Gene Expression Profiling Reveals Unique Pathways Associated with Differential Severity of Lyme Arthritis This information is current as Hillary Crandall, Diane M. Dunn, Ying Ma, R. Mark of September 25, 2021. Wooten, James F. Zachary, John H. Weis, Robert B. Weiss and Janis J. Weis J Immunol 2006; 177:7930-7942; ; doi: 10.4049/jimmunol.177.11.7930 http://www.jimmunol.org/content/177/11/7930 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2006/11/16/177.11.7930.DC1 Material http://www.jimmunol.org/ References This article cites 58 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/177/11/7930.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Gene Expression Profiling Reveals Unique Pathways Associated with Differential Severity of Lyme Arthritis1 Hillary Crandall,* Diane M. Dunn,† Ying Ma,* R. Mark Wooten,‡ James F. Zachary,§ John H. Weis,* Robert B. Weiss,† and Janis J. Weis2* The murine model of Lyme disease provides a unique opportunity to study the localized host response to similar stimulus, Borrelia burgdorferi, in the joints of mice destined to develop severe arthritis (C3H) or mild disease (C57BL/6). Pathways associated with the response to infection and the development of Lyme arthritis were identified by global gene expression patterns using oligo- nucleotide microarrays. A robust induction of IFN-responsive genes was observed in severely arthritic C3H mice at 1 wk of infection, which was absent from mildly arthritic C57BL/6 mice. In contrast, infected C57BL/6 mice displayed a novel expression profile characterized by genes involved in epidermal differentiation and wound repair, which were decreased in the joints of C3H mice. These expression patterns were associated with disease state rather than inherent differences between C3H and C57BL/6 Downloaded from mice, because C57BL/6-IL-10؊/؊ mice infected with B. burgdorferi develop more severe arthritis than C57BL/6 mice and displayed an early gene expression profile similar to C3H mice. Gene expression profiles at 2 and 4 wk postinfection revealed a common response of all strains that was likely to be important for the host defense to B. burgdorferi and mediated by NF-␬B-dependent signaling. The gene expression profiles identified in this study add to the current understanding of the host response to B. burgdorferi and identify two novel pathways that may be involved in regulating the severity of Lyme arthritis. The Journal of Immunology, 2006, 177: 7930–7942. http://www.jimmunol.org/ yme disease is a multisystem disorder caused by infection develop mild to moderate disease (8, 9). Although this difference with the tick-borne spirochete Borrelia burgdorferi (1). is not dependent on MHC alleles, it has been linked to quantitative L Signs of infection include a bull’s-eye rash at the site of trait loci (QTL)3 on chromosomes 1, 4, 5, 11, and 12 (10, 11). the tick bite, termed erythema migrans, followed by dissemination Interestingly, infected C3H and C57BL/6 mice harbor similar of bacteria to various tissues resulting in neurological abnormali- numbers of bacteria in joint tissues, indicating that differences in ties, myocarditis, and arthritis (2). Lyme arthritis occurs in ϳ60% arthritis severity are not due to differences in host defense, but of individuals not treated with antibiotics at the time of the tick rather reflect different abilities to regulate the localized inflamma- by guest on September 25, 2021 bite, is associated with the presence of B. burgdorferi in joint tis- tory response (9). B. burgdorferi lipoprotein interaction with TLR2 sue, and resolves with successful antibiotic treatment (3, 4). A results in the production of proinflammatory cytokines and che- small percentage of individuals with subacute arthritis progress to mokines, several of which have been implicated in modulating the a chronic treatment-resistant arthritis that is no longer associated development of arthritis (12–15). Furthermore, C57BL/6 mice with bacteria in joint tissue and is postulated to be autoimmune- lacking the potent anti-inflammatory cytokine IL-10 (C57BL/6 IL- mediated (5, 6). 