Identification of Autoantibodies against Transthyretin for the Screening and Diagnosis of Rheumatoid Arthritis

Saurabh Sharma,Rajesh Malhotra,Radhey Shyam Sharma,Taposh Kumar Das,Lalit Kumar Singh,Sreejoyee Ghosh,Ashish Sarkar,Yogendra Singh,Darshan Singh Bhakuni,Onkar Prasad Garg,Sagarika Biswas,Anthony Wi Lo

doi:10.1371/journal.pone.0093905

Abstract

Rheumatoid arthritis (RA) is a chronic, autoimmune, systemic and inflammatory rheumatic disease that leads to inflammation of the joints and surrounding tissues. Identification of novel protein(s) associated with severity of RA is a prerequisite for better understanding of pathogenesis of this disease that may also have potential to serve as novel biomarkers in the diagnosis of RA. Present study was undertaken to compare the amount of autoantigens and autoantibodies in the plasma of RA patients in comparison to healthy controls. Plasma samples were collected from the patients suffering from RA, Osteoarthritis (OA), Systemic lupus erythematosus (SLE) and healthy volunteers. The screening of plasma proteins were carried out using 2-dimensional gel electrophoresis followed by identification of differentially expressed protein by MALDI-TOF MS/MS. Among several differentially expressed proteins, transthyretin (TTR) has been identified as one of the protein that showed significantly up regulated expression in the plasma of RA patients. The results were further validated by Western blot analysis and ELISA. In comparison to OA synovium, an exclusive significantly high expression of TTR in RA has been validated through IHC, Western blotting and IEM studies. Most importantly, the increase in expression of TTR with the progression of severity of RA condition has been observed. The autoantibodies against TTR present in the RA plasma were identified using immunoprecipitation-Western methods. The significant production of autoantibodies was validated by ELISA and Western blot analysis using recombinant pure protein of TTR. Hence, these novel observations on increase in TTR expression with the increase in severity of RA conditions and significant production of autoantibodies against TTR clearly suggest that a systematic studies on the role TTR in the pathogenesis of RA is immediately required and TTR may be used as a serum diagnostic marker together with other biochemical parameters and clinical symptoms for RA screening and diagnosis.

Highlights

Rheumatoid arthritis (RA) is a complex, multifactorial, inflammatory autoimmune disease with the anonymous etiology that leads to cartilage destruction, joint deformation and subsequent loss of multiple joints functions which affects 0.5–1% of population [1], [2]
Out of 23 differential protein spots, 9 protein spots were successfully identified by MALDI-TOF MS/MS [Table 2].The identified proteins included haptoglobin (b-chain), haptoglobin alpha 2 chain, alpha 1 microglobulin, serum amyloid, albumin and apolipoprotein which were upregulated in the sera of RA patients as compared to healthy control
Among all the identified proteins in plasma of RA patients, TTR was found to be associated with inflammation [24] and was validated in healthy controls and RA patients using Western blot analysis.The densitometric analysis of Western blots performed (Figure 2A) with plasma of severe RA patients (n = 12) showed 3.6 fold higher TTR levels in RA patients as compared to healthy control (n = 12)

Summary

Introduction

Rheumatoid arthritis (RA) is a complex, multifactorial, inflammatory autoimmune disease with the anonymous etiology that leads to cartilage destruction, joint deformation and subsequent loss of multiple joints functions which affects 0.5–1% of population [1], [2]. The disease severity depends on the degree of the inflammation that is characterized by the increase in number of immune cells such as lymphocytes, monocytes and macrophages leading to the production of different immune mediators [3], [4]. An effective assessment of disease severity is a difficult process in an efficient clinical management of the disease [5]. Diagnosis criteria developed by American College of Rheumatology and European League against Rheumatism are primarily based on clinical symptoms. In the last few years, autoantibody reactivity against autoantigens had been described in RA diagnosis. Autoantibodies have been used as a diagnostic tool for RA, they lack specificity and sensitivity. There is an urgent need to investigate more specific disease associated proteins and autoantibodies, which would aid in better understanding of the disease

Methods

Results

Conclusion