in an miR-154-5p-dependent manner. Exosomes from antigen-stimulated RBL2H3 cells enhanced both tumorigenic and metastatic potentials of B16F1 melanoma cells in an miR-154-5p-dependent manner. Exosomes regulated both ROS level and ROS mediated cellular interactions during allergic inflammation. Our results indicate that the miR-154-5p-MCP1 axis might serve as a valuable target for the development of anti-allergy therapeutics.

Homoharringtonine (HHT) is a drug for treatment of chronic myeloid leukemia. However, the role of HHT in allergic inflammations remains unknown. Mouse model of atopic dermatitis (AD) induced by 2, 4,-dinitroflurobenzene (DNFB) and anaphylaxis employing 2,4-dinitropheny-human serum albumin (DNP-HSA) were used to examine the role of HHT in allergic inflammations. HHT inhibited in vitro allergic reactions and attenuated clinical symptoms associated with AD. DNFB induced features of allergic reactions in rat basophilic leukemia (RBL2H3) cells. HHT suppressed effect of AD on the expression of Th1/Th2 cytokines. HHT inhibited passive cutaneous anaphylaxis and passive systemic anaphylaxis. MiR-183-5p, increased by antigen stimulation, was downregulated by HHT in RBL2H3 cells. MiR-183-5p inhibitor suppressed anaphylaxis and AD. B cell translocation gene 1 (BTG1) was shown to be a direct target of miR-183-5p. BTG1 prevented antigen from inducing molecular features of in vitro allergic reactions. AD increased the expression of NF-κB, and NF-κB showed binding to the promoter sequences of miR-183-5p. NF-κB and miR-183 formed positive feedback to mediate in vitro allergic reactions. Thus, HHT can be an anti-allergy drug. We present evidence that NF-κB-miR-183-5p-BTG1 axis can serve as target for development of anti-allergy drug.

Tumor microenvironment consists of cancer cells and various stromal cells such as endothelial cells, cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs), neutrophils, macrophages, and other innate and adaptive immune cells. Of these innate immune cells, macrophages are an extremely heterogeneous population, and display both pro-inflammatory and anti-inflammatory functions. While M1 macrophages (classically activated macrophages) display anti-tumoral and pro-inflammatory functions, M2 macrophages display pro-tumoral and anti-inflammatory functions. Cellular interactions and molecular factors in the tumor microenvironment affect the polarization of macrophages. We review molecules and immune cells that influence the polarization status of macrophages. Tumor-associated macrophages (TAMs) generally express M2 phenotype, and mediate many processes that include tumor initiation, angiogenesis, and metastasis. A high number of TAMs has been associated with the poor prognosis of cancers. MicroRNAs (miRNAs) have been known to regulate cellular interactions that involve cancer cells and macrophages. Tumor-derived exosomes play critical roles in inducing the M1 or M2-like polarization of macrophages. The roles of exosomal miRNAs from tumor cells in the polarization of macrophages are also discussed and the targets of these miRNAs are presented. We review the effects of exosomal miRNAs from TAMs on cancer cell invasion, growth, and anti-cancer drug resistance. The relevance of exosomal microRNAs (miRNAs) as targets for the development of anti-cancer drugs is discussed. We review recent progress in the development of miRNA therapeutics aimed at elevating or decreasing levels of miRNAs.

The objective of this study was to investigate the relationship between autophagy and allergic inflammation. In vitro allergic inflammation was accompanied by an increased autophagic flux in rat basophilic leukemia (RBL2H3) cells. 3-MA, an inhibitor of autophagic processes, negatively regulated allergic inflammation both in vitro and in vivo. The role of p62, a selective receptor of autophagy, in allergic inflammation was investigated. P62, increased by antigen stimulation, mediated in vitro allergic inflammation, passive cutaneous anaphylaxis (PCA), and passive systemic anaphylaxis (PSA). P62 mediated cellular interactions during allergic inflammation. It also mediated tumorigenic and metastatic potential of cancer cells enhanced by PSA. TargetScan analysis predicted that miR-135-5p was a negative regulator of p62. Luciferase activity assay showed that miR-135-5p directly regulated p62. MiR-135-5p mimic negatively regulated features of allergic inflammation and inhibited tumorigenic and metastatic potential of cancer cells enhanced by PSA. MiR-135-5p mimic also inhibited cellular interactions during allergic inflammation. Extracellular vesicles mediated allergic inflammation both in vitro and in vivo. Extracellular vesicles were also necessary for cellular interactions during allergic inflammation. Transmission electron microscopy showed p62 within extracellular vesicles of antigen-stimulated rat basophilic leukemia cells (RBL2H3). Extracellular vesicles isolated from antigen-stimulated RBL2H3 cells induced activation of macrophages and enhanced invasion and migration potential of B16F1 mouse melanoma cells in a p62-dependent manner. Extracellular vesicles isolated from PSA-activated BALB/C mouse enhanced invasion and migration potential of B16F1 cells, and induced features of allergic inflammation in RBL2H3 cells. Thus, miR-135-5p-p62 axis might serve as a target for developing anti-allergy drugs.

