Cara Williams

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.

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.

Interleukin-6 neutralization alleviates pulmonary inflammation in mice exposed to cigarette smoke and poly(I:C)

Increased systemic and pulmonary levels of IL-6 (interleukin-6) are associated with the severity of exacerbations and decline of lung function in patients with COPD (chronic obstructive pulmonary disease). Whether IL-6 is directly involved or plays a bystander role in the pathophysiology of COPD remains unclear. Here we hypothesized that neutralizing circulating levels of IL-6 would modulate episodes of acute pulmonary inflammation following CS (cigarette smoke) exposure and virus-like challenges. For this purpose, we used a model where C57BL/6 mice were exposed to CS twice daily via a nose-only system, and concomitant periodic intranasal challenge with poly(I:C), a synthetic ligand for TLR3 (Toll-like receptor 3) that mimics the encounter with double stranded RNA that is carried by influenza-like viruses. This protocol recapitulates several aspects of acute pulmonary inflammation associated with COPD, including prominent airway neutrophilia, insensitivity to steroid treatment and increased levels of several inflammatory cytokines in BAL (bronchoalveolar lavage) samples. Although IL-6-deficient mice exposed to CS/poly(I:C) developed pulmonary inflammation similar to WT (wild-type) controls, WT mice exposed to CS/poly(I:C) and treated intraperitoneally with IL-6-neutralizing antibodies showed significantly lower blood counts of lymphocytes and monocytes, lower BAL levels of IL-6 and CXCL1 (CXC chemokine ligand 1)/KC (keratinocyte chemoattractant), as well as reduced numbers of BAL neutrophils, lymphocytes and macrophages. Our results thus indicate that the systemic neutralization of IL-6 significantly reduces CS/poly(I:C)-induced pulmonary inflammation, which may be a relevant approach to the treatment of episodes of acute pulmonary inflammation associated with COPD.

Acidic Mammalian Chitinase Is Not a Critical Target for Allergic Airway Disease

The expression of acidic mammalian chitinase (AMCase) is associated with Th2-driven respiratory disorders. To investigate the potentially pathological role of AMCase in allergic airway disease (AAD), we sensitized and challenged mice with ovalbumin or a combination of house dust mite (HDM) plus cockroach allergen. These mice were treated or not treated with small molecule inhibitors of AMCase, which significantly reduced allergen-induced chitinolytic activity in the airways, but exerted no apparent effect on pulmonary inflammation per se. Transgenic and AMCase-deficient mice were also submitted to protocols of allergen sensitization and challenge, yet we found little or no difference in the pattern of AAD between mutant mice and wild-type (WT) control mice. In a separate model, where mice were challenged only with intratracheal instillations of HDM without adjuvant, total bronchoalveolar lavage (BAL) cellularity, inflammatory infiltrates in lung tissues, and lung mechanics remained comparable between AMCase-deficient mice and WT control mice. However BAL neutrophil and lymphocyte counts were significantly increased in AMCase-deficient mice, whereas concentrations in BAL of IL-13 were significantly decreased compared with WT control mice. These results indicate that, although exposure to allergen stimulates the expression of AMCase and increased chitinolytic activity in murine airways, the overexpression or inhibition of AMCase exerts only a subtle impact on AAD. Conversely, the increased numbers of neutrophils and lymphocytes in BAL and the decreased concentrations of IL-13 in AMCase-deficient mice challenged intratracheally with HDM indicate that AMCase contributes to the Th1/Th2 balance in the lungs. This finding may be of particular relevance to patients with asthma and increased airway neutrophilia.

