A Rabbit Model of Systemic Lupus Erythematosus, Useful for Studies of Neuropsychiatric SLE

The aim of this review is to present in one place a summary of the development of a rabbit model of SLE conducted using pedigreed rabbits bred and selected at the National Institute of Allergy and Infectious Diseases (NIAID), NIH. We provide an overview of the knowledge gained by using rabbits (Oryctolagus cuniculus) as models for SLE, and eliciting autoantibodies typical of those produced by patients with SLE. We present here summaries of work by ourselves and coauthors that can contribute to improved understanding of neuropsychiatric SLE (NPSLE) (Sanches-Guerro et al., 2008). Our gene expression studies (Rai, et al., 2010) and extensive evaluations of autoantibody responses coupled with observed clinical symptoms of animals immunized against peptides from the Smith antigen (Sm) or the NMDA glutamate receptor have shown to the of and specific on a by This book provides a comprehensive overview of the basic and clinical sciences of Systemic Lupus Erythematosus. It is suitable for basic scientists looking for detailed coverage of their areas of interest. It describes how advances in molecular biology have increased our understanding of this disease. It is a valuable clinical resource for practicing clinicians from different disciplines including rheumatologists, rheumatology fellows and residents. This book provides convenient access to information you need about cytokines, genetics, Fas pathway, toll like receptors and atherogenesis in SLE. Animal models have been reviewed as well. How to avoid delay in SLE diagnosis and management, in addition to various clinical manifestations including pregnancy and SLE have all been explained thoroughly in this book.


Introduction
The aim of this review is to present in one place a summary of the development of a rabbit model of SLE conducted using pedigreed rabbits bred and selected at the National Institute of Allergy and Infectious Diseases (NIAID), NIH. We provide an overview of the knowledge gained by using rabbits (Oryctolagus cuniculus) as models for SLE, and eliciting autoantibodies typical of those produced by patients with SLE. We present here summaries of work by ourselves and coauthors that can contribute to improved understanding of neuropsychiatric SLE (NPSLE) (Sanches-Guerro et al., 2008). Our gene expression studies (Rai, et al., 2010) and extensive evaluations of autoantibody responses coupled with observed clinical symptoms of animals immunized against peptides from the Smith antigen (Sm) or the NMDA glutamate receptor have shown promise to improve understanding NPSLE. An overview of immune system development, genetic diversity of immunoglobulin genes and somatic diversification during B-cell development in rabbits can be found in a review by Mage et al., (2006) and reference therein. Investigations of autoantibodies found in patients with NPSLE and the problems of diagnosis and specific treatments are addressed in other chapters in this volume.

