Benzo(a)pyrene Enhanced Dermatophagoides Group 1 (Der f 1)-Induced TGFß1 Signaling Activation Through the Aryl Hydrocarbon Receptor-RhoA Axis in Asthma.

We have previously demonstrated that benzo(a)pyrene (BaP) co-exposure with dermatophagoides group 1 allergen (Der f 1) can potentiate Der f 1-induced airway inflammation. The underlying mechanism, however, remains undetermined. Here we investigated the molecular mechanisms underlying the potentiation of BaP exposure on Der f 1-induced airway inflammation in asthma. We found that BaP co-exposure potentiated Der f 1-induced TGFß1 secretion and signaling activation in human bronchial epithelial cells (HBECs) and the airways of asthma mouse model. Moreover, BaP exposure alone or co-exposure with Der f 1-induced aryl hydrocarbon receptor (AhR) activity was determined by using an AhR-dioxin-responsive element reporter plasmid. The BaP and Der f 1 co-exposure-induced TGFß1 expression and signaling activation were attenuated by either AhR antagonist CH223191 or AhR knockdown in HBECs. Furthermore, AhR knockdown led to the reduction of BaP and Der f 1 co-exposure-induced active RhoA. Inhibition of RhoA signaling with fasudil, a RhoA/ROCK inhibitor, suppressed BaP and Der f 1 co-exposure-induced TGFß1 expression and signaling activation. This was further confirmed in HBECs expressing constitutively active RhoA (RhoA-L63) or dominant-negative RhoA (RhoA-N19). Luciferase reporter assays showed prominently increased promoter activities for the AhR binding sites in the promoter region of RhoA. Inhibition of RhoA suppressed BaP and Der f 1 co-exposure-induced airway hyper-responsiveness, Th2-associated airway inflammation, and TGFß1 signaling activation in asthma. Our studies reveal a previously unidentified functional axis of AhR-RhoA in regulating TGFß1 expression and signaling activation, representing a potential therapeutic target for allergic asthma.

CaMKII oxidation regulates cockroach allergen-induced mitophagy in asthma.

BACKGROUND: Autophagy plays an important role in causing inflammatory responses initiated by environmental pollutants and respiratory tract infection. OBJECTIVE: We sought to investigate the role of cockroach allergen-induced excessive activation of autophagy in allergic airway inflammation and its underlying molecular mechanisms. METHODS: Environmental allergen-induced autophagy was investigated in the primary human bronchial epithelial cells (HBECs) and lung tissues of asthmatic mouse model and patients. The role of autophagy in asthma development was examined by using autophagy inhibitor 3-methyladenine in an asthma mouse model. Furthermore, the involvements of reactive oxygen species (ROS) and oxidized Ca2+/calmodulin-dependent protein kinase II (ox-CaMKII) signaling in regulating autophagy during asthma were examined in allergen-treated HBECs and mouse model. RESULTS: Cockroach allergen activated autophagy in HBECs and in the lung tissues from asthmatic patients and mice. Autophagy inhibitor 3-methyladenine significantly attenuated airway hyperresponsiveness, TH2-associated lung inflammation, and ROS generation. Mechanistically, we demonstrated a pathological feedforward circuit between cockroach allergen-induced ROS and autophagy that is mediated through CaMKII oxidation. Furthermore, transgenic mice with ROS-resistant CaMKII MM-VVδ showed attenuation of TH2-associated lung inflammation and autophagy. Mitochondrial ox-CaMKII inhibition induced by adenovirus carrying mitochondrial-targeted inhibitor peptide CaMKIIN suppresses cockroach allergen-induced autophagy, mitochondrial dysfunction, mitophagy, and cytokine production in HBECs. Finally, mitochondrial CaMKII inhibition suppressed the expression of one of the key ubiquitin-binding autophagy receptors, optineurin, and its recruitment to fragmented mitochondria. Optineurin knockdown inhibited cockroach allergy-induced mitophagy. CONCLUSIONS: Our data suggest a previously uncovered axis of allergen-ROS-ox-CaMKII-mitophagy in the development of allergic airway inflammation and asthma.

miR-511-3p protects against cockroach allergen-induced lung inflammation by antagonizing CCL2.

