Potent Eosinophil Chemoattractant Research Articles

Basophil-Derived IL-4 and IL-13 Protect Intestinal Barrier Integrity and Control Bacterial Translocation during Malaria.

Our previous work demonstrated that basophils regulate a suite of malaria phenotypes, including intestinal mastocytosis and permeability, the immune response to infection, gametocytemia, and parasite transmission to the malaria mosquito Anopheles stephensi. Given that activated basophils are primary sources of the regulatory cytokines IL-4 and IL-13, we sought to examine the contributions of these mediators to basophil-dependent phenotypes in malaria. We generated mice with basophils depleted for IL-4 and IL-13 (baso IL-4/IL-13 (-)) and genotype controls (baso IL-4/IL-13 (+)) by crossing mcpt8-Cre and Il4/Il13fl/fl mice and infected them with Plasmodium yoelii yoelii 17XNL. Conditional deletion was associated with ileal mastocytosis and mast cell (MC) activation, increased intestinal permeability, and increased bacterial 16S levels in blood, but it had no effect on neutrophil activation, parasitemia, or transmission to A. stephensi. Increased intestinal permeability in baso IL-4/IL-13 (-) mice was correlated with elevated plasma eotaxin (CCL11), a potent eosinophil chemoattractant, and increased ileal MCs, proinflammatory IL-17A, and the chemokines MIP-1α (CCL3) and MIP-1β (CCL4). Blood bacterial 16S copies were positively but weakly correlated with plasma proinflammatory cytokines IFN-γ and IL-12p40, suggesting that baso IL-4/IL-13 (-) mice failed to control bacterial translocation into the blood during malaria infection. These observations suggest that basophil-derived IL-4 and IL-13 do not contribute to basophil-dependent regulation of parasite transmission, but these cytokines do orchestrate protection of intestinal barrier integrity after P. yoelii infection. Specifically, basophil-dependent IL-4/IL-13 control MC activation and prevent infection-induced intestinal barrier damage and bacteremia, perhaps via regulation of eosinophils, macrophages, and Th17-mediated inflammation.

Abstract A025: Autotaxin-lysolipid signaling suppresses a CCL11- eosinophil axis to promote pancreatic cancer progression

Abstract Among all cancer types, pancreatic ductal adenocarcinoma (PDAC) has one of the worst outcomes. In recent studies, metabolic and microenvironmental reprogramming in PDAC has been shown to facilitate tumor progression by promoting immune escape mechanisms, however these mechanisms are still poorly understood. Recently, our lab established that autotaxin (ATX) - lysophosphatidic acid (LPA) signaling in PDAC enhances tumorigenicity, but the immunomodulatory function of this signal pathway remains largely unexplored. We now show that ablation of the LPA-producing enzyme autotaxin (ATX) in PDAC cells can reduce tumor burden in mice and reprogram the tumor immune microenvironment by increasing tumor eosinophil infiltration. The functional role of eosinophils in the context of PDAC is a poorly understood area. The presence of eosinophils in pancreatic cancer has been reported in a few case reports: for example, prior studies identified neutrophils and eosinophils together as a group, expressing high levels of CD66b. We confirmed the presence of eosinophils in PDAC in mice as well as patients, and we found that rare patient samples with the highest intratumoral eosinophil abundance had the longest survival rates. Genetic or pharmacologic ATX inhibition increased intratumoral eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. When eosinophils were present, primary tumor growth was modestly reduced, but metastatic spread was greatly reduced. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop wherein ATX-LPA signaling negatively regulates activity of AP-1 transcription factor c-Jun, in turn suppressing expression of potent eosinophil chemoattractant CCL11 (Eotaxin-1). This is the first study that highlights the anti-tumor potential of eosinophils in pancreatic cancer. Citation Format: Sohinee Bhattacharyya, Chet Oon, Holly Sandborg, Terry Morgan, Mara H. Sherman. Autotaxin-lysolipid signaling suppresses a CCL11- eosinophil axis to promote pancreatic cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A025.

Autotaxin-lysolipid signaling suppresses a CCL11-eosinophil axis to promote pancreatic cancer progression.

