Experimental Neurology 287 (2017) 14–20 Contents lists available at ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr Research Paper Identification of KRT16 as a target of an autoantibody response in complex regional pain syndrome Maral Tajerian, PhD a,b,c,⁎, Victor Hung, BSc a,c, Hamda Khan, BSc a,c, Lauren J Lahey, BSc a,c,d,YuanSun,PhDa,b,c, Frank Birklein, MD e, Heidrun H. Krämer, PhD f, William H Robinson, PhD a,c,d, Wade S Kingery, MD a,c,g, J David Clark, MD, PhD a,b,c a Veterans Affairs Palo Alto Health Care System Palo Alto, CA, USA b Department of Anesthesiology, Stanford University School of Medicine, Stanford, CA, USA c Palo Alto Veterans Institute for Research, Palo Alto, CA, USA d Division of Immunology and Rheumatology, Stanford University, Stanford, CA, USA e Department of Neurology, University Medical Center, Mainz, Germany f Department of Neurology, Justus Liebig University, Giessen, Germany g Physical Medicine and Rehabilitation Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA article info abstract Article history: Objective: Using a mouse model of complex regional pain syndrome (CRPS), our goal was to identify autoantigens Received 6 September 2016 in the skin of the affected limb. Received in revised form 12 October 2016 Methods: A CRPS-like state was induced using the tibia fracture/cast immobilization model. Three weeks after Accepted 18 October 2016 fracture, hindpaw skin was homogenized, run on 2-d gels, and probed by sera from fracture and control mice. Available online 20 October 2016 Spots of interest were analyzed by liquid chromatography-mass spectroscopy (LC-MS) and the list of targets val- idated by examining their abundance and subcellular localization. In order to measure the autoantigenicity of se- Keywords: lected protein targets, we quantified the binding of IgM in control and fracture mice sera, as well as in control and Complex regional pain syndrome Autoimmunity CRPS human sera, to the recombinant protein. Antigen identification Results: We show unique binding between fracture skin extracts and fracture sera, suggesting the presence of Keratin 16 auto-antigens. LC-MS analysis provided us a list of potential targets, some of which were upregulated after frac- Skin ture (KRT16, eEF1a1, and PRPH), while others showed subcellular-redistribution and increased membrane local- Chronic pain ization (ANXA2 and ENO3). No changes in protein citrullination or carbamylation were observed. In addition to Animal models of pain increased abundance, KRT16 demonstrated autoantigenicity, since sera from both fracture mice and CRPS pa- tients showed increased autoantibody binding to recombinant kRT16 protein. Conclusions: Pursuing autoimmune contributions to CRPS provides a novel approach to understanding the condi- tion and may allow the development of mechanism-based therapies. The identification of autoantibodies against KRT16 as a biomarker in mice and in humans is a critical step towards these goals, and towards redefining CRPS as having an autoimmune etiology. © 2016 Elsevier Inc. All rights reserved. 1. Introduction understanding of the syndrome, thus often preventing effective treat- ment or cure. Complex Regional Pain Syndrome (CRPS) is a chronic and debilitat- Historically, CRPS research has focused on areas such as sympathetic ing condition comprised of a host of seemingly unrelated signs and nervous system dysfunction, neurogenic inflammation, and peripheral symptoms including bone demineralization, skin growth changes, vas- and spinal nociceptive sensitization and brain reorganization as poten- cular dysfunction and pain. There are an estimated 50,000 new cases tial mechanisms (Tajerian and Clark, 2016). More recently, however, a in the US each year, and in most cases, the symptoms are limited to a collection of clinical and preclinical observations has suggested an auto- single extremity (de Mos et al., 2007). Despite the fact that CRPS was de- immune etiology. Agonistic anti-sympathetic autoantibodies have been scribed over a century ago, we still lack a comprehensive mechanistic shown in a subgroup of CRPS patients (Kohr et al., 2011) and, clinically, low dose intravenous immunoglobulin controlled CRPS symptoms in some patients (Goebel et al., 2010). In an animal model of mild tissue ⁎ Corresponding author at: Anesthesia Service Veterans Affairs Palo Alto Health Care System, 3801 Miranda Ave., Palo Alto, CA 94304, USA. trauma, IgG from CRPS patients worsened the existing nociceptive sen- E-mail address: [email protected] (M. Tajerian). sitization (Tekus et al., 2014). Moreover, in a well-characterized mouse http://dx.doi.org/10.1016/j.expneurol.2016.10.011 0014-4886/© 2016 Elsevier Inc. All rights reserved. M. Tajerian et al. / Experimental Neurology 287 (2017) 14–20 15 tibia fracture/cast model of CRPS, we reported attenuated CRPS-like Table 1 symptoms in subjects treated with anti-CD20 and in mu-MT mice lack- CRPS serum donor information. ing mature B cells (Li et al., 2014). Finally, we observed increased