Detection of Multiple Autoantibodies in Patients with Ankylosing Spondylitis Using Nucleic Acid Programmable Protein Arrays.

Ankylosing spondylitis (AS) is a common, inflammatory rheumatic disease that primarily affects the axial skeleton and is associated with sacroiliitis, uveitis, and enthesitis. Unlike other autoimmune rheumatic diseases, such as rheumatoid arthritis or systemic lupus erythematosus, autoantibodies have not yet been reported to be a feature of AS. We therefore wished to determine whether plasma from patients with AS contained autoantibodies and, if so, characterize and quantify this response in comparison to patients with rheumatoid arthritis (RA) and healthy controls. Two high density nucleic acid programmable protein arrays expressing a total of 3498 proteins were screened with plasma from 25 patients with AS, 17 with RA, and 25 healthy controls. Autoantigens identified were subjected to Ingenuity Pathway Analysis to determine the patterns of signaling cascades or tissue origin. 44% of patients with ankylosing spondylitis demonstrated a broad autoantibody response, as compared with 33% of patients with RA and only 8% of healthy controls. Individuals with AS demonstrated autoantibody responses to shared autoantigens, and 60% of autoantigens identified in the AS cohort were restricted to that group. The autoantibody responses in the AS patients were targeted toward connective, skeletal, and muscular tissue, unlike those of RA patients or healthy controls. Thus, patients with AS show evidence of systemic humoral autoimmunity and multispecific autoantibody production. Nucleic acid programmable protein arrays constitute a powerful tool to study autoimmune diseases.


Introduction
Ankylosing Spondylitis (AS) is a chronic, debilitating, rheumatic disease with a predilection for the axial skeleton and large joints. It affects in excess of 0.1% of the population and can be associated with uveitis, apical pulmonary fibrosis and cardiac disease [1,2]. AS is difficult to diagnose and patients can suffer symptoms for years before receiving appropriate treatment [2]. The aetiology is unknown, but is thought to be immune mediated. The extremely strong association with the Class I Human Leukocyte Antigen allotype HLA-B27 has led to hypotheses involving CD8 T cellmediated immunity [3]. More recently additional genetic associations, including IL23R, have suggested a role for Th17 T cells [4].
Autoantibodies are a common characteristic of many rheumatic autoimmune diseases [5]. Rheumatoid Factor (RF), an autoantibody against the Fc portion of IgG, occurs in more than 85% of patients with Rheumatoid Arthritis (RA). Although not specific to RA, RF is used routinely as a diagnostic test for RA and other autoimmune disorders [6]. More recently, autoantibodies to cyclic citrullinated peptides have proven more specific than rheumatoid factor in diagnosing RA and have been shown to have prognostic value [7].
Autoantibodies are not commonly considered to be a feature of AS. However, anti-leukocyte [8], anti-neutrophil [9], and autoantibodies to some collagen proteins have been reported [10]. Increased levels of circulating plasma cells have also been reported in AS patients [11], as well as evidence of hypergammaglobulinemia [12].
Aside from these findings, no comprehensive investigation into the presence of autoantibodies in patients with AS has been performed to date.
Protein microarrays are commonly used as tools for detecting protein-protein interactions, such as the binding of autoantibodies to their cognate antigens. However, technical issues involving the cloning and purification of thousands of proteins, protein folding and stability, and the shelf life of protein arrays have, until now, made this a challenging task. A novel technology, referred to as the Nucleic Acid Programmable Protein Array (NAPPA) has recently surmounted these issues [13].
NAPPA involves the in situ transcription-translation of thousands of glycosylated proteins in close spatial proximity. Biotinylated cDNAs containing GST-tagged query proteins are immobilized onto glass slides. Anti-GST antibodies spotted adjacent the cDNA are used as capture molecules. To synthesize the proteins, slides are covered in a continuous layer of reticulocyte lysate. The C-terminal GST tag ensures that only full-length proteins are captured. Therefore, this novel protein microarray technology provides an ideal platform for the characterization of autoantibody responses in autoimmune diseases.
In this discovery stage study we wished to determine whether patients with Ankylosing Spondylitis demonstrated autoantibody responses, using two different NAPPA arrays expressing a total of 3498 proteins. We show that AS patients demonstrate multispecific autoantibody responses to several autoantigens, predominantly targeted towards connective tissue and skeletal proteins. Blood Samples and Plasma Isolation. Venesection was performed using a 21-gauge needle and a 20 ml syringe. Heparinized blood samples were transferred into 50 ml falcon tubes. To isolate the plasma, the samples were centrifuged at 500 g for 10 minutes at room temperature. Isolated plasma was aliquoted in 1 ml fractions into 1.5 ml microcentrifuge tubes, and stored at -80ºC for one to two years. All samples underwent one freeze thaw cycle prior to analysis. All samples were collected contemporaneously.
Preparation of NAPPA slides and DNA preparation. Glass slides were treated with 2% aminosilane in acetone, washed with acetone and then water, and dried using forced air. Slides were then stored in a dry container with silica packs until ready for printing. Bacteria harbouring the expression plasmids were cultured in 1.5 ml of Terrific Broth containing 10% potassium phosphate and 100 µg/ml ampicillin in 96well plates for 24 hours, then pelleted by centrifugation at 3000 g for 30 minutes.
DNA was prepared according to published protocols [16]. DNA concentrations were measured by spectrophotometry at 260 nm and plates were deemed acceptable if 90% of the wells had a total of 15 µg or more.
Preparation of DNA Samples and Array Printing. DNA samples were precipitated by the addition of 0.8x volume of isopropanol and centrifugation at 4000 g for 30 minutes. They were then washed with 80% ethanol and allowed to air dry. Each well was resuspended in 20 µl of spotting buffer (50 µg/ml capture antibody, 3.6 mg/ml BSA, 2mM Bis Sulfosuccinimidyl Suberate) and mixed for 30 minutes. Sets of four 96 well plates were then transferred to one 384 well plate which was used for printing. Arrays were printed on the aminosilane treated glass slides using a Genetix Q Array2 printer. Standard conditions of 60% humidity were applied.

