Emergence of Autoimmunity in C57BL/6.NOD-Aec1Aec2 mice p r e a - u t t o o i s m t u i m m b u c e l n i l n i i t n i y c e a l f r o m s u b 4 c l s t i o n t a i c b 1 a l 6 l e p w h e a s e e k s S s u o j ö b f g c a r l e in g n i e c ’ s a l s t y o n d o r v o e m r t e transcriptional landscape of salivary glands targeted by autoimmunity innate immunity (darkened = dependence on susceptibility regions) adaptive immunity (darkened = dependence on susceptibility regions) Transcriptional landscapes of emerging autoimmunity: transient aberrations in the targeted tissue’s extracellular milieu precede immune responses in Sjögren’s syndrome Delaleu et al. Delaleu et al. Arthritis Research & Therapy 2013, 15:R174 http://arthritis-research.com/content/15/5/R174 Delaleu et al. Arthritis Research & Therapy 2013, 15:R174 http://arthritis-research.com/content/15/5/R174 RESEARCH ARTICLE Open Access Transcriptional landscapes of emerging autoimmunity: transient aberrations in the targeted tissue’s extracellular milieu precede immune responses in Sjögren’s syndrome Nicolas Delaleu1*, Cuong Q Nguyen2, Kidane M Tekle3, Roland Jonsson1,4 and Ammon B Peck2 Abstract Introduction: Our understanding of autoimmunity is skewed considerably towards the late stages of overt disease and chronic inflammation. Defining the targeted organ’s role during emergence of autoimmune diseases is, however, critical in order to define their etiology, early and covert disease phases and delineate their molecular basis. Methods: Using Sjögren’s syndrome (SS) as an exemplary rheumatic autoimmune disease and temporal global gene-expression profiling, we systematically mapped the transcriptional landscapes and chronological interrelationships between biological themes involving the salivary glands’ extracellular milieu. The time period studied spans from pre- to subclinical and ultimately to onset of overt disease in a well-defined model of spontaneous SS, the C57BL/6. NOD-Aec1Aec2 strain. In order to answer this aim of great generality, we developed a novel bioinformatics-based approach, which integrates comprehensive data analysis and visualization within interactive networks. The latter are computed by projecting the datasets as a whole on apriori-defined consensus-based knowledge. Results: Applying these methodologies revealed extensive susceptibility loci-dependent aberrations in salivary gland homeostasis and integrity preceding onset of overt disease by a considerable amount of time. These alterations coincided with innate immune responses depending predominantly on genes located outside of the SS-predisposing loci Aec1 and Aec2. Following a period of transcriptional stability, networks mapping the onset of overt SS displayed, in addition to natural killer, T- and B-cell-specific gene patterns, significant reversals of focal adhesion, cell-cell junctions and neurotransmitter receptor-associated alterations that had prior characterized progression from pre- to subclinical disease. Conclusions: This data-driven methodology advances unbiased assessment of global datasets an allowed comprehensive interpretation of complex alterations in biological states. Its application delineated a major involvement of the targeted organ during the emergence of experimental SS. Introduction of the molecular basis of autoimmunity is skewed toward Common to autoimmune diseases is a long and clinically late and overt disease phases. To conclusively assign etio- silent phase. As a consequence, affected individuals are di- logical relevance to any biological process altered in such agnosed only after immune system–mediated functional specimens is difficult, considering the causality di- deficiencies of the affected tissues result in overt disease lemma. Nevertheless, stratifying the chronology of these [1,2]. Hence, owing to the unavailability of human speci- events is crucial in estimating whether genetic predis- mens reflecting subclinical disease stages, understanding position to develop a specific autoimmune disease might also involve genes associated with tissue develop- * Correspondence: [email protected] ment and homeostasis or if the genes exclusively cluster 1Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, Laboratory Building, Jonas Liesvei 65, 5021 Bergen, Norway in processes associated with specific phases of innate Full list of author information is available at the end of the article adaptive immune maturation [3-5]. © 2013 Delaleu et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Delaleu et al. Arthritis Research & Therapy 2013, 15:R174 Page 2 of 19 http://arthritis-research.com/content/15/5/R174 