MicroRNA‐27b Targets Gremlin 1 to Modulate Fibrotic Responses in Pulmonary Cells

Fibrosis is a chronic disease characterized by an excessive deposition of scar tissue in the affected organs. A central mediator of this process is transforming growth factor‐β (TGF‐β), which stimulates the production of extracellular matrix proteins such as collagens. MicroRNAs (miRNAs) have been implicated in both fibrosis as well as in TGF‐β signaling, but the extent of their regulation has not been fully defined. A functional screen was conducted using a library of miRNA inhibitors to identify miRNAs that affect TGF‐β‐induced type I collagen expression, a key event in the development of fibrosis. The inhibition of one miRNA in particular, miR‐27b, caused a significant increase in type I collagen expression. We found that miR‐27b directly targets Gremlin 1 by binding to its 3′‐UTR, reducing its mRNA levels. TGF‐β signaling decreased miR‐27b expression and caused a corresponding increase in Gremlin 1 levels, suggesting that TGF‐β regulates Gremlin 1 expression in part by modulating miR‐27b expression. Reducing Gremlin 1 levels by either siRNA‐mediated gene silencing or by using the miR‐27b mimic inhibited the expression of several genes known to be involved in fibrosis, while increasing Gremlin 1 levels by the addition of either recombinant protein or the miR‐27b inhibitor enhanced the expression of these genes. In summary, we have demonstrated that miR‐27b targets Gremlin 1, and that this regulation likely represents an important control point in fibrotic pathways. J. Cell. Biochem. 115: 1539–1548, 2014.

Preclinical evaluation of an inhibitor of cytosolic phospholipase A2α for the treatment of asthma

Asthma is a chronic inflammatory lung disease with considerable unmet medical needs for new and effective therapies. Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme that is ultimately responsible for the production of eicosanoids implicated in the pathogenesis of asthma. We investigated a novel cPLA2α inhibitor, PF-5212372, to establish the potential of this drug as a treatment for asthma. PF-5212372 was a potent inhibitor of cPLA2α (7 nM) and was able to inhibit prostaglandin (PG)D2 and cysteinyl leukotriene release from anti-IgE-stimulated human lung mast cells (0.29 and 0.45 nM, respectively). In a mixed human lung cell population, PF-5212372 was able to inhibit ionomycin-stimulated release of leukotriene B4, thromboxane A2, and PGD2 (2.6, 2.6, and 4.0 nM, respectively) but was significantly less effective against PGE2 release (>301 nM; p < 0.05). In an in vitro cell retention assay, PF-5212372 retained its potency up to 24 h after being washed off. In a sheep model of allergic inflammation, inhalation of PF-5212372 significantly inhibited late-phase bronchoconstriction (78% inhibition; p < 0.001) and airway hyper-responsiveness (94% inhibition; p < 0.001), and isolated sheep lung mast cell assays confirmed species translation via effective inhibition of PGD2 release (0.78 nM). Finally, PF-5212372 was assessed for its ability to inhibit the contraction of human bronchi induced by AMP. PF5212372 significantly inhibited AMP-induced contraction of human bronchi (81% inhibition; p < 0.001); this finding, together with the ability of this drug to be effective in a wide range of preclinical asthma models, suggests that inhibition of cPLA2α with PF-5212372 may represent a new therapeutic option for the treatment of asthma.

Discovery of potent and selective matrix metalloprotease 12 inhibitors for the potential treatment of chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with irreversible progressive airflow limitation. Matrix metalloproteinase-12 (MMP-12) has been characterized to be one of the major proteolytic enzymes to induce airway remodeling, destruction of elastin and the aberrant remodeling of damaged alveoli in COPD and asthma. The goal of this project is to develop and identify an orally potent and selective small molecule inhibitor of MMP-12 for treatment of COPD and asthma. Syntheses and structure-activity relationship (SAR) studies of a series of dibenzofuran (DBF) sulfonamides as MMP-12 inhibitors are described. Potent inhibitors of MMP-12 with excellent selectivity against other MMPs were identified. Compound 26 (MMP118), which exhibits excellent oral efficacy in the MMP-12 induced ear-swelling inflammation and lung inflammation mouse models, had been successfully advanced into Development Track status.