NAPPA Protein Expression.
Printed slides were blocked for 1 hour at room temperature on a rocking platform in SuperBlock (Thermo Scientific Pierce) using 30 ml for four slides to wash away any unbound NAPPA reagents (plasmid, BSA, or capture antibody). The slides were then rinsed with water and dried with filtered compressed air. 100 µL of rabbit reticulocyte lysate in vitro transcription-translation mix (IVTT) (Promega, Wisconsin) was prepared per slide (4 µL TNT buffer, 2 µL T7 polymerase, 1 µL of -Met, 1 µL of -Leu, 2 µL of RNaseOUT and 90 µL of DEPC water) and Hybriwell gaskets (Grace Biolabs, Oregon) were applied. IVTT mix was pipetted onto the array through the hole in the gasket. Port seals were applied to both ports to avoid evaporation. The arrays were incubated for 1.5 hours at 30ºC then 30 minutes at 15ºC for protein expression and binding the immobilized capture antibody.
The HybriWells were removed and the arrays were washed with milk three times for 3 minutes on a rocking platform. The protein arrays were then blocked with milk at room temperature for 1 hour. Between-plate replicate protein spot concentrations had a correlation coefficient of 0.96 under these conditions [16].
Antibody capture and visualization. Plasma samples were diluted between 1/170 and 1/2000 in 2 ml of 5% milk in PBS to achieve an equal amount of non-specific background binding. To avoid any biases from irregularities in the spotting procedure only arrays from a single batch were used. Furthermore, the 67 serum samples from all three groups were coded, mixed and randomized to the 67 arrays. The samples were pipetted onto the arrays, assembled into gaskets (Corning) and incubated overnight while rotating at 4ºC. The arrays were then disassembled and washed three times in milk for 5 minutes on a rocking platform. Secondary antibody (HRPconjugated anti-human IgG) was applied to the slide under a cover slip and incubated for 1 hour at room temperature. The slides were washed in PBS three times for 5 minutes, once with water, and dried. Tyramide signal amplification solution covering the arrays was applied under a cover slip and incubated for 10 minutes at room temperature. Arrays were rinsed with water and dried with filtered compressed air.
Arrays were then scanned in a micro array scanner, using settings for Cy3.

NAPPA Data analysis.
For each protein target query on the NAPPA arrays, the mean and standard deviation (SD) of the signal intensity of the 25 healthy controls were calculated. Z-scores were then calculated for every query protein spot in every individual in the three groups. Zscore = (Signal -Mean)/(Standard Deviation). A positive "hit" was attributed if the Z-score was greater than three standard deviations above the healthy control mean intensity.

