Haizhen Hu

IL‐13 induced increases in nitrite levels are primarily driven by increases in inducible nitric oxide synthase as compared with effects on arginases in human primary bronchial epithelial cells

Background Exhaled nitric oxide is increased in asthma, but the mechanisms controlling its production, including the effects of T‐helper type 2 (Th2) cytokines, are poorly understood. In mouse and submerged human epithelial cells, Th2 cytokines inhibit expression of inducible nitric oxide synthase (iNOS). Arginases have been proposed to contribute to asthma pathogenesis by limiting the arginine substrate available to NOS enzymes, but expression of any of these enzymes has not been extensively studied in primary human cells.

Interleukin-13–induced MUC5AC Is Regulated by 15-Lipoxygenase 1 Pathway in Human Bronchial Epithelial Cells

RATIONALE 15-Lipoxygenase-1 (15LO1) and MUC5AC are highly expressed in asthmatic epithelial cells. IL-13 is known to induce 15LO1 and MUC5AC in human airway epithelial cells in vitro. Whether 15LO1 and/or its product 15-HETE modulate MUC5AC expression is unknown. OBJECTIVES To determine the expression of 15LO1 in freshly harvested epithelial cells from subjects with asthma and normal control subjects and to determine whether IL-13-induced 15LO1 expression and activation regulate MUC5AC expression in human bronchial epithelial cells in vitro. METHODS Human airway epithelial cells from subjects with asthma and normal subjects were evaluated ex vivo for 15LO1 and MUC5AC expression. The impact of 15LO1 on MUC5AC expression in vitro was analyzed by inhibiting 15LO1 through pharmacologic (PD146176) and siRNA approaches in human bronchial epithelial cells cultured under air-liquid interface. We analyzed 15 hydroxyeicosatetraenoic acid (15-HETE) by liquid chromatography/UV/mass spectrometry. MUC5AC and 15LO1 were analyzed by real-time RT-PCR, immunofluoresence, and Western blot. MEASUREMENTS AND MAIN RESULTS Epithelial 15LO1 expression increased with asthma severity (P < 0.0001). 15LO1 significantly correlated with MUC5AC ex vivo and in vitro. IL-13 increased 15LO1 expression and stimulated formation of two molecular species of 15-HETE esterified to phosphotidylethanolamine (15-HETE-PE). Inhibition of 15LO1 suppressed 15-HETE-PE and decreased MUC5AC expression in the presence of IL-13 stimulation. The addition of exogenous 15-HETE partially restored MUC5AC expression. CONCLUSIONS Epithelial 15LO1 expression increases with increasing asthma severity. IL-13 induction of 15-HETE-PE enhances MUC5AC expression in human airway epithelial cells. High levels of 15LO1 activity could contribute to the increases of MUC5AC observed in asthma.

MAPK Regulation of IL-4/IL-13 Receptors Contributes to the Synergistic Increase in CCL11/Eotaxin-1 in Response to TGF-β1 and IL-13 in Human Airway Fibroblasts

CCL11/eotaxin-1 is a potent eosinophilic CC chemokine expressed by primary human fibroblasts. The combination of TGF-β1 and IL-13 synergistically increases CCL11 expression, but the mechanisms behind the synergy are unclear. To address this, human airway fibroblast cultures from normal and asthmatic subjects were exposed to IL-13 alone or TGF-β1 plus IL-13. Transcriptional (nuclear run-on) and posttranscriptional (mRNA stability) assays confirmed that transcriptional regulation is critical for synergistic expression of CCL11. TGF-β1 plus IL-13 synergistically increased STAT-6 phosphorylation, nuclear translocation, and binding to the CCL11 promoter as compared with IL-13 alone. STAT-6 small interfering RNA significantly knocked down both STAT-6 mRNA expression and phosphorylation and inhibited CCL11 mRNA and protein expression. Regulation of the IL-4Rα complex by TGF-β1 augmented IL-13 signaling by dampening IL-13Rα2 expression, overcoming IL-13’s autoregulation of its pathway and enhancing the expression of CCL11. Our data suggest that TGF-β1 induced activation of the MEK/ERK pathway reduces IL-13Rα2 expression induced by IL-13. Thus, TGF-β1, a pleiotropic cytokine upregulated in asthmatic airways, can augment eosinophilic inflammation by interfering with IL-13’s negative feedback autoregulatory loop under MEK/ERK-dependent conditions.