Our model 2.1 Earlier studies of SLE models by other laboratories using rabbit
We set out to develop a model of SLE in pedigreed rabbits because an earlier report showed that immunization of non-pedigreed rabbits with peptides such as PPPGMRPP, derived from the Sm B/B' subunit of the spliceosomal Smith autoantigen led to epitope spreading, SLE-like autoantibody production and clinically observed seizures. This peptide sequence is one of the major regions of reactivity in SLE patients and may mimic the peptide PPPGRRP from the EBNA-1 component of Epstein-Barr virus (EBV) (James et al., 1995). Another study attempted to reproduce this report but only found some evidence for epitope spreading with no suggestion of induced autoimmunity (Mason et al., 1999). We hypothesized that the different results may have been obtained because small numbers of rabbits were studied, secondary antibody. Biotinylated antibodies were visualized by PE-conjugated streptavidin (Jackson ImmunoResearch laboratories, Inc.). After washing, cells were analyzed using a FACS-Calibur flow cytometer (BD Pharmingen) and FlowJo analytical software (Tree Star). Cells were gated on the side scatter x forward scatter (SSCxFSC) profiles to include both small and large lymphocytes, as well as monocytes but exclude red blood cells and granulocytes; dead cells were excluded by propidium iodide staining. Rabbit IgM + B cells were detected by FITC-conjugated goat anti-rabbit IgM (Southern Biotechnology Associates). Gene Expression studies: RNA extraction and synthesis of cDNA and cRNA Peripheral white blood cells (PWBCs) were lysed with TRIzol (Invitrogen, CA) and total RNA was extracted using RNAeasy Mini columns following the manufacturer's instructions (Qiagen, CA). The cRNA probes were prepared from mRNA using the Affymetrix gene chip eukaryotic small sample target labeling protocol assay version II (Affymetrix, Santa Clara, CA) using 2 cycles of cDNA synthesis and in vitro transcription (IVT) reactions. The cRNA thus obtained was used in the final IVT cycle for obtaining biotinylated cRNA using CTP and UTP (EnzoBioarray, Enzo Life Sciences, Farmingdale, NY) (Rai et al 2010).
Microarray analysis Affymetrix U95A human microarray chips were used and hybridization of the labeled cRNA was carried out according to the manufacturer's recommended protocol. Non-normalized MAS5 signals were used to compare raw probeset intensity values between human and rabbit samples. Final rabbit study analyses were conducted with expression values summarized using dChip, log2 transformed and Loess normalized using an R package (http://www.elwood9.net/spike). Analyses of the gene sets were done using Database for Annotation, Visualization and Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov/knowledgebase/) and Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Mountain View, CA; www.ingenuity.com). Quantitative real time PCR: Quantitative real time PCR analysis of mRNAs was performed on a 7900HT Sequence Detection System (Applied Biosystems). The cDNA synthesized from isolated PWBCs was directly used as template for real-time PCR by using TaqMan 2x PCR Master Mix Reagents Kit (Applied Biosystems). Each sample from three independent experiments was run in duplicate. The unit number showing relative mRNA levels in each sample was determined as a value of mRNA normalized against Peptidylprolyl isomerase A (PPIA). RT-PCR data were analyzed by using the 2 −  C T method. Based on its uniform expression among rabbit groups in the microarray analysis, rabbit peptidylprolyl isomerase A (PPIA; cyclophilin A) was selected as the housekeeping gene control and used for the calculation of C T . Where rabbit sequences were unavailable, primers were designed after searching for rabbit sequences with corresponding human gene sequences in the database containing the trace archives of the whole genome shotgun sequence of the rabbit (Oryctolagus cuniculus) generated by the Broad Institute of MIT and Harvard University (NCBI trace archive: cross-species Megablast at http://www.ncbi.nlm.nih.gov/blast/ tracemb.shtml) and in assemblies of rabbit scaffolds at Ensembl and UCSC (see NCBI Rabbit Genome Resources site) at: http://www.ncbi.nlm.nih.gov/projects/genome/guide/rabbit/ Figure 1 shows the pedigree and an overview of antibody responses of the rabbits immunized and selectively bred during the project to develop a rabbit model of SLE. There were 31 1-to 2-year-old rabbits in the initial studies (Rai et al., 2006). Rabbits of groups 2 or www.intechopen.com A Rabbit Model of Systemic Lupus Erythematosus, Useful for Studies of Neuropsychiatric SLE 207 3 were descendants of rabbits of groups 1 or 2 and/or their siblings. Rabbits that did not respond with autoantibody production after immunization during this initial study by Rai et al., (2006) are not shown. The fourth group was described in Rai et al., (2010) and their mRNA included along with mRNA from the first three groups for gene expression profiling of a total of 46 pedigreed control-or immunized-rabbits as detailed below (section 2.2.3). Controls that received only phosphate buffered saline are designated PB. Because the GR peptide generally elicited better autoantibody responses than the SM peptide, the two subsequent groups [5 (Puliyath et al., 2008)  The selective breeding led to subsequent progeny (groups 5 and 6) exhibiting more consistent autoantibody production. In the pedigree, we can trace the ancestry of some responder rabbits back to the first high responders (SM1 and GR9) that also exhibited seizures. For example, GR54 and GR55 from litter 2YY299 had high-responder grandsires SM1 and SM15 (Puliyath et al. 2008). The model developed using selectively bred pedigreed rabbits remains a promising one for further genetic investigations. As is found in human sera (Li et al., 2011), some rabbits had detectable pre-immune antinuclear antibodies (ANA) by ELISA. ANA of sixteen of twenty-four rabbits in group 5, including four immunized with only MAP-8 backbone had an increased ELISA value (delta OD) above pre-immune of 1.0 or more optical density units after the third boost. Anti-dsDNA increased in 12/24 rabbits after the fifth or seventh boost (Puliyath et al., 2008). Figure 2 shows examples of indirect immunofluorescence (ANA-IFA) studies of some sera from group 6 (Yang et al. 2009b). As in human SLE sera, the ANA-IFA patterns reflect responses to one or more autoantigens in different individuals. Littermates that received GR peptide such as UA269-3 and -1 (GR73 and GR77) developed similar patterns after the 3 rd boost. ANA staining with sera of littermates XA346-1 and -2 (GR85 and BB82) resulted in different patterns. GR85 serum exhibited some cytoplasmic and peripheral nuclear staining not seen with the serum of BB82. Puliyath et al., (2008) also noticed that GR-immunized littermates had similar ANA-IFA staining patterns but that BB immunized animals' patterns generally differed.