Screening of AS and control plasma samples using two dedicated NAPPA arrays.
Prior to the NAPPA screening plasma samples were coded, blinded and randomized.
Each sample was screened on a mini-array, consisting of 100 proteins, to measure background signal. All plasma samples were diluted to at least 1/170 to normalize for background intensity. Figure 1 shows the workflow of this study. After the proteins were synthesized in situ using a transcription-translation coupled rabbit reticulocyte lysate system, the slides were washed and blocked to minimize non-specific interactions between plasma proteins and those present on the array. The slides were incubated with plasma overnight at four degrees to permit binding of autoantibodies to their target antigens, and were then incubated with a secondary HRP-conjugated anti-human IgG antibody. Visualization was performed using a Tyramide-Cyanine 3 conjugated amplification system, and scanned using a slide fluorescence scanner.
Signals for each spot were averaged over the cohort and a statistical distribution of the intensities of those signals among the controls was calculated. From these distributions means and standard deviations were calculated for each spot and individual. On each array positive controls included the Epstein-Barr virus EBNA protein and human IgG protein (to which the HRP-anti-human secondary antibody would bind). Negative controls included the parental expression vector containing the GST-tag but lacking a cDNA, as well as spots that carry the spotting mix but lacked any DNA.  NAPPA method validation. Although the NAPPA method is a highly reproducible and robust system for detecting protein-protein interactions [16], we wished to validate one of the NAPPA results via an independent experimental method. Eight AS patient plasma samples were pooled, in half of which IL-6 had been identified as a putative autoantigen, as were eight plasma samples from healthy control individuals.
From these two pools IgG molecules were immunoprecipitated using protein A beads.
These IgG immunoprecipitates were then tested in a dose-dependent manner for recovery efficiency of recombinant IL-6. After blotting 10% of the immunoprecipitated material for the presence of IL-6, densitometry analysis showed that IgG from the AS plasma pool recovered more than 60% of recombinant IL-6, compared with just over 5% recovery from the healthy control plasma pool (see figure   3).
Multiple AS patients show autoantibody responses to shared autoantigens. We next asked whether any of the autoantibodies were present in multiple plasma samples within the AS patient group. From the screening of NAPPA array GST#1, 193 autoantigens were shared by three AS patients, 82 autoantigens were shared by four AS patients, 30 autoantigens were shared by five AS patients and three autoantigens were shared by six AS patients (figure 4A). Data from array GST#2 was similarly analysed. Figure 4B shows that 130 autoantigens were detected in at least two AS patients, 18 autoantigens were detected in three AS patients and two autoantigens were detected in four AS patients. Fewer autoantibodies were present in multiple plasma samples in the RA patients and no autoantibodies were found in multiple healthy control individuals from the screening of either the GST#1 or GST#2 arrays.
60% of autoantibodies detected are specific to the AS cohort. We then asked if the detected autoantibodies were largely common between AS and RA patients, or specific to the AS group. From the screening of array GST#1, 482 (62%) were specific to the AS patients ( Figure 5A). The AS and RA cohort shared 256 common autoantigens. Results from the screening of array GST #2 ( Figure 5B) were comparable, with 436 (58%) of these autoantibodies restricted to the AS cohort and 281 shared by the AS and RA groups.

Autoantibodies from AS patients show a bias towards antigens involved in
skeletal and connective tissue disorders. We next asked if autoantigens detected in the AS cohort showed a bias towards any particular biological pathway. For this purpose we used Ingenuity Pathway Analysis © (IPA), which is based on a comprehensive collection of literature data on protein networks. Figure 6A shows that, when compared to the pathways assigned to the 1749 target proteins expressed by the array itself, the autoantigens identified in the AS patients demonstrated a distinct bias towards the pathway involving connective tissue development and function. The GST#1 array expressed 95 proteins involved in connective tissue development and function. Of these, 65 proteins (68%) were detected as autoantigens in the AS cohort. By contrast, only 0-37% of pathway proteins were recognized in the other 26 pathways assigned to the array. This was expressed by IPA as a more significant p-value for connective tissue development and function in the AS autoantigen analysis (p-value = 4.09 x 10 -11 ) compared to the analysis of the entire GST#1 array (p-value = 1.54 x10 -10 ) after correction for multiple comparisons. The same analysis, conducted on results from the independent screening of NAPPA array GST #2, was consistent with the analysis from NAPPA array GST #1, and showed that the autoantigens detected in the AS cohort were specifically biased towards connective tissue disorder and skeletal and muscular disorder pathways (see figure   6B). We next explored whether autoantigens from these significant pathways occurred in multiple AS patients and were restricted to the AS cohort. 83 such proteins were identified. Table 3 lists a subset of these autoantigens, specifically those involved in extra-cellular matrix and bone remodelling.

