Samuel J. Goldman

Role of IL-17A, IL-17F, and the IL-17 Receptor in Regulating Growth-Related Oncogene-α and Granulocyte Colony-Stimulating Factor in Bronchial Epithelium: Implications for Airway Inflammation in Cystic Fibrosis

IL-17R signaling is critical for pulmonary neutrophil recruitment and host defense against Gram-negative bacteria through the coordinated release of G-CSF and CXC chemokine elaboration. In this study, we show that IL-17R is localized to basal airway cells in human lung tissue, and functional IL-17R signaling occurs on the basolateral surface of human bronchial epithelial (HBE) cells. IL-17A and IL-17F were potent inducers of growth-related oncogene-α and G-CSF in HBE cells, and significant synergism was observed with TNF-α largely due to signaling via TNFRI. The activities of both IL-17A and IL-17F were blocked by a specific anti-IL-17R Ab, but only IL-17A was blocked with a soluble IL-17R, suggesting that cell membrane IL-17R is required for signaling by both IL-17A and IL-17F. Because IL-17A and IL-17F both regulate lung neutrophil recruitment, we measured these molecules as well as the proximal regulator IL-23p19 in the sputum of patients with cystic fibrosis (CF) undergoing pulmonary exacerbation. We found significantly elevated levels of these molecules in the sputum of patients with CF who were colonized with Pseudomonas aeruginosa at the time of pulmonary exacerbation, and the levels declined with therapy directed against P. aeruginosa. IL-23 and the downstream cytokines IL-17A and IL-17F are critical molecules for proinflammatory gene expression in HBE cells and are likely involved in the proinflammatory cytokine network involved with CF pathogenesis.

An IL-17F/A Heterodimer Protein Is Produced by Mouse Th17 Cells and Induces Airway Neutrophil Recruitment

IL-17A and IL-17F are related homodimeric proteins of the IL-17 family produced by Th17 cells. In this study, we show that mouse Th17 cells also produce an IL-17F/A heterodimeric protein. Whereas naive CD4+ T cells differentiating toward the Th17 cell lineage expressed IL-17F/A in higher amounts than IL-17A/A homodimer and in lower amounts than IL-17F/F homodimer, differentiated Th17 cells expressed IL-17F/A in higher amounts than either homodimer. In vitro, IL-17F/A was more potent than IL-17F/F and less potent than IL-17A/A in regulating CXCL1 expression. Neutralization of IL-17F/A with an IL-17A-specific Ab, and not with an IL-17F-specific Ab, reduced the majority of IL-17F/A-induced CXCL1 expression. To study these cytokines in vivo, we established a Th17 cell adoptive transfer model characterized by increased neutrophilia in the airways. An IL-17A-specific Ab completely prevented Th17 cell-induced neutrophilia and CXCL5 expression, whereas Abs specific for IL-17F or IL-22, a cytokine also produced by Th17 cells, had no effects. Direct administration of mouse IL-17A/A or IL-17F/A, and not IL-17F/F or IL-22, into the airways significantly increased neutrophil and chemokine expression. Taken together, our data elucidate the regulation of IL-17F/A heterodimer expression by Th17 cells and demonstrate an in vivo function for this cytokine in airway neutrophilia.

Enhanced Interleukin (IL)-13 Responses in Mice Lacking IL-13 Receptor α 2

Interleukin (IL)-13 has recently been shown to play important and unique roles in asthma, parasite immunity, and tumor recurrence. At least two distinct receptor components, IL-4 receptor (R)α and IL-13Rα1, mediate the diverse actions of IL-13. We have recently described an additional high affinity receptor for IL-13, IL-13Rα2, whose function in IL-13 signaling is unknown. To better appreciate the functional importance of IL-13Rα2, mice deficient in IL-13Rα2 were generated by gene targeting. Serum immunoglobulin E levels were increased in IL-13Rα2−/− mice despite the fact that serum IL-13 was absent and immune interferon γ production increased compared with wild-type mice. IL-13Rα2–deficient mice display increased bone marrow macrophage progenitor frequency and decreased tissue macrophage nitric oxide and IL-12 production in response to lipopolysaccharide. These results are consistent with a phenotype of enhanced IL-13 responsiveness and demonstrate a role for endogenous IL-13 and IL-13Rα2 in regulating immune responses in wild-type mice.