miR-511-3p, encoded by CD206/Mrc1, was demonstrated to reduce allergic inflammation and promote alternative (M2) macrophage polarization. Here, we sought to elucidate the fundamental mechanism by which miR-511-3p attenuates allergic inflammation and promotes macrophage polarization. Compared with WT mice, the allergen-challenged Mrc1-/- mice showed increased airway hyperresponsiveness (AHR) and inflammation. However, this increased AHR and inflammation were significantly attenuated when these mice were pretransduced with adeno-associated virus-miR-511-3p (AAV-miR-511-3p). Gene expression profiling of macrophages identified Ccl2 as one of the major genes that was highly expressed in M2 macrophages but antagonized by miR-511-3p. The interaction between miR-511-3p and Ccl2 was confirmed by in silico analysis and mRNA-miR pulldown assay. Further evidence for the inhibition of Ccl2 by miR-511-3p was given by reduced levels of Ccl2 in supernatants of miR-511-3p-transduced macrophages and in bronchoalveolar lavage fluids of AAV-miR-511-3p-infected Mrc1-/- mice. Mechanistically, we demonstrated that Ccl2 promotes M1 macrophage polarization by activating RhoA signaling through Ccr2. The interaction between Ccr2 and RhoA was also supported by coimmunoprecipitation assay. Importantly, inhibition of RhoA signaling suppressed cockroach allergen-induced AHR and lung inflammation. These findings suggest a potentially novel mechanism by which miR-511-3p regulates allergic inflammation and macrophage polarization by targeting Ccl2 and its downstream Ccr2/RhoA axis.

Benzo(a)pyrene facilitates dermatophagoides group 1 (Der f 1)-induced epithelial cytokine release through aryl hydrocarbon receptor in asthma.

BACKGROUND: Environmental pollutants, which coexist with allergens, have been associated with the exacerbation of asthma. However, the underlying molecular mechanisms remain elusive. We sought to determine whether benzo(a)pyrene (BaP) co-exposure with dermatophagoides group 1 allergen (Der f 1) can potentiate Der f 1-induced asthma and its underlying mechanisms. METHODS: The effect of BaP was investigated in Der f 1-induced mouse model of asthma, including airway hyper-responsiveness, allergic inflammation, and epithelial-derived cytokines. The impact of BaP on Der f 1-induced airway epithelial cell oxidative stress (ROS) and cytokine release was further analyzed. The role of aryl hydrocarbon receptor (AhR) signaling in BaP-promoted Der f 1-induced ROS, cytokine production, and allergic inflammation was also investigated. RESULTS: Compared with Der f 1, BaP co-exposure with Der f 1 led to airway hyper-responsiveness and increased lung inflammation in mouse model of asthma. Increased expression of TSLP, IL-33, and IL-25 was also found in the airways of these mice. Moreover, BaP co-exposure with Der f 1 activated AhR signaling with increased expression of AhR and CYP1A1 and promoted airway epithelial ROS generation and TSLP and IL-33, but not IL-25, expression. Interestingly, AhR antagonist CH223191 or cells with AhR knockdown abrogated the increased expression of ROS, TSLP, and IL-33. Furthermore, ROS inhibitor N-acetyl-L-cysteine (NAC) also suppressed BaP co-exposure-induced expression of epithelial TSLP, IL-33, and IL-25. Finally, AhR antagonist CH223191 and NAC inhibited BaP co-exposure with Der f 1-induced lung inflammation. CONCLUSIONS: Our findings suggest that BaP facilitates Der f 1-induced epithelial cytokine release through the AhR-ROS axis.

Ras homolog family member A/Rho-associated protein kinase 1 signaling modulates lineage commitment of mesenchymal stem cells in asthmatic patients through lymphoid enhancer-binding factor 1.