IgM Subject ID deposition in the skin of the affected hindpaw not explained by vascular 12 345 leak of immune complexes, thereby suggesting the presence of auto-an- tigens in skin tissue, which could be responsible for regionalized trophic Age 24 43 44 45 50 Sex Female Female Male Female Male changes, allodynia, and other signs of the syndrome (Li et al., 2014). CRPS duration 4 months 11 months 2 weeks 7 years 4 weeks In the current study we employed the mouse tibia fracture/cast im- CRPS location Right foot Left hand Left hand Right foot Left hand mobilization model to address the critical issue of identifying the target CRPS cause Surgery Fracture Fracture Fracture Contusion antigen(s) of potentially CRPS-related autoantibodies in skin. The iden- Edema Yes Yes Yes No Yes Allodynia Yes No No Yes No tification of such proteins would both support the autoimmune hypoth- Pain NRS 6 9 7 8 8.5 esis and provide biomarkers useful in following disease activity and responses to treatments. NRS = Numeric Rating Scale. 2. Materials and methods 2.4. Gel electrophoresis 2.1. Animals 2.4.1. 2-Dimensional 200 μg of protein was precipitated in acetone, centrifuged at 7800 g, – Male C57/B6J mice aged 12 14 weeks (Jackson Labs) were housed in and the pellet was rehydrated and incubated with the IPG strips (PH 5– groups of 4 (12-h light/dark cycle, ambient temperature of 22 ± 3 °C, 8, 11 cm; BioRad). One-dimensional isoelectric focusing was carried out with food and water available ad libitum). All animal procedures and using an IEF protean cell (Biorad). The IPG strips were then equilibrated experimental designs were approved by the Veterans Affairs Palo Alto and subjected to two-dimensional separation according to standard Health Care System Institutional Animal Care and Use Committee and protocols. followed the “animal subjects” guidelines of the International Association for the Study of Pain. 2.4.2. 1-Dimensional (vertical) Vertical gel electrophoresis was performed (in preparation for West- 2.2. Limb fracture and cast immobilization ern blotting) according to standard sodium dodecyl sulfate polyacryl- amide gel electrophoresis procedures. Mice were anesthetized with 1.5% isoflurane and underwent a distal tibial fracture in the right hind limb. Immediately following fracture and 2.5. Mass spectrometry while still under anesthesia, a cast was placed around the injured hindlimb as previously described (Li et al., 2014). Three weeks after sur- 2.5.1. In-gel digestion fl gery, the mice were anesthetized (iso urane) and the casts were Per standard procedures, gel bands were washed with MilliQ water, removed. destained, dehydrated, and dried in a speed-vac (Thermo Savant). The gel pieces were rehydrated and incubated at 56 °C for 20 min. After discarding the supernatant, the gel pieces were incubated in 15 mM 2.3. Sample preparation iodoacetamide, washed with water and dehydrated and dried as before. The dried gel pieces were rehydrated and the reaction mixture was then All analysis was done blind to the identity and experimental condi- acidified and desalted. Peptides were eluted and lyophilized in a tion of the subject or tissue. SpeedVac (Thermo Savant). 2.3.1. Mice Mice were deeply anesthetized using isoflurane, and blood was Table 2 collected via cardiac puncture. The ventral hindpaw skin was then List of antibodies used. − dissected and stored at 20 °C. Following clotting at room tempera- Antibody Supplier; cat. # Dilution Application ture, blood was centrifuged at 10,000 ×g and the supernatant serum Primary antibodies was collected. Rabbit monoclonal Abcam; 157,455 1:20,000 WB Total protein from skin and serum samples: Skin was homogenized anti eEF1A1 using T-PER Protein Extraction Reagent (Thermo Scientific) with pro- Mouse monoclonal Abcam; 129,007 1:5000 WB teinase and phosphatase inhibitors (Roche Applied Science), centri- anti PRPH Rabbit Polyclonal anti Abcam; 41,803 1:1000 WB fuged at 12,000 ×g, and supernatant fractions were collected. ANXA2 Protein extracts from subcellular fractions: Cytoplasmic, membrane, Rabbit polyclonal anti ENO3 Abcam; 96,334 1:500 WB nuclear soluble, chromatin-bound, and cytoskeletal protein extracts Rabbit monoclonal anti Abcam; 181,602 1:5000 WB from skin samples were separated using a subcellular fractionation kit GAPDH (Thermo Scientific). Rabbit polyclonal anti KRT16 Abcam; 182,791 1:500 WB Secondary antibodies IrDye 800CW Goat anti LI-COR Biosciences; 1:20,000 WB/DB 2.3.2. Human sera mouse IgM 926–32,280 The study protocol was approved by the institutional review board DyLight 800 Goat anti Thermo Fisher; 1:10,000 WB/DB human IgM SA5–10,108 at the Justus Liebig University (Gießen, Germany). Sera were collected IrDye 800CW Goat anti LI-COR Biosciences; 1:20,000 WB from CRPS patients, and 5 serum samples were chosen at random to rabbit IgG 925–32,211 represent both acute and chronic stages of the syndrome (see Table 1 IrDye 800CW Goat anti LI-COR Biosciences; 1:20,000 WB for details; all patients fulfilled the CRPS research criteria).