Differential Proteomic Analysis of Bronchoalveolar Lavage Fluid in Asthmatics following Segmental Antigen Challenge

Allergic asthma is characterized by persistent airway inflammation and remodeling. Bronchoalveolar lavage conducted with fiberoptic bronchoscopy has been widely used for investigating the pathogenesis of asthma and other lung disorders. Identification of proteins in the bronchoalveolar lavage fluid (BALF) and their expression changes at different stages of asthma could provide further insights into the complex molecular mechanisms involved in this disease. In this report, we describe the first comprehensive differential proteomic analysis of BALF from both asthmatic patients and healthy subjects before and 24 h after segmental allergen challenge. Our proteomic analysis involves affinity depletion of six abundant BALF proteins, SDS-PAGE fractionation, protein in-gel digestion, and subsequent nano-LC-MS/MS analysis in conjunction with database searching for protein identification and semiquantitation. More than 1,500 distinct proteins were identified of which about 10% displayed significant up-regulation specific to the asthmatic patients after segmental allergen challenge. The differentially expressed proteins represent a wide spectrum of functional classes such as chemokines, cytokines, proteases, complement factors, acute phase proteins, monocyte-specific granule proteins, and local matrix proteins, etc. The majority of these protein expression changes are closely associated with many aspects of the pathophysiology of asthma, including inflammation, eosinophilia, airway remodeling, tissue damage and repair, mucus production, and plasma infiltration. Importantly a large portion of these proteins and their expression changes were identified for the first time from BALF, thus providing new insights for finding novel pathological mediators and biomarkers of asthma.

Pathways Activated during Human Asthma Exacerbation as Revealed by Gene Expression Patterns in Blood

Background Asthma exacerbations remain a major unmet clinical need. The difficulty in obtaining airway tissue and bronchoalveolar lavage samples during exacerbations has greatly hampered study of naturally occurring exacerbations. This study was conducted to determine if mRNA profiling of peripheral blood mononuclear cells (PBMCs) could provide information on the systemic molecular pathways involved during asthma exacerbations. Methodology/Principal Findings Over the course of one year, gene expression levels during stable asthma, exacerbation, and two weeks after an exacerbation were compared using oligonucleotide arrays. For each of 118 subjects who experienced at least one asthma exacerbation, the gene expression patterns in a sample of peripheral blood mononuclear cells collected during an exacerbation episode were compared to patterns observed in multiple samples from the same subject collected during quiescent asthma. Analysis of covariance identified genes whose levels of expression changed during exacerbations and returned to quiescent levels by two weeks. Heterogeneity among visits in expression profiles was examined using K-means clustering. Three distinct exacerbation-associated gene expression signatures were identified. One signature indicated that, even among patients without symptoms of respiratory infection, genes of innate immunity were activated. Antigen-independent T cell activation mediated by IL15 was also indicated by this signature. A second signature revealed strong evidence of lymphocyte activation through antigen receptors and subsequent downstream events of adaptive immunity. The number of genes identified in the third signature was too few to draw conclusions on the mechanisms driving those exacerbations. Conclusions/Significance This study has shown that analysis of PBMCs reveals systemic changes accompanying asthma exacerbation and has laid the foundation for future comparative studies using PBMCs.

IL-33 synergizes with IgE-dependent and IgE-independent agents to promote mast cell and basophil activation

ObjectiveMast cell and basophil activation contributes to inflammation, bronchoconstriction, and airway hyperresponsiveness in asthma. Because IL-33 expression is inflammation inducible, we investigated IL-33-mediated effects in concert with both IgE-mediated and IgE-independent stimulation.MethodsBecause the HMC-1 mast cell line can be activated by GPCR and RTK signaling, we studied the effects of IL-33 on these pathways. The IL-33- and SCF-stimulated HMC-1 cells were co-cultured with human lung fibroblasts and airway smooth muscle cells in a collagen gel contraction assay. IL-33 effects on IgE-mediated activation were studied in primary mast cells and basophils.ResultIL-33 synergized with adenosine, C5a, SCF, and NGF receptor activation. IL-33-stimulated and SCF-stimulated HMC-1 cells demonstrated enhanced collagen gel contraction when cultured with fibroblasts or smooth muscle cells. IL-33 also synergized with IgE receptor activation of primary human mast cells and basophils.ConclusionIL-33 amplifies inflammation in both IgE-independent and IgE-dependent responses.

IgE Generation and Mast Cell Effector Function in Mice Deficient in IL-4 and IL-13

IL-4 and IL-13 are potent cytokines that drive production of IgE, which is critical to the development of atopic disease. In this study, we directly compared IgE generation and IgE-dependent mast cell effector function in mouse strains lacking IL-4, IL-13, IL-4 + IL-13, or their common receptor component, IL-4Rα. Although serum IgE was undetectable under resting conditions in most animals deficient in one or both cytokines, peritoneal mast cells from mice lacking IL-4 or IL-13 had only partial reductions in surface IgE level. In contrast, peritoneal mast cells from IL-4/13−/− and IL-4Rα−/− animals were severely deficient in surface IgE, and showed no detectable degranulation following treatment with anti-IgE in vitro. Surprisingly, however, intradermal challenge with high concentrations of anti-IgE Ab induced an ear-swelling response in these strains, implying some capacity for IgE-mediated effector function in tissue mast cells. Furthermore, upon specific immunization with OVA, both IL-4/IL-13−/− and IL-4Rα−/− mice produced detectable levels of serum IgE and Ag-specific IgG1, and generated strong ear-swelling responses to intradermal administration of anti-IgE. These findings suggest that a mechanism for IgE production exists in vivo that is independent of IL-4 or IL-13.

