Autoantibodies against citrullinated-lipopolysaccharide binding protein as a novel biomarker in seronegative rheumatoid arthritis

Background A specific feature of rheumatoid arthritis is the presence of citrullinated antigen and production of anti-citrullinated protein autoantibodies (ACPA) which can appear years prior to disease onset to trigger immune responses. In this study, the serum citrulline-containing antigens from RA patients were screened and the significance of the antibodies against citrullinated lipopolysaccharide binding protein (anti-cLBP) was studied. Methods Polypeptides isolated from the serum of patients with RA were identified by the Orbitrap high-precision proteomic technology. And then the citrulline-containing proteins was demonstrated. We synthesized citrullinated LBP peptide based on its richness and possible antigenity. The levels of anti-cLBP were determined in sera of 100 RA, 27 OA, 20 SLE and 50 healthy controls by indirect enzyme-linked immunosorbent assay (ELISA). A total of 11 citrulline-containing antigens were identified in proteins from sera of RA patients. By using citrullinated LBP, the antibodies (anti-cLBP) was detected in RA patients, healthy and disease controls. We found that the levels of anti-cLBP were significantly increased in RA patients. The sensitivity and specificity of anti-cLBP antibody were 28.00% and 95.92%, respectively. In anti-CCP-negative and RF-negative RA patients, the prevalences of anti-cLBP were 19.05% (4/21) and 20.59% (7/34), respectively. In addition, in RA patients of anti-CCP and RF-double negative, anti-cLBP was also detectable in 16.67% (3/18) of the patients. Further analysis of the clinical relevance, we found that anti-cLBP antibody was associated with disease activities in RA. It was noticed that the level of anti-cLBP was closely related with a high incidence of infection in patients with RA. autoantibody novel biomarker in RA, especially in seronegative RA, and associated with disease severity. organ involvements, treatments, history of smoke, infection and metabolic disorders. ESR was evaluated by the Westergren method. Serum levels of immunoglobulins (IgG, IgM and IgA), complements (C3, C4), CRP, and rheumatoid factor (RF)-IgM units; LPS: ESR: erythrocyte sedimentation CRP: C-reaction protein; ASO: Anti-streptococcus hemolysin O; WBC: TG: triglyceride; TCHO: LDL: HDL: lipoprotein; ALT: glutamic-pyruvic transaminase; AST: aspartate amino transferase; ERY: erythrocyte; PRO: protein.


Background
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease that affects 0.5-1.0% of the global population and 0.28% in China [1][2][3]. It is characterized by systemic inflammation resulting in damaged cartilage and bone, leading to restricted movement and disability. Several factors have been proposed to play a role in the pathogenesis of RA, including infection, genetic and hormonal factors [4][5]. Retrospective analyses have shown that ACPAs begin to accumulate in RA patient sera several years before clinical onset of symptoms [6][7] and the detection of ACPAs is the most specific diagnostic test for RA [8]. Importantly, higher ACPA titers correlate with a more severe disease course. Because of the limited sensitivity (67%) and specificity (95%) of ACPAs, RA patients, especially seronegative cases are still facing delayed diagnosis and treatment [9]. We attempted to study potential biomarkers or antigens in serum in RA to facilitate clinical diagnosis.
Serum proteins contains a variety of antigens that may associate with underlying diseases.
Citrullinated polypeptides incorporated into serum proteins are potential candidates for diagnostic biomarker screening in RA. At present, proteomics with mass spectroscopy has been applied to the exploration of novel biomarkers in vaious diseases [10]. Van Beers et al. applied it in RA synovial fluid and identified citrullinated apolipoprotein E as a novel autoantigen in RA, which suggested that apolipoprotein E might play an important role in the pathogenesis of RA [11].
In this study, we applied high resolution mass spectrometry with nano-LC combined with Orbitrap Q Exactive mass spectrometer to identify novel citrullinated autoantigens present in the serum of RA patients. The autoantibody against citrulline-containing lipopolysaccharide binding protein (cLBP), one of the identified citrullinated proteins, is significantly increased in RA, especially in seronegative patients and associated with disease severity and infection incidence in RA patients.

Study population and serum samples
Serum samples were obtained from 100 patients with RA [12].
OA patients were grouped according to the criteria of the 1995 ACR criteria [13][14][15]. The 2009 SLICC revision of the ACR classification criteria for SLE [16] was used for the diagnosis of SLE.
The healthy volunteers were free from any symptoms of infections. The study was approved by the Ethics Committee of Peking University People's Hospital according to the declaration of Helsinki.
All patients had been informed and signed the consent for participation in the study. antiperinuclear factor (APF) and RF-IgG were tested by indirect immunofluorescence assay. Anticitrullinated peptide (anti-CCP) antibodies and glucose phosphate isomerase (GPI) were tested by ELISA. The 28-joint count Disease Activity Score (DAS28) was evaluated as described [17]. The AU value was considered positive if it was greater than the mean+2×standard deviation of the healthy group.

Data analysis
Data analyses were performed using SPSS 19.0 for Windows. The distribution of numerical data was evaluated by Shapiro-Wilk test. Numerical data with normal distribution were expressed as the mean ± standard and differences between two groups were analyzed by independent -test.
Numerical data with skewed distribution were expressed as median (P25, P75) and differences between two groups were analyzed by Mann-Whitney test. Ranked data were expressed as percentage and differences between two groups were analyzed by chi-square test. Spearman's rank correlation coefficient was applied to determine the correlations. A difference between groups was considered to be significant if p < 0.05.

