Shuichiro Maruoka

In utero supplementation with methyl donors enhances allergic airway disease in mice

Asthma is a complex heritable disease that is increasing in prevalence and severity, particularly in developed countries such as the United States, where 11% of the population is affected. The contribution of environmental and genetic factors to this growing epidemic is currently not well understood. We developed the hypothesis, based on previous literature, that changes in DNA methylation resulting in aberrant gene transcription may enhance the risk of developing allergic airway disease. Our findings indicate that in mice, a maternal diet supplemented with methyl donors enhanced the severity of allergic airway disease that was inherited transgenerationally. Using a genomic approach, we discovered 82 gene-associated loci that were differentially methylated after in utero supplementation with a methyl-rich diet. These methylation changes were associated with decreased transcriptional activity and increased disease severity. Runt-related transcription factor 3 (Runx3), a gene known to negatively regulate allergic airway disease, was found to be excessively methylated, and Runx3 mRNA and protein levels were suppressed in progeny exposed in utero to a high-methylation diet. Moreover, treatment with a demethylating agent increased Runx3 gene transcription, further supporting our claim that a methyl-rich diet can affect methylation status and consequent transcriptional regulation. Our findings indicate that dietary factors can modify the heritable risk of allergic airway disease through epigenetic mechanisms during a vulnerable period of fetal development in mice.

ASK1 regulates influenza virus infection-induced apoptotic cell death.

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.

Role of mitogen-activated protein kinases in influenza virus induction of prostaglandin E2 from arachidonic acid in bronchial epithelial cells

Background Influenza virus (IV) infection causes airway inflammation; however, it has not been determined whether IV infection could catabolize arachidonic acid cascade in airway epithelial cells. In addition, the responsible intracellular signalling molecules that catabolize arachidonic acid cascade have not been determined.

Apoptosis signal-regulating kinase 1-mediated signaling pathway regulates hydrogen peroxide-induced apoptosis in human pulmonary vascular endothelial cells

ObjectiveReactive oxygen species initiate pulmonary vascular endothelial cell damage leading to an increase in endothelial permeability resulting in the production of pulmonary edema. Apoptosis signal-regulating kinase (ASK)-1 is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the MKK3/MKK6-p38 MAPK and the SEK1-c-Jun N-terminal kinase (JNK) signaling cascade. ASK1 has been implicated in cytokine- and stress-induced apoptosis. However, little is known about the role of ASK1 in apoptosis in hydrogen peroxide (H2O2)-stimulated pulmonary vascular endothelial cells and how ASK1-mediated apoptosis is executed. To clarify this issue, we examined the role of ASK1-p38 MAPK/JNK cascade in apoptosis and caspase-3 activation in H2O2-stimulated pulmonary vascular endothelial cells. DesignExperimental laboratory study. SettingUniversity laboratory. SubjectsNormal human pulmonary artery endothelial cells. InterventionsWestern blot analysis and quantification of apoptosis in cells. Measurements and Main ResultsThe results showed that H2O2 induced ASK1 phosphorylation and concomitantly p38 MAPK and JNK phosphorylation as well as induced caspase-3 activation in pulmonary vascular endothelial cells. To further characterize the role of ASK1 cascade in H2O2-induced apoptosis of pulmonary vascular endothelial cells, the dominant negative form of ASK1-stably transfected porcine artery endothelial cells was used. p38 MAPK and JNK phosphorylation, caspase-3 activation, and apoptosis in the dominant negative form of ASK1-stably transfected porcine artery endothelial cells were depressed compared with those in the parental porcine artery endothelial cells. ConclusionASK1-p38 MAPK/JNK cascade regulates apoptosis of H2O2-stimulated human pulmonary vascular endothelial cells.