BACKGROUND: Numbers of mesenchymal stem cells (MSCs) are increased in the airways after allergen challenge. Ras homolog family member A (RhoA)/Rho-associated protein kinase 1 (ROCK) signaling is critical in determining the lineage fate of MSCs in tissue repair/remodeling. OBJECTIVES: We sought to investigate the role of RhoA/ROCK signaling in lineage commitment of MSCs during allergen-induced airway remodeling and delineate the underlying mechanisms. METHODS: Active RhoA expression in lung tissues of asthmatic patients and its role in cockroach allergen-induced airway inflammation and remodeling were investigated. RhoA/ROCK signaling-mediated MSC lineage commitment was assessed in an asthma mouse model by using MSC lineage tracing mice (nestin-Cre; ROSA26-EYFP). The role of RhoA/ROCK in MSC lineage commitment was also examined by using MSCs expressing constitutively active RhoA (RhoA-L63) or dominant negative RhoA (RhoA-N19). Downstream RhoA-regulated genes were identified by using the Stem Cell Signaling Array. RESULTS: Lung tissues from asthmatic mice showed increased expression of active RhoA when compared with those from control mice. Inhibition of RhoA/ROCK signaling with fasudil, a RhoA/ROCK inhibitor, reversed established cockroach allergen-induced airway inflammation and remodeling, as assessed based on greater collagen deposition/fibrosis. Furthermore, fasudil inhibited MSC differentiation into fibroblasts/myofibroblasts but promoted MSC differentiation into epithelial cells in asthmatic nestin-Cre; ROSA26-EYFP mice. Consistently, expression of RhoA-L63 facilitated differentiation of MSCs into fibroblasts/myofibroblasts, whereas expression of RhoA-19 switched the differentiation toward epithelial cells. The gene array identified the Wnt signaling effector lymphoid enhancer-binding factor 1 (Lef1) as the most upregulated gene in RhoA-L63-transfected MSCs. Knockdown of Lef1 induced MSC differentiation away from fibroblasts/myofibroblasts but toward epithelial cells. CONCLUSIONS: These findings uncover a previously unrecognized role of RhoA/ROCK signaling in MSC-involved airway repair/remodeling in the setting of asthma.

miR-155 Modulates Cockroach Allergen- and Oxidative Stress-Induced Cyclooxygenase-2 in Asthma.

Exposure to cockroach allergen is a strong risk factor for developing asthma. Asthma has been associated with allergen-induced airway epithelial damage and heightened oxidant stress. In this study, we investigated cockroach allergen-induced oxidative stress in airway epithelium and its underlying mechanisms. We found that cockroach extract (CRE) could induce reactive oxygen species (ROS) production, particularly mitochondrial-derived ROS, in human bronchial epithelial cells. We then used the RT2 Profiler PCR array and identified that cyclooxygenase-2 (COX-2) was the most significantly upregulated gene related to CRE-induced oxidative stress. miR-155, predicted to target COX-2, was increased in CRE-treated human bronchial epithelial cells, and was showed to regulate COX-2 expression. Moreover, miR-155 can bind COX-2, induce COX-2 reporter activity, and maintain mRNA stability. Furthermore, CRE-treated miR-155-/- mice showed reduced levels of ROS and COX-2 expression in lung tissues and PGE2 in bronchoalveolar lavage fluid compared with wild-type mice. These miR-155-/- mice also showed reduced lung inflammation and Th2/Th17 cytokines. In contrast, when miR-155-/- mice were transfected with adeno-associated virus carrying miR-155, the phenotypic changes in CRE-treated miR-155-/- mice were remarkably reversed, including ROS, COX-2 expression, lung inflammation, and Th2/Th17 cytokines. Importantly, plasma miR-155 levels were elevated in severe asthmatics when compared with nonasthmatics or mild-to-moderate asthmatics. These increased plasma miR-155 levels were also observed in asthmatics with cockroach allergy compared with those without cockroach allergy. Collectively, these findings suggest that COX-2 is a major gene related to cockroach allergen-induced oxidative stress and highlight a novel role of miR-155 in regulating the ROS-COX-2 axis in asthma.

Mannose receptor modulates macrophage polarization and allergic inflammation through miR-511-3p.