Genomic Differences Distinguish the Myofibroblast Phenotype of Distal Lung Fibroblasts from Airway Fibroblasts

Primary human distal lung/parenchymal fibroblasts (DLFs) exhibit a different phenotype from airway fibroblasts (AFs), including the expression of high levels of α-smooth muscle actin (α-SMA). The scope of the differences between these anatomically differentiated fibroblasts, or the mechanisms driving them, has remained unknown. To determine whether the different characteristics of regional fibroblasts are predicted by distinct genomic differences in AFs versus DLFs, matched human fibroblast pairs were isolated from proximal and distal lung tissue and evaluated. Microarray analysis was performed on 12 matched fibroblast pairs (four normal and eight asthmatic samples) and validated by quantitative real-time PCR. The potential functional implications of these differences were analyzed using computational approaches. Four hundred seventy-four transcripts were up-regulated in AFs, and 611 were up-regulated in DLFs via microarray analysis. No differences in normal and asthmatic fibroblasts were evident, and the data were combined for subsequent analyses. Gene ontology and network analyses suggested distinct patterns of pathway activation between AFs and DLFs. The up-regulation of extracellular matrix-associated molecules in AFs was observed, whereas genes associated with actin binding and cytoskeletal organization were up-regulated in DLFs. The up-regulation of activated/total SMAD3 and c-Jun N-terminal kinase in DLFs may partly explain these myofibroblast-like characteristics in DLFs. Thus, marked genomic differences exist between these two populations of regional lung fibroblasts. These striking differences may help identify potential mechanisms by which AFs and DLFs differ in their responses to injury, regeneration, and remodeling in the lung.

IL-27 and type 2 immunity in asthmatic patients: Association with severity, CXCL9, and signal transducer and activator of transcription signaling

BACKGROUND Severe asthma (SA) can involve both innate and type 2 cytokine-associated adaptive immunity. Although IL-27 has been reported to potentiate TH1 responses (including the chemokine CXCL9) and suppress TH2 responses, its function in asthmatic patients is unknown. OBJECTIVE We sought to evaluate IL-27 expression in human asthma alone and in combination with type 2 immunity to determine the relationship to disease severity and CXCL9 expression. We also sought to model these interactions in vitro in human bronchial epithelial cells. METHODS Bronchoalveolar lavage cells from 87 participants were evaluated for IL-27 mRNA and protein alone and in association with epithelial CCL26 (a marker of type 2 activation) in relation to asthma severity and CXCL9 mRNA. Human bronchial epithelial cells cultured at the air-liquid interface and stimulated with IL-27 (1-100 ng/mL) with or without IL-13 (1 ng/mL) were evaluated for CXCL9 expression by using quantitative real-time PCR and ELISA. Phosphorylated and total signal transducer and activator of transcription (STAT) 1/3 were detected by means of Western blotting. Small interfering RNA knockdown of STAT1 or STAT3 was performed. RESULTS Bronchoalveolar lavage cell IL-27 mRNA and protein levels were increased in asthmatic patients. Patients with evidence for type 2 pathway activation had higher IL-27 expression (P = .02). Combined IL-27 and CCL26 expression associated with more SA and higher CXCL9 expression (P = .004 and P = .007 respectively), whereas IL-27 alone was associated with milder disease. In vitro IL-13 augmented IL-27-induced CXCL9 expression, which appeared to be due to augmented STAT1 activation and reduced STAT3 activation. CONCLUSIONS IL-27, in combination with a type 2/CCL26 signature, identifies a more SA phenotype, perhaps through combined effects of IL-27 and IL-13 on STAT signaling. Understanding these interactions could lead to new targets for asthma therapy.