One approach to delineate, and to a certain extent strat- General Medical Sciences, Bethesda, MD, USA) [17] and ify, the molecular events associated with subclinical phases design of an advanced visualization methodology. By of autoimmune diseases is the use of adequate experimen- exploiting this approach, we sought to significantly im- tal models [6,7]. For this purpose, a suitable experimental prove our ability to analyze such “-omics” data sets com- strain must, in correspondence with humans, develop its prehensively and systematically and, in turn, to minimize relevant autoimmune phenotype over an extended period the introduction of personal bias. of time and in the context of its genetic background. C57BL/6.NOD-Aec1Aec2 mice fulfill these criteria as a Methods model of primary Sjögren’s syndrome (SS) because they Animals develop, in the absence of other inflammatory conditions, C57BL/6.NOD-Aec1Aec2 and C57BL/6 male mice were all major features relevant to the diagnosis of SS in bred and maintained under specific pathogen-free condi- humans spontaneously and over a period of several tions at the Department of Pathology mouse facility at the months [7,8]. University of Florida, Gainesville, FL, USA. To dissect the With a prevalence of 0.1% to 0.3% in the total popula- SGs, mice were killed by cervical dislocation after deep tion, SS is considered a relatively common autoimmune anesthetization. All procedures were approved by the disease. It mainly involves the exocrine glands. Nearly all University of Florida’s Institutional Animal Care and Use patients complain about persistent symptoms of dry Committee (protocols B317-2007 and 2008011756). mouth, and many present with hyposalivation. Severe disease outcomes also include disabling fatigue and de- Isolation of RNA from salivary glands velopment of non-Hodgkin’s lymphoma. To date, all Total RNA was isolated according to the protocol de- therapies tested have been ineffective in reversing the scribed in detail elsewhere [18]. When the mice were 4, 8, course of SS [9,10]. Similar to patients with systemic 12 and 16 weeks of age, the SGs free of lymph nodes were lupus erythematosus, a subpopulation of individuals with excised in parallel from five C57BL/6.NOD-Aec1Aec2 and SS exhibit a type 1 IFN signature, suggesting that a viral five C57BL/6 mice, then snap-frozen in liquid nitrogen. agent may be involved in triggering the disease [11]. As Total RNA from each mouse was isolated concurrently a consequence, studies designed to discover genetic as- using the RNeasy Mini Kit (QIAGEN, Valencia, CA, sociations have focused either on innate immunity [12] USA), then RNA concentrations and purities were evalu- or on genes that might explain the dominant role of B ated using UV spectroscopy. The ratio of absorbance cells in the pathogenesis of SS [10]. Unfortunately, these (260 nm and 280 nm) of the RNA samples averaged 1.976. studies have yet to yield results that allow estimation of Subsequently, each sample was hybridized separately on a an individual’s risk of developing SS. GeneChip Mouse Genome 430 2.0 Array and 3′ IVT Ex- Histological evaluations of minor salivary glands (SGs) press Kit (Affymetrix, Santa Clara, CA, USA) according to obtained from patients with SS commonly show focal in- the manufacturer’s instructions (annotation: build 32; 6 flammation that may coincide with epithelial cell atro- September 2011). Microarrays were assessed using Affy- phy and the presence of adipose tissue and fibrosis. metrix Expression Console Software 1.1 without changing Morphologically, these glands may also display structural the default settings (Affymetrix), and the data quality was disorganization, including loss of cell–cell and cell– deemed adequate for further analyses. extracellular matrix (ECM) adhesion [13,14]. However, organizing these findings chronologically and conclu- Submission of data to Gene Expression Omnibus sively as etiological, pathogenic or bystander processes All the data sets reported herein have been deposited has not yet been possible [9]. and are publicly available in the Gene Expression Omni- Thus, the aim of this study was to delineate the tran- bus [GSE15640, GSE36378]. scriptional landscape associated with the extracellular milieu (EM) of the SGs during spontaneous emergence of Verification of microarray data experimental SS. The global scope of our aim favors inte- In addition to the experiments performed to validate the gration over reduction and is ideally based on a data- quality of the microarray data presented previously driven approach that ensures impartial interpretation of [19,20], verification