Correlation of plasma monocyte chemoattractant protein-1 (MCP-1) and monocyte inflammatory protein-1α (MIP-1α) levels with disease activity and clinical course of sarcoidosis

MCP‐1 and MIP‐1α exhibit chemotactic activity toward macrophages/monocytes and induce the production of inflammatory cytokines affecting granuloma formation. Up‐regulated expression of MCP‐1 and MIP‐1α in the affected organ of sarcoidosis has been shown; however, the relationship between their plasma levels and the clinical course of this disease has not been determined. In the present study we measured plasma MCP‐1 and MIP‐1α levels in 26 patients with active sarcoidosis by ELISA in order to assess the state of MCP‐1 and MIP‐1α in this disease. Most patients in this study (21/26) had clinical evidence of extrathoracic disease in addition to pulmonary involvement. In addition, a high proportion of patients (n = 15) showed spontaneous remission of disease, whereas five patients showed no spontaneous remission and six patients were treated with corticosteroids over the 2‐year period of study. At the time of diagnosis, both plasma MCP‐1 and MIP‐1α levels in patients with active sarcoidosis were significantly higher than in the normal controls. The levels of these cytokines in patients with extrathoracic disease were compatible with those in patients without extrathoracic disease. A longitudinal evaluation of plasma MCP‐1 and MIP‐1α levels showed that the changes in both cytokines were closely related to the clinical course of sarcoidosis. These results suggest that plasma MCP‐1 and MIP‐1α may be useful parameters for monitoring the clinical course of sarcoidosis. In addition, plasma MCP‐1 and MIP‐1α may reflect subclinical evidence of extrathoracic sarcoidosis and may play a role in initiating monocyte migration into the tissue.

Cyclooxygenase-2 Regulates Th17 Cell Differentiation during Allergic Lung Inflammation

RATIONALE Th17 cells comprise a distinct lineage of proinflammatory T helper cells that are major contributors to allergic responses. It is unknown whether cyclooxygenase (COX)-derived eicosanoids regulate Th17 cells during allergic lung inflammation. OBJECTIVES To determine the role of COX metabolites in regulating Th17 cell differentiation and function during allergic lung inflammation. METHODS COX-1(-/-), COX-2(-/-), and wild-type mice were studied in an in vivo model of ovalbumin-induced allergic inflammation and an in vitro model of Th17 differentiation using flow cytometry, cytokine assays, confocal microscopy, real-time polymerase chain reaction, and immunoblotting. In addition, the role of specific eicosanoids and their receptors was examined using synthetic prostaglandins (PGs), selective inhibitors, and siRNA knockdown. MEASUREMENTS AND MAIN RESULTS Th17 cell differentiation in lung, lymph nodes, and bronchoalveolar lavage fluid was significantly lower in COX-2(-/-) mice after ovalbumin sensitization and exposure in vivo. In vitro studies revealed significantly impaired Th17 cell differentiation of COX-2(-/-) naive CD4(+) T cells with decreased Stat3 phosphorylation and RORγt expression. Synthetic PGF(2α) and PGI(2) enhanced Th17 cell differentiation of COX-2(-/-) CD4(+) T cells in vitro. The selective COX-2 inhibitor, NS-398, and PGF(2α) receptor and PGI(2) receptor siRNA knockdown significantly decreased Th17 cell differentiation in vitro. Administration of synthetic PGs restored accumulation of Th17 cells in lungs of allergic COX-2(-/-) mice in vivo. CONCLUSIONS COX-2 is a critical regulator of Th17 cell differentiation during allergic lung inflammation via autocrine signaling of PGI(2) and PGF(2α) through their respective cell surface receptors.

Glucocorticoids enhance airway epithelial barrier integrity.