Brain-Derived Neurotrophic Factor Expression in Asthma. Association with Severity and Type 2 Inflammatory Processes

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, exists in several isoforms, which differentially impacts neuronal and immune cell survival and differentiation. The role of BDNF and its isoforms in asthma remains unclear. The objectives of this study were to compare the BDNF protein isoforms and specific splice variant expression in sputum and bronchoscopic samples from healthy control subjects and participants with asthma, and to relate these changes to findings in IL-13-stimulated human airway epithelial cells. Sputum and bronchoscopic samples from healthy control subjects and participants with asthma were evaluated for BDNF protein (ELISA and Western blot) and BDNF mRNA (gel and quantitative real-time PCR) in relation to asthma severity and type 2 inflammatory processes. BDNF mRNA was measured in cultured primary human airway epithelial cells after IL-13 stimulation. Total BDNF protein differed among the groups, and its mature isoform was significantly higher in sputum from subjects with severe asthma compared with healthy control subjects (overall P = 0.008, P = 0.027, respectively). Total BDNF was higher in those with elevated fractional exhaled nitric oxide and sputum eosinophilia. In vitro, IL-13 increased BDNF exon VIb splice variant and the ratio to BDNF common exon IX mRNA (P < 0.001, P = 0.003, respectively). Epithelial brushing exon VIb mRNA and total BDNF protein differed among the groups and were higher in subjects with severe asthma than in healthy control subjects (overall P = 0.01, P = 0.02, respectively). The mature BDNF isoform and the exon VIb splice variant are increased in human asthmatic airways. The in vitro increase in response to IL-13 suggests that type 2 cytokines regulate BDNF levels and activity in asthma.

IL-13 desensitizes β2-adrenergic receptors in human airway epithelial cells through a 15-lipoxygenase/G protein receptor kinase 2 mechanism

BACKGROUND β2-Adrenergic receptor (β2AR) agonists are critical treatments for asthma. However, receptor desensitization can lead to loss of therapeutic effects. Although desensitization to repeated use of β2-agonists is well studied, type 2 inflammation could also affect β2AR function. OBJECTIVE We sought to evaluate the effect of the type 2 cytokine IL-13 on β2AR desensitization in human airway epithelial cells (HAECs) and determine whether 15-lipoxygenase-1 (15LO1) binding with phosphatidylethanolamine-binding protein 1 (PEBP1) contributes to desensitization through release of G protein receptor kinase 2 (GRK2). METHODS HAECs in air-liquid interface culture with or without IL-13 (48 hours) or isoproterenol hydrochloride (ISO; 30 minutes) pretreatment were stimulated with ISO (10 minutes). Cyclic adenosine 3, 5-monophosphate (cAMP) levels were measured using ELISA, and β2AR and GRK2 phosphorylation was measured using Western blotting. Short interfering RNA was used for 15LO1 knockdown. Interactions of GRK2, PEBP1, and 15LO1 were detected by means of immunoprecipitation/Western blotting and immunofluorescence. HAECs and airway tissue from control subjects and asthmatic patients were evaluated for I5LO1, PEBP1, and GRK2. RESULTS Pretreatment with ISO or IL-13 decreased ISO-induced cAMP generation compared with ISO for 10 minutes alone paralleled by increases in β2AR and GRK2 phosphorylation. GRK2 associated with PEBP1 after 10 minutes of ISO in association with low phosphorylated GRK2 (pGRK2) levels. In contrast, in the presence of IL-13 plus ISO (10 minutes), binding of GRK2 to PEBP1 decreased, whereas 15LO1 binding and pGRK2 levels increased. 15LO1 knockdown restored ISO-induced cAMP generation. These findings were recapitulated in freshly brushed HAECs from cells and tissue of asthmatic patients. CONCLUSION IL-13 treatment of HAECs leads to β2AR desensitization, which involves 15LO1/PEBP1 interactions to free GRK2, and allows it to phosphorylate (and desensitize) β2ARs, suggesting that the beneficial effects of β2-agonists could be blunted in patients with type 2 associated asthma.