Lipids and their modifying enzymes regulate diverse features of the tumor microenvironment and cancer progression. The secreted enzyme autotaxin (ATX) hydrolyzes extracellular lysophosphatidylcholine to generate the multifunctional lipid mediator lysophosphatidic acid (LPA) and supports the growth of several tumor types, including pancreatic ductal adenocarcinoma (PDAC). Here we show that ATX suppresses the accumulation of eosinophils in the PDAC microenvironment. Genetic or pharmacologic ATX inhibition increased the number of intratumor eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop, wherein ATX-LPA signaling negatively regulates the activity of the AP-1 transcription factor c-Jun, in turn suppressing the expression of the potent eosinophil chemoattractant CCL11 (eotaxin-1). Eosinophils were identified in human PDAC specimens, and rare individuals with high intratumor eosinophil abundance had the longest overall survival. Together with recent findings, this study reveals the context-dependent, immune-modulatory potential of ATX-LPA signaling in cancer.

Concise Syntheses of Microsomal Metabolites of a Potent OXE (Oxoeicosanoid) Receptor Antagonist.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most potent eosinophil chemoattractant among lipid mediators, and its actions are mediated by the selective oxoeicosanoid (OXE) receptor. Our group previously developed a highly potent indole-based OXE antagonist, S-C025, with an IC50 value of 120 pM. S-C025 was converted to a number of metabolites in the presence of monkey liver microsomes. Complete chemical syntheses of authentic standards enabled us to identify that the four major metabolites were derived by the oxidation at its benzylic and N-methyl carbon atoms. Herein we report concise syntheses of the four major metabolites of S-C025.

Type-2 Cytokines Promote the Secretion of the Eosinophil-Attractant CCL26 by Intestinal Epithelial Cells in Food-Sensitized Patients.

Several inflammatory processes of the bowel are characterized by an accumulation of eosinophils at inflammation sites. The mechanisms that govern mucosal infiltration with eosinophils are not fully understood. In this work, we studied the colorectal polyp-confined tissue containing eosinophils and we hypothesized that intestinal epithelial cells are the cell source of eotaxin-3 or CCL26, a potent chemoattractant for eosinophils. We analyzed colorectal polyps (n=50) from pediatric patients with rectal bleeding by H&E staining and eosin staining, and different pro-inflammatory cytokines were assessed by RT-qPCR and ELISA. IgE and CCL26 were investigated by RT-qPCR, ELISA and confocal microscopy. Finally, the intracellular signaling pathway that mediates the CCL26 production was analyzed using a kinase array and immunoblotting in human intestinal Caco-2 cell line. We found a dense cell agglomeration within the polyps, with a significantly higher frequency of eosinophils than in control adjacent tissue. IL-4 and IL-13 were significantly up-regulated in polyps and CCL26 was elevated in the epithelial compartment. Experiments with Caco-2 cells showed that the type-2 cytokine IL-13 increased STAT3 and STAT6 phosphorylation and eotaxin-3 secretion. The addition of the blocking antibody Dupilumab or the inhibitor Ruxolitinib to the cytokine-stimulated Caco-2 cells diminished the CCL26 secretion to basal levels in a dose-dependent manner. In conclusion, our findings demonstrate a high frequency of eosinophils, and elevated levels of type-2 cytokines and eotaxin-3 in the inflammatory stroma of colorectal polyps from pediatric patients. Polyp epithelial cells showed to be the main cell source of CCL26, and IL-13 was the main trigger of this chemokine through the activation of the STAT3/STAT6/JAK1-2 pathway. We suggest that the epithelial compartment actively participates in the recruitment of eosinophils to the colonic polyp-confined inflammatory environment.

Targeting the OXE receptor with a selective antagonist inhibits allergen‐induced pulmonary inflammation in non‐human primates

The 5-lipoxygenase product, 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid), is a potent chemoattractant for eosinophils and neutrophils. However, little is known about its pathophysiological role because of the lack of a rodent ortholog of the oxoeicosanoid (OXE) receptor. The present study aimed to determine whether the selective OXE receptor antagonist S-Y048 can inhibit allergen-induced pulmonary inflammation in a monkey model of asthma. Monkeys sensitized to house dust mite antigen (HDM) were treated with either vehicle or S-Y048 prior to challenge with aerosolized HDM, and bronchoalveolar (BAL) fluid was collected 24 h later. After 6weeks, animals that had initially been treated with vehicle received S-Y048 and vice versa for animals initially treated with S-Y048. Eosinophils and neutrophils in BAL and lung tissue samples were evaluated, as well as mucus-containing cells in bronchi. HDM significantly increased the numbers of eosinophils, neutrophils, and macrophages in BAL fluid 24 h after challenge. These responses were all significantly inhibited by S-Y048, which also reduced the numbers of eosinophils and neutrophils in lung tissue 24 h after challenge with HDM. S-Y048 also significantly reduced the numbers of bronchial epithelial cells staining for mucin and MUC5AC after antigen challenge. This study provides the first evidence that 5-oxo-ETE may play an important role in inducing allergen-induced pulmonary inflammation and could also be involved in regulating MUC5AC in goblet cells. OXE receptor antagonists such as S-Y048 may useful therapeutic agents in asthma and other eosinophilic as well as neutrophilic diseases.