Asthma is a chronic inflammatory disorder of the airways, but its pathogenesis is incompletely understood. While asthma is a complex disease caused by multiple factors, epithelial barrier damage is a cardinal feature. Glucocorticoids (GCs) are the most effective anti-inflammatory drugs in the treatment of asthma. However, the effects of GCs on the airway epithelial barrier have not been evaluated. Epithelial barrier functions were evaluated in cultured human airway epithelial cell monolayers, Calu-3 and 16HBE. Then, the cells were treated with dexamethasone (Dex), fulticasone propionate (FP), or budesonide (BD) for 5 days. Permeability measured by transepithelial electrical resistance was increased by treatment with Dex, FP, and BD in a dose-dependent manner. Permeability to fluorescein isothiocyanate-labeled dextran was markedly reduced by these treatments. Immunocytostaining revealed that Dex treatment potentiated tight junction formation in these polarized epithelial cells. Knockdown of epidermal growth factor receptor (EGFR) by small interference RNA blunted the effects of Dex on barrier integrity. Although EGFR expression was not affected by Dex treatment, EGFR phosphorylation was enhanced in Dex-treated cells. This is suggesting that EGFR are important for this phenomenon. These findings suggest that GC inhalation therapy can improve epithelial barrier integrity and might contribute to the therapeutic effects of GCs for treating asthma.

Mitogen-activated protein kinase involves neutrophil elastase-induced morphological changes in human bronchial epithelial cells.

Neutrophil elastase (NE) promotes the detachment of airway epithelial cells; however, changes in overall morphology of NE-stimulated bronchial epithelial cell (BEC) monolayer are different from trypsin stimulation. Ras/Raf-initiated-mitogen activated protein kinase (MAPK) also known as extracellular signal-regulated kinase, pathway regulates integrin functions which participate in regulating attachment and detachment of cell and cellular morphology. However, little is known about the role of MAPK in NE-induced changes in overall morphology of BEC. In the present study, we examined the role of MAPK in NE-induced changes in overall morphology of BEC monolayer. To this end, we examined changes in cellular morphology and MAPK activation in NE-stimulated BEC monolayer, and the effect of PD 98059 as the specific inhibitor for MAPK kinase-1 (MEK-1, the upstream regulator of MAPK) on NE-induced changes in cellular morphology and MAPK activation. The results showed that in stimulation of NE, BECs detached and gaps developed, and MAPK activation was observed. PD 98059 attenuated NE-induced changes in cellular morphology as well as MAPK activation. These results indicated that in addition to proteolytic activity of NE on extracellular matrix (ECM), NE-activated MAPK pathway, at least in part, is involved in NE-induced changes in overall morphology and the detachment of BEC monolayer.

Retinoic acid differentially regulates interleukin-1β and interleukin-1 receptor antagonist production by human alveolar macrophages

Mechanism in the pathogenesis of acute respiratory distress syndrome which is the clinical feature of pulmonary involvement in retinoic acid (RA) syndrome has been investigated. Pulmonary infiltration of matured neutrophils and leukemic cells is thought to be associated with the pathogenesis of pulmonary involvement in RA syndrome; however. Little is known about the mechanism in pulmonary infiltration of these cells. In the present study, we examined the effect of RA on IL-1beta and IL-1ra production by human alveolar macrophages in order to clarify the mechanism in pulmonary infiltration of neutrophils, since IL-1 has been shown to initiate neutrophil recruitment into the lung through up-regulated expression of adhesion molecules on vascular endothelium. RA enhanced IL-1beta and inhibited IL-1ra production by 4beta phorbol 12beta-myristate-13alpha acetate (PMA)- and lipopolysaccharide (LPS)-stimulated human alveolar macrophages. These results show that RA differentially regulates IL-1beta and IL-1ra production by alveolar macrophages and indicate that an imbalanced production between IL-1beta and IL-1ra may contribute to initiating neutrophil recruitment into the lung through up-regulated expression of adhesion molecules.

Grepafloxacin inhibits tumor necrosis factor- α-induced interleukin-8 expression in human airway epithelial cells

We examined the effect of grepafloxacin (GPFX), a new fluoroquinolone antimicrobial agent, on interleukin-8 (IL-8) expression in tumor necrosis factor-alpha (TNF-alpha)-stimulated human airway epithelial cells (AEC). GPFX inhibited IL-8 protein production as well as mRNA expression in a concentration-dependent manner (2.5 - 25 micro g/ml), but the inhibition of IL-8 expression by corresponding concentrations of GPFX to serum and airway lining fluids was not complete. We discuss the modulatory effect of GPFX on IL-8 production in the context of its efficacy on controlling chronic airway inflammatory diseases.