Gene expression studies
We extended the information about the rabbit model of SLE by microarray-based expression profiling of mRNA from peripheral blood leukocytes following peptide immunization (Rai et al., 2010). Data obtained in studies of gene expression in the first four groups of immunized rabbits were deposited in the Gene Expression Omnibus and became At the time of the gene expression studies, microarrays specific for study of gene expression profiles were not available for rabbits. We therefore first conducted comparisons of identically prepared rabbit and human cRNA binding to the Affymetrix U95 microarray available for human gene expression analyses. We showed that the human microarray could be used with rabbit cRNA to yield information on genetic pathways activated and/or suppressed in autoantibody-producing immunized rabbits. After demonstrating that human expression arrays could be used with rabbit RNA to yield information on molecular pathways, we designed a study evaluating gene expression profiles in a total of 46 rabbits from 4 groups of the pedigreed control and immunized rabbits. We discovered unique gene expression changes associated with lupus-like serological patterns in immunized rabbits.
Our results also demonstrated that caution must be applied when choosing the structure of the carrier Multiple Antigen Peptide (MAP-peptide) for immunization. We discovered that using MAP-4 rather than MAP-8 significantly altered patterns of immune response and gene expression. www.intechopen.com

Figure 3 shows that distinct patterns and clusters of functionally related genes were found t o b e u p r e g u l a t e d w h e n p e p t i d e s S M o r G R o n M A P -4 b a c k b o n e ( A ) w e r e u s e d a s
immunogens compared to when MAP-8 backbone was used (B) (Rai et al., 2010). Validation of gene expression data by quantitative real-time PCR was conducted for two genes for which primer sequences were available beta2-microglobulin (B2M) and p-21-protein (Cdc42/Rac)-activated kinase 1 (PAK1) (Figure 6 in Rai et al., 2010). These genes appear in the interactive pathway shown in Figure 4 below. Among the genes significantly upregulated in SLE rabbits were those associated with NK cytotoxicity, antigen presentation, leukocyte migration, cytokine activity, protein kinases, RNA spliceosomal ribonucleoproteins, intracellular signaling cascades, and glutamate receptor activity (Rai et al., 2010).   Table 2 summarize the patterns of upregulated gene expression found in the rabbits from the three groups immunized with MAP-8-peptides that made anti-dsDNA compared to those that only made other anti-nuclear antibodies. Twenty-five genes associated with inflammatory disorders were significantly upregulated in expression. Subsets of these were associated with various immunological disorders in the IPA databases including Autoimmune, Rheumatic, and inflammatory diseases. The results linked increased immune activation with up-regulation of components associated with neurological and anti-RNP responses, demonstrating the utility of the rabbit SLE model to uncover biological pathways related to SLE-induced clinical symptoms, including NPSLE. We suggested that our finding of distinct gene expression patterns in rabbits that made anti-dsDNA should be further investigated in subsets of SLE patients with different autoantibody profiles (Rai et al., 2010). In Figure 4, the connecting lines indicate direct interactions among the products of these genes. The shapes classify the proteins found as transmembrane receptors e.g. CD 40, cytokines/growth factors, e.g. CCL2, kinases, e.g. TYK2, peptidases, e.g. MMP9, other enzymes, e.g. ARF1 and transcriptional regulators, e.g. STAT5B.