Altered Regulation of Aquaporin Gene Expression in Allergen and IL-13-Induced Mouse Models of Asthma

IL-13 is known to affect many processes that contribute to an asthmatic phenotype, including inflammation, fibrosis, and mucus production. Members of the aquaporin (AQP) family of transmembrane water channels are targets of regulation in models of lung injury and inflammation. Therefore, we examined AQP mRNA and protein expression in allergen and IL-13-induced mouse models of asthma. Lungs from ovalbumin sensitized and ovalbumin challenged (OVA/OVA) and IL-13 treated mice showed airway thickening, increased mucus production, and pulmonary eosinophilia. Pulmonary function tests showed a significant increase in methacholine-induced airway hyperreactivity in OVA/OVA and IL-13-treated mice as compared with controls. Quantitative PCR analysis revealed differential regulation of AQPs in these two models. AQP1 and AQP4 mRNA expression was downregulated in the OVA/OVA model, but not in the IL-13 model. AQP5 mRNA was reduced in both models, whereas AQP3 was upregulated only in the IL-13 model. Western analysis showed that diminished expression of an apically localized aquaporin, (AQP5), and concomitant upregulation of a basolateral aquaporin (AQP3 or AQP4) are characteristic features of both inducible asthma models. These results demonstrate that aquaporins are common targets of gene expression in both allergen and IL-13 induced mouse models of asthma.

Therapeutic activity of an interleukin-4/interleukin-13 dual antagonist on oxazolone-induced colitis in mice.

Interleukin‐4 (IL‐4) and IL‐13 are critical drivers of immune activation and inflammation in ulcerative colitis, asthma and other diseases. Because these cytokines may have redundant function, dual targeting holds promise for achieving greater efficacy. We have recently described a bifunctional therapeutic targeting IL‐4 and IL‐13 developed on a novel protein scaffold, generated by combining specific binding domains in an optimal configuration using appropriate linker regions. In the current study, the bifunctional IL‐4/IL‐13 antagonist was evaluated in the murine oxazolone‐induced colitis model, which produces disease with features of ulcerative colitis. The bifunctional IL‐4/IL‐13 antagonist reduced body weight loss throughout the 7‐day course of the model, and ameliorated the increased colon weight and decreased colon length that accompany disease. Colon tissue gene expression was modulated in accordance with the treatment effect. Concentrations of serum amyloid P were elevated in proportion to disease severity, making it an effective biomarker. Serum concentrations of the bifunctional IL‐4/IL‐13 antagonist were inversely proportional to disease severity, colon tissue expression of pro‐inflammatory genes, and serum amyloid P concentration. Taken together, these results define a panel of biomarkers signifying engagement of the IL‐4/IL‐13 pathway, confirm the T helper type 2 nature of disease in this model, and demonstrate the effectiveness of dual cytokine blockade.

Design and chemoproteomic functional characterization of a chemical probe targeted to bromodomains of BET family proteins

Bromodomain-containing proteins form the signal-reading element of a principal system for the control of gene expression in eukaryotes. Their potential as targets for selective drug action is increasingly being assessed and exploited. Deep characterization of the specificity, potency and other attributes of prototypical agents is an essential element of this process. Continuing studies of a dihydroquinazolinone-based series (prototype: PFI-1) with specificity for members of the BET (bromodomain and extra terminal) family led to the discovery of quinolin-2(1H)-one inhibitors with similar potency and selectivity, but increased chemical stability. Structure-guided design then led to the elaboration of a desthiobiotinylated analog retaining a high fraction of the potency of its parent compound and therefore suitable for chemoproteomic affinity capture experiments. These experiments, conducted using nuclear extracts of THP-1 cells, extended confidence in the selectivity of the series as first proposed. An additional and subsequent evaluation of specificity performed with a panel of recombinant bromodomains (BROMOscan™, DiscoveRx) supported the BET family specificity of the dihydroquinazolinone and quinolin-2(1H)-one series while adding appreciation of weaker effects shown at other bromodomains.