Immunomodulator Galectin-9 is Increased in Blood and Skin of Patients with Bullous Pemphigoid.

Massive recruitment of eosinophils into the dermis is a hallmark of bullous pemphigoid pathogenesis. Identifying the chemoattractant(s) guiding eosinophils into the skin in bullous pemphigoid is a prerequisite to therapeutic targeting of eosinophil recruitment. Galectin-9 is a potent chemoattractant for eosinophils, but its potential role in bullous pemphigoid is unknown. The aim of this study was to determine the expression levels of galectin-9 in serum and skin of patients with bullous pemphigoid. Galectin-9 levels were significantly elevated in serum of patients with bullous pemphigoid compared with age- and sex-matched controls, but did not correlate with disease activity assessed with the Bullous Pemphigoid Disease Area Index. Galectin-9 expression was also increased in lesional skin of patients with bullous pemphigoid, and was expressed predominantly in eosinophils, neutrophils and keratinocytes. In conclusion, these results support the notion that galectin-9 may play a role in the pathogenesis of bullous pemphigoid.

Potentiation of IL-4 Signaling by Retinoic Acid in Intestinal Epithelial Cells and Macrophages-Mechanisms and Targets.

We previously demonstrated that IL4, IL13, CLCA1, and CCL26 mRNA were significantly upregulated in the lungs of pigs given a low dose of all trans-retinoic acid (ATRA) and infected with Ascaris suum. We also demonstrated that in vitro ATRA induced a state of partial alternative activation in porcine macrophages (Mφs) and amplified certain aspects of M2a activation induced by IL-4. Given these results, we tested the effect of ATRA on IL-4 responses in two porcine intestinal epithelial cell lines, IPEC1 and IPEC-J2 and observed that ATRA increased mRNA for the IL-4 receptor alpha chain. ATRA also increased IL-4 induced phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mRNA expression of the chloride channel, calcium activated, family member 1 (CLCA1), important for mucus formation, and chemokine (C-C motif) ligand 26 (CCL26), a potent eosinophil chemoattractant. We extended these findings to human Mφ THP-1 cells and showed that ATRA synergistically increased IL-4–induced CCL2, CCL13, and CCL26 mRNA and protein levels. Transglutaminase 2 mRNA, protein, and enzyme activity were synergistically induced in THP-1 cells pretreated with ATRA and then treated with IL-4, thus, ATRA increased signaling in response to IL-4 in porcine epithelial cells and porcine and human Mφs. Given the prevalence of allergic and parasitic diseases worldwide and the close similarities in the porcine and human immune responses, these findings have important implications for the nutritional regulation of allergic inflammation at mucosal surfaces.

TSLP regulates eotaxin-1 production by nasal epithelial cells from patients with eosinophilic CRSwNP.

Eosinophilic chronic rhinosinusitis with nasal polyps (eCRSwNP) is characterized by Th2-skewed inflammation with eosinophilic infiltration. Thymic stromal lymphopoietin (TSLP) promotes the development of allergic inflammation. Although increased TSLP is found in eCRSwNP, little is known about whether TSLP regulates eotaxin-1 production, a potent eosinophil chemoattractant that recruits and activates eosinophils. The aim of this study was to investigate the effects and mechanisms of TSLP in eotaxin-1 production in the eosinophilic inflammation of eCRSwNP. Human nasal epithelial cells (HNECs) from eCRSwNP patients were stimulated with recombinant human TSLP in the presence or absence of CYT387 (Janus kinase 1/2 inhibitor). Phosphorylated signal transducer activator of transcription 3 (p-STAT3) was measured by using immunocytochemistry. Eotaxin-1 expression was determined by using real-time PCR. Western blotting was used to detect the levels of p-STAT3 and eotaxin-1 protein. The treatment with TSLP induced STAT3 phosphorylation in HNECs, and promoted p-STAT3 nuclear translocation, leading to a time-dependent increase of eotaxin-1 expression. However, these effects were attenuated by CYT387 pretreatment. TSLP regulated eotaxin-1 production in HNECs via JAK1/2-STAT3 signaling, which might contribute to the eosinophilic inflammation of eCRSwNP.