A Selective Matrix Metalloprotease 12 Inhibitor for Potential Treatment of Chronic Obstructive Pulmonary Disease (COPD): Discovery of (S)-2-(8-(Methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methylbutanoic acid (MMP408)

Matrix metalloprotease 12 plays a significant role in airway inflammation and remodeling. Increased expression and production of MMP-12 have been found in the lung of human COPD patients. MMP408 (14), a potent and selective MMP-12 inhibitor, was derived from a potent matrix metalloprotease 2 and 13 inhibitor via lead optimization and has good physical properties and bioavailability. The compound blocks rhMMP-12-induced lung inflammation in a mouse model and was advanced for further development for the treatment of COPD.

Interleukin-13 Neutralization by Two Distinct Receptor Blocking Mechanisms Reduces Immunoglobulin E Responses and Lung Inflammation in Cynomolgus Monkeys

Interleukin (IL)-13 is a key cytokine driving allergic and asthmatic responses and contributes to airway inflammation in cynomolgus monkeys after segmental challenge with Ascaris suum antigen. IL-13 bioactivity is mediated by a heterodimeric receptor (IL-13Rα1/IL-4Rα) and can be inhibited in vitro by targeting IL-13 interaction with either chain. However, in cytokine systems, in vitro neutralization activity may not always predict inhibitory function in vivo. To address the efficacy of two different IL-13 neutralization mechanisms in a primate model of atopic disease, two humanized monoclonal antibodies to IL-13 were generated, with highly homologous properties, differing in epitope recognition. Ab01 blocks IL-13 interaction with IL-4Rα, and Ab02 blocks IL-13 interaction with IL-13Rα1. In a cynomolgus monkey model of IgE responses to A. suum antigen, both Ab01 and Ab02 effectively reduced serum titers of Ascaris-specific IgE and diminished ex vivo Ascaris-triggered basophil histamine release, assayed 8 weeks after a single administration of antibody. The two antibodies also produced comparable reductions in pulmonary inflammation after lung segmental challenge with Ascaris antigen. Increased serum levels of IL-13, lacking demonstrable biological activity, were seen postchallenge in animals given either anti-IL-13 antibody but not in control animals given human IgG of irrelevant specificity. These findings demonstrate a potent effect of IL-13 neutralization on IgE-mediated atopic responses in a primate system and show that IL-13 can be efficiently neutralized by targeting either the IL-4Rα-binding epitope or the IL-13Rα1-binding epitope.

Matrix metalloproteinase-12 is a therapeutic target for asthma in children and young adults.

BACKGROUND Matrix metalloproteinase (MMP)-12-mediated pathologic degradation of the extracellular matrix and the subsequent repair cycles influence the airway changes in patients with asthma and chronic obstructive pulmonary disease (COPD). The common serine variant at codon 357 of the MMP12 gene (rs652438) is associated with clinical manifestations consistent with more aggressive matrix degradation in other tissues. OBJECTIVE We sought to explore the hypothesis that MMP12 represents a novel therapeutic target in asthma. METHODS The role of the rs652438 variant on clinical phenotype was explored in young asthmatic patients and patients with COPD. Candidate MMP-12 inhibitors were identified on the basis of potency and selectivity against a panel of other MMPs. The role of MMP-12-specific inhibition was tested in vitro, as well as in animal models of allergic airway inflammation. RESULTS The odds ratio for having greater asthma severity was 2.00 (95% CI, 1.24-3.24; P = .004) when comparing asthmatic patients with at least 1 copy of the serine variant with those with none. The carrier frequency for the variant increased in line with asthma treatment step (P = .000). The presence of the variant nearly doubled the odds in favor of asthmatic exacerbations (odds ratio, 1.90; 95% CI, 1.19-3.04; P = .008) over the previous 6 months. The serine variant was also associated with increased disease severity in patients with COPD (P = .016). Prior administration of an MMP-12-specific inhibitor attenuated the early airway response and completely blocked the late airway response with subsequent Ascaris suum challenge in sheep. CONCLUSION Studies on human participants with asthma and COPD show that the risk MMP12 gene variant is associated with disease severity. In allergen-sensitized sheep pharmacologic inhibition of MMP12 downregulates both early and late airway responses in response to allergic stimuli.