Discussion
We have used a novel type of protein array screening tool to characterize the autoantibody response in patients with Ankylosing Spondylitis. In total, 44% of AS patients demonstrated a broad autoantibody response, with over 750 reactivities seen at plasma dilutions of greater than 1/170, which is considered clinically significant for autoimmune disease [17]. AS patients demonstrated autoantibody responses to several shared autoantigens, and 60% of the autoantibodies in AS patients appeared specific to that group, in that they were not found in RA or healthy controls. Further evidence of biological relevance is provided by the fact that only autoantibodies from AS patients showed a bias towards autoantigens involved in skeletal and connective tissue. Our studies indicate that NAPPA is a powerful new technique to screen for autoantibodies in human autoimmune diseases. The use of NAPPA arrays has the advantage that large numbers of proteins can be screened and that these proteins are translated and transcribed in a eukaryotic cell extract, promoting proper folding and glycosylation. However, a disadvantage is that antibodies to proteins with posttranslational modifications would not be detected. Similarly, epitopes whose conformation may be membrane-dependent might not be detected.
Autoantibodies are associated with many systemic autoimmune diseases and can be highly specific (e.g. myasthenia gravis) [18] or broadly recognize multiple specificities (e.g. systemic lupus erythematosus) [19]. Our data show for the first time that AS patients' plasma contains multiple autoantibodies recognizing a variety of antigens, with a bias towards proteins expressed in connective tissue.
We propose two possible interrelated mechanisms for this. Firstly production of IL-17 by T cells in AS [20] may directly stimulate B cell maturation and Ig production, as shown for systemic lupus erythematosus by Doreau and colleagues [21]. Secondly, the presence of professional antigen presenting cells together with T and B cells within inflamed areas of connective tissue, as demonstrated histopathologically in AS [22], may lead to autoimmunity in the presence of appropriate cytokine stimulation. Interestingly, we identified and validated Interleukin-6, a cytokine implicated in both AS and the Th17 response, as a target autoantigen. Accumulating evidence from immune mediated diseases supports the hypothesis that the tissue damage caused by immune responses can result in "epitope spreading" following priming of self-reactive lymphocytes. Here presentation of pathogenic epitopes in draining lymph nodes leads to the migration of activated lymphocytes to the site of inflammation, recruiting more phagocytes, and contributing to further tissue destruction. The debris from this destruction can result in extracellular matrix and inflammatory self-proteins being proteolytically digested and their resultant peptides (inappropriately) presented by professional antigen presenting cells. This may lead to the activation of autoreactive T and B lymphocytes, perpetuating the cycle of inflammation and tissue destruction [23].
Autoantibodies to extracellular matrix (ECM) components such as collagen I, II, III, IV and V have previously been reported in AS patients [24,25], and elevated levels of IgA antibodies to keratin proteins have been detected in patients with spondyloarthropathy [26]. Also, higher levels of IgA antibodies have been detected in immune complexes precipitated from AS patient plasma [27]. We were not able to look for autoantibodies to collagen proteins I-IV in the screening of the NAPPA GST#1 and GST#2 arrays, as they were not expressed (Table S1). Unfortunately, the design of this study was not appropriate for the identification of known RA antigens for the following reasons: First, we were not able to identify Rheumatoid factor (an autoantibody against the Fc portion of IgG) in the RA cohort, as IgG proteins were included as positive controls for the function of the secondary antibody in the NAPPA system. Secondly, antibodies to citrullinated proteins and peptides (ACPA) are enzymatically post-translationally modified in vivo during inflammation, and therefore not detected in the NAPPA system. However, we did identify several extracellular matrix proteins as autoantigens in multiple AS patients. These included connective tissue growth factor [28], glypican 3 [29], glypican 4 [30], matrix Gla protein [31], and SMOC1, a protein involved in extracellular matrix assembly [32].
These proteins were all specific to the AS cohort (see Table 3). Furthermore, in addition to proteins involved in extracellular matrix remodelling, proteins involved in ossification and bone remodelling were also identified as autoantigens in multiple AS patients, and were restricted to the AS cohort. For example, chondromodulin is a bone remodelling factor [33] that is thought to function by allowing cartilaginous tissue to be vascularized and replace by bone. The purinergic receptor P2RX7 is involved in ossification [34] and has been shown to regulate bone formation [35]. Similarly, the melanocortin 4 receptor has been shown to increase bone resorption [36].    blue < green < yellow < orange < red; most intense.        IPA Pathways Assigned to NAPPA Array GST#1