Independent Pathways Asthma
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Transcript Signatures in Experimental Asthma: Identification of STAT6-Dependent and -Independent Pathways This information is current as Nives Zimmermann, Anil Mishra, Nina E. King, Patricia C. of September 29, 2021. Fulkerson, Matthew P. Doepker, Nikolaos M. Nikolaidis, Laura E. Kindinger, Elizabeth A. Moulton, Bruce J. Aronow and Marc E. Rothenberg J Immunol 2004; 172:1815-1824; ; doi: 10.4049/jimmunol.172.3.1815 Downloaded from http://www.jimmunol.org/content/172/3/1815 Supplementary http://www.jimmunol.org/content/suppl/2004/01/16/172.3.1815.DC1 Material http://www.jimmunol.org/ References This article cites 63 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/172/3/1815.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Transcript Signatures in Experimental Asthma: Identification of STAT6-Dependent and -Independent Pathways1 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. Rothenberg2* 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 Downloaded from 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. http://www.jimmunol.org/ 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. espite intense ongoing asthma research, there is cur- Mice with targeted deletion of IL-4, IL-13, or STAT6 develop rently an epidemic of this disease in the western world attenuation of certain features of asthma, including inflammatory D and the incidence is on the rise (1, 2). Experimentation cell infiltrates and airway hyperresponsiveness (AHR)3 (14, 15). in the asthma field has largely focused on analysis of the cellular Although these studies have provided the rationale for the devel- and molecular events induced by allergen exposure in sensitized opment of multiple therapeutic agents that interfere with specific animals (primarily mice) and humans. These studies have identi- inflammatory pathways (16–19), the development of the asthma by guest on September 29, 2021 fied elevated production of IgE, mucus hypersecretion, airways phenotype is likely to be related to the complex interplay of a large obstruction, eosinophilic inflammation, and enhanced bronchial re- number of genes. In this study, we aimed to gain critical insight activity to spasmogens in the asthmatic response (3–5). Clinical into the spectrum of genes involved in the pathogenesis of exper- and experimental investigations have demonstrated a strong cor- imental asthma, to define the genetic variability between two “phe- relation between the presence of CD4ϩ Th2 lymphocytes (Th2 notypically similar” asthmatic states, and to use transcript profiling cells) and disease severity, suggesting an integral role for these to further uncover the importance of STAT6 pathways in the cells in the pathophysiology of asthma (6, 7). Th2 cells are thought pathogenesis of disease. 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 Materials and Methods asthmatic lung and are thought to be central regulators of many of Experimental asthma induction the hallmark features of disease (11). They share a common re- ␣ BALB/c mice (National Cancer Institute, Frederick, MD) and STAT6-de- ceptor subunit, the IL-4R and signaling through STAT6 (12, 13). ficient mice (20) (BALB/c background, The Jackson Laboratory, Bar Har- bor, ME) were housed under specific pathogen-free conditions. Asthma Divisions of *Allergy and Immunology and †Pediatric Informatics, Department of models were induced by two i.p. injections of OVA and aluminum hy- Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati droxide, followed by two OVA or saline intranasal challenges 3 days apart, College of Medicine, Cincinnati, OH 45229 as previously described (21). Mice were sacrificed 3 or 18 h following the first or second allergen challenge. Aspergillus fumigatus Ag-associated Received for publication August 6, 2003. Accepted for publication November 17, 2003. 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 costs of publication of this article were defrayed in part by the payment of page the last challenge. In some experiments, bronchoalveolar lavage fluid charges. This article must therefore be hereby marked advertisement in accordance (BALF) was collected and infiltrating cells differentiated. However, in ex- with 18 U.S.C. Section 1734 solely to indicate this fact. periments in which RNA was collected for microarray analysis, BALF was 1 This work was supported in part by the American Heart Association Scientist De- not performed. 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. 3 Abbreviations used in this paper: AHR, airway hyperresponsiveness; BALF, bron- 2 Address correspondence and reprint requests to Dr. Marc E. Rothenberg, Division of choalveolar lavage fluid; CXCL, CXC chemokine ligand; CCL, CC chemokine li- Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet gand; MRP, myeloid-related protein; ADAM, a disintegrin and metalloprotease; Avenue, Cincinnati, OH 45229-3039. E-mail address: [email protected] SPRR, small, proline rich. Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 1816 TRANSCRIPT SIGNATURES IN ASTHMA Preparation of RNA and microarray hybridization Indeed, 11 of the early activation genes remained elevated, RNA was extracted using the TRIzol reagent as per the manufacturer’s whereas 46 additional genes increased (Fig. 1). Those include the instructions. Following TRIzol purification, RNA was repurified with phe- gene for ADAM-8 (a disintegrin and metalloprotease) (31) and nol-chloroform extraction and ethanol precipitation. Microarray hybridiza- acidic chitinase (32), genes not previously reported in asthma but tion was performed by the Affymetrix GeneChip Core facility at Children’s related to proteins associated with tissue remodeling. A role for Hospital Medical Center using the murine U74Av2 GeneChip (Affymetrix, Santa Clara, CA) as previously reported (21). This analysis was performed specific members of the ADAM family (ADAM-10 and ADAM- with one mouse per chip (n Ն3 for each allergen challenge condition and 17) has been demonstrated in the immune system where they are n Ն2 for each saline challenge condition). A further description of the involved in processing of the cell surface precursor form of methodology according to the Minimum Information About a Microarray TNF-␣; indeed, a member of this family of molecules (ADAM-33) Experiment guidelines (available at www.mged.org/Workgroups/MIAME/ miame.html) is provided in supplementary information. has recently