5-Oxo-ETE from Nasal Epithelial Cells Upregulates Eosinophil Cation Protein by Eosinophils in Nasal Polyps in vitro

Background: 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent eosinophil chemoattractant and activator that is synthesized not only in inflammatory cells but also in bronchial epithelial cells. The purpose of this study is to clarify whether 5-oxo-ETE can promote the production of eosinophil cation protein (ECP) by eosinophils in nasal polyps (NP) in vitro, and whether normal nasal epithelial cells can produce this lipid mediator in response to oxidative stress. Materials and Methods: Nasal biopsy samples were obtained from normal subjects or subjects with chronic rhinosinusitis with NP. The infiltration of eosinophil in NP was detected and cultured. After that, concentrations of ECP in eosinophil and NP cultures were evaluated after the treatment of 5-oxo-ETE or 5-oxo-ETE + its receptor (OXER) antagonist, pertussis toxin (PT). Then we studied the synthesis of 5-oxo-ETE after H₂O₂ stimulation by normal nasal epithelial cells and by epithelial cells of NP alone in the cultures, and also determined the OXER expression in NP. Results: The number of infiltrative eosinophils in NP was increased. The ECP levels in eosinophil and NP cultures were enhanced after the administration of 5-oxo-ETE, and decreased by the PT treatment. 5-Oxo-ETE was upregulated in the cultures of nasal epithelial cells in the presence of H₂O₂ and of NP epithelial cells alone. The OXER was expressed in inflammatory cells, and not in epithelial cells. Conclusion: 5-Oxo-ETE produced by nasal epithelial cells may play a role in the formation and development of NP.

Induction of mast cell accumulation by chymase via an enzymatic activity- and intercellular adhesion molecule-1-dependent mechanism.

Chymase is a unique, abundant secretory product of mast cells and a potent chemoattractant for eosinophils, monocytes and neutrophils, but little is known of its influence on mast cell accumulation. A mouse peritoneal inflammation model, cell migration assay and flowcytometry analysis, were used to investigate the role of chymase in recruiting mast cells. Chymase increased, by up to 5.4-fold, mast cell numbers in mouse peritoneum. Inhibitors of chymase, heat-inactivation of the enzyme, sodium cromoglycate and terfenadine, and pretreatment of mice with anti-intercellular adhesion molecule 1, anti-L-selectin, anti-CD11a and anti-CD18 antibodies dramatically diminished the chymase-induced increase in mast cell accumulation. These findings indicate that this effect of chymase is dependent on its enzymatic activity and activation of adhesion molecules. In addition, chymase provoked a significant increase in 5-HT and eotaxin release (up to 1.8- and 2.2-fold, respectively) in mouse peritoneum. Since 5-HT, eotaxin and RANTES can induce marked mast cell accumulation, these indirect mechanisms may also contribute to chymase-induced mast cell accumulation. Moreover, chymase increased the trans-endothelium migration of mast cells in vitro indicating it also acts as a chemoattractant. The finding that mast cells accumulate in response to chymase implies further that chymase is a major pro-inflammatory mediator of mast cells. This effect of chymase, a major product of mast cell granules, suggests a novel self-amplification mechanism for mast cell accumulation in allergic inflammation. Mast cell stabilizers and inhibitors of chymase may have potential as a treatment of allergic disorders.

In vivo α-hydroxylation of a 2-alkylindole antagonist of the OXE receptor for the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid in monkeys

We have developed a selective indole antagonist (230) targeting the OXE receptor for the potent eosinophil chemoattractant 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid), that may be useful for the treatment of eosinophilic diseases such as asthma. In previous studies we identified ω2-oxidation of the hexyl side chain of racemic 230 as a major metabolic route in monkeys, but also obtained evidence for another pathway that appeared to involve hydroxylation of the hexyl side chain close to the indole. The present study was designed to investigate the metabolism of the active S-enantiomer of 230 (S230) and to identify the novel hydroxy metabolite and its chirality. Following oral administration, S230 rapidly appeared in the blood along with metabolites formed by a novel and highly stereospecific α-hydroxylation pathway, resulting in the formation of αS-hydroxy-S230. The chirality of α-hydroxy-S230 was determined by the total synthesis of the relevant diastereomers. Of the four possible diastereomers of α-hydroxy-230 only αS-hydroxy-S230 has significant OXE receptor antagonist activity and only this diastereomer was found in significant amounts in blood following oral administration of S230.Other novel metabolites of S230 identified in plasma by LC–MS/MS were αS,ω2-dihydroxy-S230 and glucuronides of S230 and ω2-hydroxy-S230. Thus the alkyl side chain of S230, which is essential for its antagonist activity, is also the major target of the metabolic enzymes that terminate its antagonist activity. Modification of this side chain might result in the development of related antagonists with improved metabolic stability and efficacy.