Functional Annotation p-Value Number of molecules
Genes shown were common to the pathways listed in Table 2 that were upregulated in the anti-dsDNA positive rabbits.

Rabbit BAFF
Our laboratory described the expression and localization of rabbit B-cell activating factor (BAFF also termed BLys, TNFSF13b TALL1, zTNF4) and its receptor BR3 in cells and tissues of the rabbit (Yang et al., 2009a). In addition to its important role in B-cell development and survival, disease activity in human lupus patients has been reported to correlate with serum BAFF levels (reviewed in Groom et al., 2007) and with elevated expression of mRNA for BAFF and two BAFF receptors, BR3 and transmembrane activator and CAML interactor (TACI) in PBMC of lupus patients (Petri et al, 2008). We therefore also investigated BAFF and its receptors in our rabbit model of SLE (Yang et al., 2009b). We previously concluded that BAFF detected on B cells by flow cytometry represented BAFF bound to its receptors on the cells (Yang et al, 2009a). An independent study (Yeramilli, & Knight, 2010) also reported that BAFFbinding receptors on rabbit B-cells are occupied by endogenous soluble BAFF. These authors' studies also suggested that B cells in rabbit could produce BAFF. With the small number of total animals available in group 6 (Table 1B), and no reagents available to detect levels of serum BAFF, we could only measure BAFF on cell surfaces by flow cytometry. These studies found decreased surface expression of BAFF, BR3 and TACI after immunization and boosting www.intechopen.com in most animals. However, two rabbits that produced high anti-dsDNA responses (GR76 and GR77) developed higher percentages of BAFF/CD14 and BR3/CD14 positive cells. We did observe consistently lower mean fluorescence intensities of staining of TACI on PBMC and lower percentages of TACI positive cells. We suggested that since TACI is a negative regulator of B cells in mouse and man, perhaps the decrease in TACI in the rabbits producing autoantibodies had allowed autoreactive B cells to escape regulation. At the time these studies were conducted, clinical trials targeting BAFF/BLys and its receptors were in progress. With the FDA approval of Benlysta® (belimumab) in March, 2011, this monoclonal antibody, that inhibits binding of BLys/BAFF to receptors on B cells, became the first United States FDA approved treatment for SLE in over fifty years. Unfortunately, the clinical trials did not include SLE patients with severe active central nervous system lupus or nephritis. Post-approval trials will be required before this treatment can be recommended for these cohorts of patients.

Future prospects 2.3.1 Detection of autoantibodies to other antigens including neuroantigens in the rabbit model
In our rabbits, the development of severe symptoms may not yet have occurred because many were euthanized to make room for immunization and testing of their progeny and for tissue collection. For example, although nephritis was not observed, our gene expression studies identified upregulation of genes associated with Glomerulonephritis and also found in mice with Lupus Nephritis (Table 2 and Figure 4). Protein arrays containing microbial and autoantigens have been used to extend information on patients' serum profiles beyond the standard tests used in diagnosis (see for example, Robinson et al., 2002;Quintana et al., 2004;Li et al., 2005;Fattal et al. 2010). Recently, Li et al, (2011) used protein microarrays to determine risk factors for ANA positivity in healthy persons and concluded that serum profiles of autoantibodies can potentially identify healthy individuals with potential to develop lupus and other autoimmune diseases. Their observations extended the widely quoted earlier observations by Arbuckle et al, (2003) that autoantibodies develop as much as ten years before the clinical onset of SLE. In a NOD mouse model of cyclophosphamideaccelerated diabetes, Quintana et al (2004) used a protein microarray to predict from autoantibody repertoires, resistance or susceptibility to the development of diabetes before the induction with cyclophosphamide. Recently Fattal et al, (2010) applied the same technology to studies of SLE patients and controls. They reported highly specific SLE profiles that typically show increases in IgG binding to dsDNA, single-stranded DNA, Epstein-Barr virus, and hyaluronic acid. Interestingly, a healthy control subject who had the SLE antibody profile was later found to develop clinical SLE. Decreases in some specific IgM reactivities to autoantigens observed in this and earlier studies (Li et al., 2005) suggest that some natural IgM autoantibodies may play a protective role. A project to determine the antibody profiles of the rabbits' serum IgG and IgM, purified anti-dsDNA, and anti-peptide on protein microarrays carrying microbial and self antigens including those from the central and peripheral nervous system is in progress.

