Transcript Signatures in Experimental Asthma: Identification of STAT6-Dependent and -Independent Pathways 1 Nives Zimmermann,* Anil Mishra,* Nina E. King,* Patricia C. Fulkerson,* Matthew P. Doepker,* Nikolaos M. Nikolaidis,* Laura E. Kindinger,* Elizabeth A. Moulton,* Bruce J. Aronow, † and Marc E. Rothenberg 2 * The analysis of polygenic diseases such as asthma poses a challenging problem. In an effort to provide unbiased insight into disease pathogenesis, we took an empirical approach involving transcript expression profiling of lung tissue from mice with experimental asthma. Asthmatic responses were found to involve sequential induction of 4.7% of the tested genome; notably, there was ectopic expression of a series of genes not previously implicated in allergic or pulmonary responses. Genes were widely distributed throughout all chromosomes, but preferentially included genes involved in immunity, development, and homeostasis. When asthma was induced by two independent experimental regimens, unique gene transcript profiles were found depending upon the mode of disease induction. However, the majority of genes were common to both models representing an asthma signature genome. Analysis of STAT6-deficient mice revealed that an unexpectedly large segment of the asthma genes were STAT6 independent; this correlated with sustained inflammatory events in these mice. Notably, induction of asthma in STAT6-deficient mice resulted in gene induction not seen in wild-type mice. These results raise concern that therapeutic blockade of STAT6 in the asthmatic setting may reprogram the genetic signature, resulting in alternative lung pathology, which we indeed observed in STAT6-deficient mice. These results provide unprecedented insight into the complex steps involved in the pathogenesis of allergic airway responses; as such, these results have significant therapeutic and clinical implications. The Journal of Immunology, 2004, 172: 1815–1824. D espite intense ongoing asthma research, there is cur- rently an epidemic of this disease in the western world and the incidence is on the rise (1, 2). Experimentation in the asthma field has largely focused on analysis of the cellular and molecular events induced by allergen exposure in sensitized animals (primarily mice) and humans. These studies have identi- fied elevated production of IgE, mucus hypersecretion, airways obstruction, eosinophilic inflammation, and enhanced bronchial re- activity to spasmogens in the asthmatic response (3–5). Clinical and experimental investigations have demonstrated a strong cor- relation between the presence of CD4 + Th2 lymphocytes (Th2 cells) and disease severity, suggesting an integral role for these cells in the pathophysiology of asthma (6, 7). Th2 cells are thought to induce asthma through the secretion of an array of cytokines that activate inflammatory and residential effector pathways (8–10). In particular, IL-4 and IL-13 are produced at elevated levels in the asthmatic lung and are thought to be central regulators of many of the hallmark features of disease (11). They share a common re- ceptor subunit, the IL-4R and signaling through STAT6 (12, 13). Mice with targeted deletion of IL-4, IL-13, or STAT6 develop attenuation of certain features of asthma, including inflammatory cell infiltrates and airway hyperresponsiveness (AHR) 3 (14, 15). Although these studies have provided the rationale for the devel- opment of multiple therapeutic agents that interfere with specific inflammatory pathways (16 –19), the development of the asthma phenotype is likely to be related to the complex interplay of a large number of genes. In this study, we aimed to gain critical insight into the spectrum of genes involved in the pathogenesis of exper- imental asthma, to define the genetic variability between two “phe- notypically similar” asthmatic states, and to use transcript profiling to further uncover the importance of STAT6 pathways in the pathogenesis of disease. Materials and Methods Experimental asthma induction BALB/c mice (National Cancer Institute, Frederick, MD) and STAT6-de- ficient mice (20) (BALB/c background, The Jackson Laboratory, Bar Har- bor, ME) were housed under specific pathogen-free conditions. Asthma models were induced by two i.p. injections of OVA and aluminum hy- droxide, followed by two OVA or saline intranasal challenges 3 days apart, as previously described (21). Mice were sacrificed 3 or 18 h following the first or second allergen challenge. Aspergillus fumigatus Ag-associated asthma was induced by exposing mice intranasally three times a week for 3 wk as described elsewhere (22–24). Mice were sacrificed 18 h following the last challenge. In some experiments, bronchoalveolar lavage fluid (BALF) was collected and infiltrating cells differentiated. However, in ex- periments in which RNA was collected for microarray analysis, BALF was not performed. Divisions of *Allergy and Immunology and † Pediatric Informatics, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229 Received for publication August 6, 2003. Accepted for publication November 17, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by the American Heart Association Scientist De- velopment (to N.Z.) and postdoctorate fellowship (to N.E.K.) grants, National Insti- tutes of Health Grants R01 AI42242-05 (to M.E.R.), AI45898-04 (to M.E.R.), AI53479-01 (to M.E.R.), the Human Frontier Science Program (to M.E.R), Interna- tional Life Sciences Institute (to M.E.R.), Burroughs Wellcome Fund (to M.E.R.), and by the University of Cincinnati Center for Environmental Genetics. 2 Address correspondence and reprint requests to Dr. Marc E. Rothenberg, Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address: Rothenberg@cchmc.org 3 Abbreviations used in this paper: AHR, airway hyperresponsiveness; BALF, bron- choalveolar lavage fluid; CXCL, CXC chemokine ligand; CCL, CC chemokine li- gand; MRP, myeloid-related protein; ADAM, a disintegrin and metalloprotease; SPRR, small, proline rich. The Journal of Immunology Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00