Pharmacokinetics and Metabolism of Selective Oxoeicosanoid (OXE) Receptor Antagonists and Their Effects on 5-Oxo-6,8,11,14-eicosatetraenoic Acid (5-Oxo-ETE)-Induced Granulocyte Activation in Monkeys.

The potent eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a 5-lipoxygenase product that acts via the selective OXE receptor, which is present in many species, but not rodents. We previously reported that the indole 230 is a potent human OXE receptor antagonist. The objective of the present study was to determine whether the monkey would be a suitable animal model to investigate its pharmaceutical potential. We found that monkey leukocytes synthesize and respond to 5-oxo-ETE and that 230 is a potent antagonist of the OXE receptor in monkey eosinophils. Pharmacokinetic studies revealed that 230 appears rapidly in the blood following oral administration. Using chemically synthesized standards, we identified the major microsomal and plasma metabolites of 230 as products of ω2-hydroxylation of the alkyl side chain. These studies demonstrate that the monkey is a promising animal model to investigate the drug potential of OXE receptor antagonists.

Design and synthesis of affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase

Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles. To achieve this task, we designed and synthesized affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase (5-HEDH), the enzyme responsible for the formation of 5-oxo-ETE.

CCL11 enhances excitotoxic neuronal death by producing reactive oxygen species in microglia.

The chemokine CCL11 (also known as eotaxin-1) is a potent eosinophil chemoattractant that mediates allergic diseases such as asthma, atopic dermatitis, and inflammatory bowel diseases. Previous studies demonstrated that concentrations of CCL11 are elevated in the sera and cerebrospinal fluids (CSF) of patients with neuroinflammatory disorders, including multiple sclerosis. Moreover, the levels of CCL11 in plasma and CSF increase with age, and CCL11 suppresses adult neurogenesis in the central nervous system (CNS), resulting in memory impairment. However, the precise source and function of CCL11 in the CNS are not fully understood. In this study, we found that activated astrocytes release CCL11, whereas microglia predominantly express the CCL11 receptor. CCL11 significantly promoted the migration of microglia, and induced microglial production of reactive oxygen species by upregulating nicotinamide adenine dinucleotide phosphate-oxidase 1 (NOX1), thereby promoting excitotoxic neuronal death. These effects were reversed by inhibition of NOX1. Our findings suggest that CCL11 released from activated astrocytes triggers oxidative stress via microglial NOX1 activation and potentiates glutamate-mediated neurotoxicity, which may be involved in the pathogenesis of various neurological disorders.

Elevated serum galectin-9 levels in patients with atopic dermatitis.

Galectin-9 is a member of the galectin family that has a wide spectrum of biological functions. Among them, galectin-9 has been known mainly as a potent chemoattractant for eosinophils. In addition, galectin-9 alters the T-cell balance by negatively regulating T-helper (Th)1 and Th17 cells, resulting in Th2 polarization. Atopic dermatitis (AD) is a skin allergic disease characterized by peripheral eosinophilia, mast cell activation and predominance of Th2 cells. To investigate possible roles of galectin-9 in AD, we measured serum galectin-9 levels in AD patients and investigated galectin-9 expression in lesional skin by immunohistochemistry. Serum galectin-9 levels in patients with AD were significantly higher than those in healthy controls and correlated with the Eczema Area and Severity Index. Serum galectin-9 levels were decreased after treatment, accompanied by improvement of skin lesions. Immunohistochemical study revealed that galectin-9 was expressed on epidermal keratinocytes and mast cells in lesional skin of AD. Our results suggest that elevated galectin-9 expression is associated with progression of AD and that galectin-9 could be a therapeutic target in AD.

The flavone eupatilin inhibits eotaxin expression in an NF-κB-dependent and STAT6-independent manner.