BACKGROUND: Mannose receptor (MRC1/CD206) has been suggested to mediate allergic sensitization and asthma to multiple glycoallergens, including cockroach allergens. OBJECTIVE: We sought to determine the existence of a protective mechanism through which MRC1 limits allergic inflammation through its intronic miR-511-3p. METHODS: We examined MRC1-mediated cockroach allergen uptake by lung macrophages and lung inflammation using C57BL/6 wild-type (WT) and Mrc1-/- mice. The role of miR-511-3p in macrophage polarization and cockroach allergen-induced lung inflammation in mice transfected with adeno-associated virus (AAV)-miR-511-3p (AAV-cytomegalovirus-miR-511-3p-enhanced green fluorescent protein) was analyzed. Gene profiling of macrophages with or without miR-511-3p overexpression was also performed. RESULTS: Mrc1-/- lung macrophages showed a significant reduction in cockroach allergen uptake compared with WT mice, and Mrc1-/- mice had an exacerbated lung inflammation with increased levels of cockroach allergen-specific IgE and TH2/TH17 cytokines in a cockroach allergen-induced mouse model compared with WT mice. Macrophages from Mrc1-/- mice showed significantly reduced levels of miR-511-3 and an M1 phenotype, whereas overexpression of miR-511-3p rendered macrophages to exhibit a M2 phenotype. Furthermore, mice transfected with AAV-miR-511-3p showed a significant reduction in cockroach allergen-induced inflammation. Profiling of macrophages with or without miR-511-3p overexpression identified 729 differentially expressed genes, wherein expression of prostaglandin D2 synthase (Ptgds) and its product PGD2 were significantly downregulated by miR-511-3p. Ptgds showed a robust binding to miR-511-3p, which might contribute to the protective effect of miR-511-3p. Plasma levels of miR-511-3p were significantly lower in human asthmatic patients compared with nonasthmatic subjects. CONCLUSION: These studies support a critical but previously unrecognized role of MRC1 and miR-511-3p in protection against allergen-induced lung inflammation.

Macrophage polarization and allergic asthma.

Allergic asthma is associated with airway inflammation and airway hyperresponsiveness. Macrophage polarization has been shown to have a profound impact on asthma pathogenesis. On exposure to local microenvironments, recruited macrophages can be polarized into either classically activated (or M1) or alternatively activated (or M2) phenotypes. Macrophage polarization has been heavily associated with development of asthma. The process of regulation of macrophage polarization involves an intricate interplay between various cytokines, chemokines, transcriptional factors, and immune-regulatory cells. Different signals from the microenvironment are controlled by different receptors on the macrophages to initiate various macrophage polarization pathways. Most importantly, there is an increased attention on the epigenetic changes (eg, microRNAs, DNA methylation, and histone modification) that impact macrophage functional responses and M1/M2 polarization through modulating cellular signaling and signature gene expression. Thus, modulation of macrophage phenotypes through molecular intervention by targeting some of those potential macrophage regulators may have therapeutic potential in the treatment of allergic asthma and other allergic diseases. In this review, we will discuss the origin of macrophages, characterization of macrophages, macrophage polarization in asthma, and the underlying mechanisms regarding allergen-induced macrophage polarization with emphasis on the regulation of epigenetics, which will provide new insights into the therapeutic strategy for asthma.

Functional role of kynurenine and aryl hydrocarbon receptor axis in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with mast cell-mediated inflammation and heightened oxidant stress. Kynurenine (KYN), an endogenous tryptophan metabolite, can promote allergen-induced mast cell activation through the aryl hydrocarbon receptor (AhR). OBJECTIVES: We sought to determine the role of the KYN/AhR axis and oxidant stress in mast cell activation and the development of CRSwNP. METHODS: We measured the expression of indoleamine 2,3-dioxygenase 1, tryptophan 2,3-dioxygenase, KYN, and oxidized calmodulin-dependent protein kinase II (ox-CaMKII) in nasal polyps and controls. KYN-potentiated ovalbumin (OVA)-induced ROS generation, cell activation, and ox-CaMKII expression were investigated in wild-type and AhR-deficient (AhR-/-) mast cells. The role of ox-CaMKII in mast cell activation was further investigated. RESULTS: Nasal polyps in CRSwNP showed an increased expression of indoleamine 2,3-dioxygenase 1, tryptophan2,3-dioxygenase, and KYN compared with controls. AhR was predominantly expressed in mast cells in nasal polyps. Activated mast cells and local IgE levels were substantially increased in eosinophilic polyps compared with noneosinophilic polyps and controls. Furthermore, KYN potentiated OVA-induced ROS generation, intracellular Ca2+ levels, cell activation, and expression of ox-CaMKII in wild-type, but not in AhR-/- mast cells. Compared with noneosinophilic polyps and controls, eosinophilic polyps showed increased expression of ox-CaMKII in mast cells. Mast cells from ROS-resistant CaMKII MMVVδ mice or pretreated with CaMKII inhibitor showed protection against KYN-promoted OVA-induced mast cell activation. CONCLUSIONS: These studies support a potentially critical but previously unidentified function of the KYN/AhR axis in regulating IgE-mediated mast cell activation through ROS and ox-CaMKII in CRSwNP.