NPSLE and anti-NMDA glutamate receptors
The suggestion from extensive studies in the laboratory of Betty Diamond that some anti-dsDNA antibodies may react with the NMDA receptor and contribute to neurological manifestations in some lupus patients (DeGiorgio et al. 2001;Kowal et al, 2004), has led to numerous follow-up studies by the Diamond group,  and others. A recent editorial (Appenzeller, 2011) provides an updated overview of controversies in the field and discusses the accompanying paper by Gono et al., (2011) who report new analyses of 107 patients' sera for cross-reactivities of anti-dsDNA with a peptide derived from the sequence of the human NMDA receptor 2A (NR2A) compared with the similar peptide from human NR2B. They suggest that the sensitivity for detection of autoantibodies is greater with the NR2A peptide although their ELISA results directly comparing serum reactivities with each peptide were correlated with high significance (r = 0.94; P<0.0001). They conclude that assays of sera for anti-NR2A antibodies may be a better predictor of NPSLE than assays for NR2B and suggest that mixed results from other similar studies may be explained by small numbers of patients (Husebye et al., 2005) or less sensitive assays. We chose the GR peptide used in our immunization protocol based on the human sequence of NR2B because the rabbit sequence was not yet known. However, we knew that this sequence was highly conserved in several species including mouse, rat, dog, cow and chicken.

Rabbit genomics
Future studies of rabbit autoimmune and infectious diseases will benefit from the availability of a high quality draft rabbit genome sequence and assembly at ~7 x coverage recently completed at the Broad Institute, Boston (OryCun2.0). The donor was from a partially inbred strain. NCBI maintains a Rabbit Genome Resources website: http://www.ncbi.nlm.nih.gov/projects/genome/guide/rabbit/ Rabbit genomic sequences and assemblies from the ENCODE Project, with ~ 1% of rabbit genomic sequence from a different, outbred NZW animal are also available in GenBank. The selection of peptides for future immunization studies in rabbits can benefit from searching these resources.

Conclusion
The work described in this review documents that rabbits have a strong genetic component that leads to predisposition to production of autoantibodies similar to those found in SLE patients including those with NPSLE. Breeding and selection for consistent autoantibody production in the rabbit model can be accomplished over a few generations. When one of us (RGM) retired to Emeritus status at NIAID, the pedigreed colony was no longer maintained. Some animals related to those studied were distributed to others. In addition, although the pedigreed colony was dispersed, there is sperm available from two male breeders rabbits LL191-1 (SM13) and 1UA344-1 (GR49). In particular male SM13 and his progeny in the breeding scheme shown in Figure 2 generated numerous responders that made autoantibodies similar to those found in human Lupus patients. Cryovials of sperm from these animals are currently stored at the Twinbrook 3 facility of the Comparative Medicine Branch (CMB) of NIAID in liquid nitrogen storage tanks, and monitored weekly by their personnel. Further contact information can be obtained at the website of the CMB, of the NIAID, NIH at: http://www.niaid.nih.gov/LabsAndResources/labs/aboutlabs/ cmb/Pages/default.aspx.

Acknowledgment
This research was supported by the Intramural Research Program of the NIH, NIAID. All coauthors of the papers from the laboratory made valuable contributions to the current www.intechopen.com understanding of the rabbit model of SLE. We appreciate the major contributions of Cornelius Alexander, Laboratory of Immunology, NIAID as well as the veterinary staff at Spring Valley Laboratories who provided invaluable technical assistance. We thank Jeff Skinner for statistical analyses, Mariam Quiñones for help with IPA analyses and figures, and Folake Soetan and Rami Zahr for assistance with preparation of some figures. We dedicate this chapter to the memory of Dr. Barbara A. Newman who was a major contributor to this research.