The CC chemokine eotaxin contributes to epithelium-induced inflammation in airway diseases such as asthma. Eupatilin (5,7-dihydroxy-3',4',6'-trimethoxyflavone), a bioactive component of Artemisia asiatica Nakai (Asteraceae), is reported to inhibit the adhesion of eosinophils to bronchial epithelial cells. However, little is known about the molecular mechanism of eupatilin-induced attenuation of bronchial epithelium-induced inflammation. In this study, we investigated the effect of eupatilin on expression of eotaxin-1 (CCL11), a potent chemoattractant for eosinophils. Eupatilin significantly inhibited eotaxin expression in bronchial epithelial cells stimulated with TNF-α, while NF-κB and IκBα kinase (IKK) activities declined concurrently. Eupatilin also inhibited mitogen-activated protein kinase (MAPK) activity; however, all of these anti-inflammatory activities were reversed by MAPK overexpression. In contrast, eupatilin did not affect the signal transducer and activator of transcription 6 (STAT6) signalling in bronchial epithelial cells stimulated with IL-4. Furthermore, eupatilin significantly attenuated TNF-α-induced eosinophil migration. These results suggest that the eupatilin inhibits the signalling of MAPK, IKK, NF-κB and eotaxin-1 in bronchial epithelial cells, leading to inhibition of eosinophil migration.

Wogonin, a plant flavone from Scutellariae radix, attenuated ovalbumin-induced airway inflammation in mouse model of asthma via the suppression of IL-4/STAT6 signaling.

Bronchial asthma is a chronic inflammatory disease of the airways characterized by a marked infiltration of eosinophils at the site of inflammation. Eotaxins are potent chemoattractants for eosinophils and play important roles in pathogenesis of asthma. In the course of screening for eotaxin-3 inhibitors, we found that wogonin showed potent inhibitory activity of interleukin-4 (IL-4)-induced eotaxin-3 expression in BEAS-2B cells. In this study, we examined the effects of wogonin on IL-4/STAT6 signaling pathway and biological implication in a mouse model of asthma. Wogonin inhibited IL-4-induced activation and nuclear translocation of STAT6 which plays a key role in either the transcription of STAT6-response genes or Th2 cytokine-mediated inflammation. Oral administration of wogonin significantly reduced activation of STAT6 in the lung and the expression of eotaxin and RANTES in bronchoalveolar lavage fluids. Histological examination of lung tissue demonstrated that wogonin significantly inhibited allergen-induced eosinophilic inflammation. Administration of wogonin reduced the total IgE and ovalbumin-specific IgE levels compared with the ovalbumin-challenged group. All of these data demonstrated that wogonin could alleviate airway inflammation through inhibition of STAT6 activation induced by Th2 cytokines. Our finding implicates a potential therapeutic value of wogonin in the treatment of asthma through regulation of IL-4/STAT6 signaling pathway.

Synephrine inhibits eotaxin-1 expression via the STAT6 signaling pathway.

Citrus contain various flavonoids and alkaloids that have multiple biological activities. It is known that the immature Citrus contains larger amounts of bioactive components, than do the mature plants. Although Citrus flavonoids are well known for their biological activities, Citrus alkaloids have not previously been assessed. In this study, we identified synephrine alkaloids as an active compound from immature Citrus unshiu, and investigated the effect of synephrine on eotaxin-1 expression. Eotaxin-1 is a potent chemoattractant for eosinophils, and a critical mediator, during the development of eosinophilic inflammation. We found that synephrine significantly inhibited IL-4-induced eotaxin-1 expression. This synephrine effect was mediated through the inhibition of STAT6 phosphorylation in JAK/STAT signaling. We also found that eosinophil recruitment induced by eotaxin-1 overexpression was inhibited by synephrine. Taken together, these findings indicate that inhibiting IL-4-induced eotaxin-1 expression by synephrine occurs primarily through the suppression of eosinophil recruitment, which is mediated by inhibiting STAT6 phosphorylation.

Base-dependent formation of cis and trans olefins and their application in the synthesis of 5-oxo-ETE receptor antagonists

5-Oxo-ETE is the most potent eosinophil chemoattractant among lipid mediators. We have developed two 5-oxo-ETE receptor antagonists. In the course of the work, we have developed a procedure to selectively introduce a cis and trans double bond in an alkyl side chain. Reacting indolecarboxaldehydes with alkyl ylides using the Li base affords the trans olefins, whereas using the K base yields the cis olefins.