10Ϫ/Ϫ) develop more severe arthritis than wild-type C57BL/6 Infection-associated Lyme arthritis has been studied in the mice while more effectively controlling bacterial growth (16–18). mouse, where arthritis develops 3–4 wk following intradermal in- The mouse model of Lyme arthritis provides a unique opportu- oculation and is histopathologically similar to Lyme arthritis in nity to study contrasting responses to similar bacterial stimuli in humans (7, 8). The severity of arthritis is genetically regulated, mice developing severe or mild arthritis (8, 9, 19). Localized re- with C3H mice developing severe arthritis whereas C57BL/6 mice sponses to B. burgdorferi were assessed by global gene expression analysis in whole joint tissue from C3H, C57BL/6, and C57BL/ 6-IL-10Ϫ/Ϫ mice during the progression of disease development. *Department of Pathology, and †Department of Human Genetics, University of Utah, This analysis revealed the activation of two unexpected and diver- Salt Lake City, Utah 84112; ‡Department of Medical Microbiology and Immunology, gent pathways in response to infection in mice destined to develop § Medical University of Ohio, Toledo, Ohio 43614; and Department of Pathobiology, arthritis of different severities, and suggested that an early com- University of Illinois at Urbana-Champaign, Urbana, Illinois 61802 mitment to a gene expression phenotype in infected joint tissue Received for publication June 13, 2006. Accepted for publication September 12, 2006. could determine the severity of subsequent Lyme arthritis. 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 Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. Mice 1 This work was supported by National Institutes of Health Grants (AI-32223 to J.J.W. and J.F.Z.; AR-43521 to J.J.W.; AI-24158 to J.H.W.; and HL-072903 to Female C3H/HeNCr (C3H) and C57BL/6NCr (C57BL/6) mice were ob- R.B.W.), National Institutes of Health/National Institute of Diabetes and Digestive tained from the National Cancer Institute, whereas female B6.129P2-IL- and Kidney Diseases Training Grant (DK07115 to H.C.), the American Heart Asso- 10tm1Cgn/J (C57BL/6-IL-10Ϫ/Ϫ) mice on the closely related C57BL/6J ciation (Grant 0335148N to R.M.W.), and by funds from Associated Regional Uni- mouse were obtained from The Jackson Laboratory. Mice were housed in versity Pathologists. 2 Address correspondence and reprint requests to Dr. Janis J. Weis, Department of Pathology, University of Utah School of Medicine, 15 North Medical Drive East, 3 Abbreviations used in this paper: QTL, quantitative trait loci; SAM, significance Room 2100, Salt Lake City, Utah 84112-5650. E-mail address: janis.weis@path. analysis of microarray; F, forward; R, reverse; KO, knockout; NC, not changed; utah.edu MMP, matrix metalloproteinase; TIMP, tissue inhibitor of MMP. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 7931 the Animal Resource Center at the University of Utah Health Science Cen- ter according to the guidelines of the National Institutes of Health for the care and use of laboratory animals. B. burgdorferi culture and infection Mice were infected by intradermal injection at 6 wk of age with 2 ϫ 103 B. burgdorferi clone N40 (provided by S. Barthold, University of Califor- nia, Davis, CA) that had been cultured for 4 days in Barbour-Stoenner- Kelly II medium containing 6% rabbit serum (Sigma-Aldrich). Assessment of infection status and arthritis severity Infection of mice was confirmed by culture of spirochetes from bladders, production of B. burgdorferi-specific Abs, and detection of B. burgdorferi recA in ear tissues by quantitative PCR (7, 20). Ankle swelling was used as a relative indicator of arthritis development in the actual tissue collected for microarray analysis and was determined from measurements made of the rear ankle joints with a metric caliper. Increases in ankle measurement were similar to those in previous studies, where complete histological as- sessment of arthritis severity was performed (9, 16). Isolation of RNA Downloaded from Total RNA was isolated from tissues and cells using acid guanidine ex- traction (21). Skin was removed from the rear ankles, and tissue extending ϳ5 mm in each direction was collected from infected and control mice at the indicated times. Joint tissues were flash frozen and homogenized in cold acid guanidine using an Ultra-Turrax disperser (IKA Works), and RNA was separated by cesium chloride cushion centrifugation. RNA was recovered by ethanol precipitation and applied to a RNeasy kit (Qiagen). http://www.jimmunol.org/ Gene expression analysis Equal amounts of total RNA from the more swollen ankle of five individual mice of each genotype from each time point were pooled into a single sample that was prepared for Affymetrix array hybridization according to the manufacturer’s instructions (Affymetrix) (22).