The objective of this study was to investigate the effect of human adipose tissue-derived mesenchymal stem cells (AdMSCs) on atopic dermatitis (AD) in the BALB/c mouse model. The AdMSCs attenuated clinical symptoms associated with AD, decreased numbers of degranulated mast cells (MCs), IgE level, amount of histamine released, and prostaglandin E2 level. Atopic dermatitis increased the expression levels of cytokines/chemokines, such as interleukin-5 (IL-5), macrophage inflammatory protein-1ß (MIP-1ß), MIP-2, chemokine (C-C motif) ligand 5 (CCL5), and IL-17, in BALB/c mouse. The AdMSCs showed decreased expression levels of these cytokines in the mouse model of AD. In vivo downregulation of MIP-2 attenuated the clinical symptoms associated with AD. Atopic dermatitis increased the expression levels of hallmarks of allergic inflammation, induced interactions of Fc𝜀RIβ with histone deacetylase 3 (HDAC3) and Lyn, increased ß-hexosaminidase activity, increased serum IgE level, and increased the amount of histamine released in an MIP-2-dependent manner. Downregulation of MIP-2 increased the levels of several miRNAs, including miR-122a-5p. Mouse miR-122a-5p mimic inhibited AD, while suppressor of cytokine signaling 1 (SOCS1), a predicted downstream target of miR-122a-5p, was required for AD. The downregulation of SOCS1 decreased the expression levels of MIP-2 and chemokine (C-X-C motif) ligand 13 (CXCL13) in the mouse model of AD. The downregulation of CXCL13 attenuated AD and allergic inflammation such as passive cutaneous anaphylaxis. The role of T cell transcription factors in AD was also investigated. Atopic dermatitis increased the expression levels of T-bet and GATA-3 [transcription factors of T-helper 1 (Th1) and T-helper 2 (Th2) cells, respectively] but decreased the expression of Foxp3, a transcription factor of regulatory T (Treg) cells, in an SOCS1-dependent manner. In addition to this, miR-122a-5p mimic also prevented AD from regulating the expression of T-bet, GATA-3, and Foxp3. Atopic dermatitis increased the expression of cluster of differentiation 163 (CD163), a marker of M2 macrophages, but decreased the expression of inducible nitric oxide synthase (iNOS), a marker of M1 macrophages. Additionally, SOCS1 and miR-122a-5p mimic regulated the expression of CD163 and iNOS in the mouse model of AD. Experiments employing conditioned medium showed interactions between MCs and macrophages in AD. The conditioned medium of AdMSCs, but not the conditioned medium of human dermal fibroblasts, negatively inhibited the features of allergic inflammation. In summary, we investigated the anti-atopic effects of AdMSCs, identified targets of AdMSCs, and determined the underlying mechanism for the anti-atopic effects of AdMSCs.

Cyclooxgenase-2 (COX-2) knock-out mouse experiments showed that COX-2 was necessary for in vivo allergic inflammation, such as passive cutaneous anaphylaxis, passive systemic anaphylaxis, and triphasic cutaneous allergic reaction. TargetScan analysis predicted COX-2 as a target of miR-26a and miR-26b. miR-26a/-26b decreased luciferase activity associated with COX-2-3'-UTR. miR-26a/-26b exerted negative effects on the features of in vitro and in vivo allergic inflammation by targeting COX-2. ChIP assays showed the binding of HDAC3 and SNAIL, but not COX-2, to the promoter sequences of miR-26a and miR-26b. Cytokine array analysis showed that the induction of chemokines, such as MIP-2, in the mouse passive systemic anaphylaxis model occurred in a COX-2-dependent manner. ChIP assays showed the binding of HDAC3 and COX-2 to the promoter sequences of MIP-2. In vitro and in vivo allergic inflammation was accompanied by the increased expression of MIP-2. miR-26a/-26b negatively regulated the expression of MIP-2. Allergic inflammation enhanced the tumorigenic and metastatic potential of cancer cells and induced positive feedback involving cancer cells and stromal cells, such as mast cells, macrophages, and endothelial cells. miR-26a mimic and miR-26b mimic negatively regulated the positive feedback between cancer cells and stromal cells and the positive feedback among stromal cells. miR-26a/-26b negatively regulated the enhanced tumorigenic potential by allergic inflammation. COX-2 was necessary for the enhanced metastatic potential of cancer cells by allergic inflammation. Taken together, our results indicate that the miR26a/-26b-COX-2-MIP-2 loop regulates allergic inflammation and the feedback relationship between allergic inflammation and the enhanced tumorigenic and metastatic potential. [ABSTRACT FROM AUTHOR]