N-glycan in cockroach allergen regulates human basophil function.

INTRODUCTION: Cockroach allergen exposure elicits cockroach sensitization and poses an increased risk for asthma. However, the major components in cockroach allergen and the mechanisms underlying the induction of cockroach allergen-induced allergy and asthma remain largely elusive. We sought to examine the role of cockroach-associated glycan in regulating human basophil function. METHODS: N-linked glycans from naturally purified cockroach allergen Bla g 2 were characterized by MALDI-TOF mass spectrometry. Binding of cockroach allergen to serum IgE from cockroach allergic subjects was determined by solid-phase binding immunoassays. Role of cockroach associated glycan in histamine release and IL-4 production from human basophils was examined. Expression of C-type lectin receptors (CLRs) and their role in mediating glycan-uptake in the basophils was also investigated. RESULTS: MALDI-TOF mass spectrometric analysis of N-glycan from Bla g 2 showed complex hybrid-types of glycans that terminated with mannose, galactose, and/or N-acetyl glucosamine (GlcNAc). Deglycosylated Bla g 2 showed reduced binding to IgE and was less capable of inducing histamine release from human basophils. In contrast, N-glycan derived from Bla g 2 significantly inhibited histamine release and IL-4 production from basophils passively sensitized with serum from cockroach allergic subjects. An analysis of CLRs revealed the expression of DC-SIGN and DCIR, but not MRC1 and dectin-1, in human basophils. Neutralizing antibody to DCIR, but not DC-SIGN, significantly inhibited Bla g 2 uptake by human basophils. A dose-dependent bindings of cockroach allergen to DCIR was also observed. CONCLUSIONS: These observations indicate a previously unrecognized role for cockroach allergen-associated glycans in allergen-induced immune reactions, and DCIR may play a role in mediating the regulation of glycan on basophil function.

Aryl Hydrocarbon Receptor Protects Lungs from Cockroach Allergen-Induced Inflammation by Modulating Mesenchymal Stem Cells.

Exposure to cockroach allergen leads to allergic sensitization and increased risk of developing asthma. Aryl hydrocarbon receptor (AhR), a receptor for many common environmental contaminants, can sense not only environmental pollutants but also microbial insults. Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the capacity to modulate immune responses. In this study, we investigated whether AhR can sense cockroach allergens and modulate allergen-induced lung inflammation through MSCs. We found that cockroach allergen-treated AhR-deficient (AhR(-/-)) mice showed exacerbation of lung inflammation when compared with wild-type (WT) mice. In contrast, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an AhR agonist, significantly suppressed allergen-induced mouse lung inflammation. MSCs were significantly reduced in cockroach allergen-challenged AhR(-/-) mice as compared with WT mice, but increased in cockroach allergen-challenged WT mice when treated with TCDD. Moreover, MSCs express AhR, and AhR signaling can be activated by cockroach allergen with increased expression of its downstream genes cyp1a1 and cyp1b1. Furthermore, we tracked the migration of i.v.-injected GFP(+) MSCs and found that cockroach allergen-challenged AhR(-/-) mice displayed less migration of MSCs to the lungs compared with WT. The AhR-mediated MSC migration was further verified by an in vitro Transwell migration assay. Epithelial conditioned medium prepared from cockroach extract-challenged epithelial cells significantly induced MSC migration, which was further enhanced by TCDD. The administration of MSCs significantly attenuated cockroach allergen-induced inflammation, which was abolished by TGF-ß1-neutralizing Ab. These results suggest that AhR plays an important role in protecting lungs from allergen-induced inflammation by modulating MSC recruitment and their immune-suppressive activity.