Identification of citrulline-containing proteins in serum of rheumatoid arthritis
Eleven antigens derived from serum of RA patients are identified and summarized in Table 2.
These proteins contain at least one citrulline and are involved in immune response (complement C3), blood pressure (Angiotensinogen) and lipid transportation (apolipoprotein B-100 precursor).
Eight of the above 11 proteins are possible novel antigens in RA which have not been previously reported ( Table 2). Apolipoprotein A-I, complement C3 and angiotensinogen have been identified in RA synovial fluids.

Autoantibodies to citrullinated lipopolysaccharide-binding protein peptide were increased in RA
To validate the significance of the potential autoantigen epitopes derived from serum proteins, we selected citrulline-containing lipopolysaccharide-binding protein (cLBP) with sequence as CALQSELL-Cit-ITLP, among which 5 amino acids (RITLP) are predicted to be a B cell epitope covering the citrulline site identified by mass spectrometry ( data from IMMUNE EPITOPE DATABASE AND ANALYSIS RESOURCE, http://www.immuneepitope.org).
Serum levels of autoantibodies against the cLBP were determined by indirect ELISA in RA patients, disease and healthy controls. The antibodies against citrulline-containing LBP (anti-cLBP) were significantly elevated in RA than those from healthy controls, SLE and OA patients ( Figure 1 and Table 3, p<0.05). The sensitivity and specificity of anti-cLBP antibodies in the diagnosis of RA were 28.00% and 95.92%, respectively (Table 3). Surprisingly, the prevalence of anti-cLBP was 20.59% (7/34) or 19.05% (4/21) in RF negative or anti-CCP negative RA patients respectively. Three out of 18 (16.67%) patients with high anti-cLBP were found from 18 patients with both anti-CCP and RF negative. These results revealed that anti-cLBP antibody might be a valuable biomarker when there is a need of distinguishing serological RA patients from patients with arthralgia caused by other autoimmune diseases.

Anti-cLBP antibody was associated with disease activity in rheumatoid arthritis
As shown in  Table 6, at the end of the article), which indicated that anti-cLBP antibodies were positively associated with the inflammation and disease activity of RA. Further analysis suggested that anti-cLBP antibodies were positively correlated with the levels of anti-CCP, IgG and IgA (p<0.05, Table 4 and Figure 2 B-D). We found that RA patients with higher serum levels of anti-cLBP antibodies showed higher anti-CCP, RF, APF positivity, AKA positivity and HRF-IgG (p<0.05, Table 5), and patients positive for RF, AKA, APF, anti-CCP or HRF-IgG had higher levels of anti-cLBP antibodies (p<0.05, Table 6), which suggested that anti-cLBP antibodies were positively associated with autoantibody production.

Anti-cLBP antibody was increased in RA patients with infection
As shown in Table 5, RA patients with higher anti-cLBP antibody levels showed increased infection incidence than patients with normal anti-cLBP levels ( Table 6).
To determine whether the association of anti-cLBP antibodies with infection is specific or not, we examined the association between incidence of infection in RA patients and the positivity of other RA associated autoantibodies including anti-CCP, AKA, APF, HRF and RF, and no significant association was found, which showed that anti-cLBP was the only indication of infection (Table 7).

Discussion
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic and erosive polyarthritis caused by abnormal growth of synovial tissue, and leads to irreversible joint disability.
The finding of ACPAs is a milestone in the history of RA serology [18]. In this study, we applied high resolution mass spectrometry to screen serum proteins incorporated citrulline modifications from RA patients. A total of 11 citrulline-containing proteins were identified in the serum of RA patients, including LBP, Apo A-I, et al ( Table 2). Eight of the proteins are novelly identified in RA, which have not been previously reported. Apolipoprotein A-I and its citrulline epitope have been reported in RA synovial fluid [11]. The citrulline-containing Complement C3 and angiotensinogen have also been identified in RA patients in previous studies, but the citrulline epitopes identified in this study have not been reported. These proteins were involved in physiological process including immune response, blood pressure and lipid transportation. Two (Apo B-100p and C3) of the identified polypeptides appeared to be citrullinated on more than 1 position (Table 2). Also, it is noteworthy that this set could not contain the whole collection of citrulline containing polypeptides in RA, not only because the MS data only partially covered the polypeptide sequences, but also because of the heterogeneity of citrullination patterns for different RA patients.
Besides the identification of a series of citrulline-containing proteins, we further found that the levels of anti-cLBP were significantly increased in RA patients, especially in seronegative patients.
We also confirmed that anti-cLBP antibody was associated with RA disease activity. This result confirmed the feasibility and reliability of protein analysis on novel citrulline-containing antigens in RA.
LBP, a class I acute-phase protein, could mediate innate immune responses after recognizing lipopolysaccharides (LPS) from gram-negative bacterium [19][20]. LBP could form a high-affinity complex with LPS, then LPS was delivered to cell through CD14 or TLR4-MD2 and triggered a cascade of cytokines and pro-inflammatory factors [21]. Previous studies suggested that serum LBP in sepsis significantly elevated almost seven times higher than normal levels [21]. LBP not only exerts a diagnostic value in sepsis, UTI, periodontitis, etc., but also is related to the prognosis and mortality of some types of infections [22][23][24]. In our study, we found correlation between anti-cLBP antibody and infections. Previous study showed serum LBP level was elevated in RA. The incidence of infection is higher in LBP-positive RA patients than in LBP-negative cases, but there was no statistical difference [21]. LBP can promote infection immunity, contribute to eliminating pathogens, and control the further occurrence of infection. However, anti-cLBP antibody may interfere with LBP normal physiological function. Therefore, RA patients with elevated anti-cLBP antibody are prone to infection. It is necessary to elucidate the pathological roles of anti-cLBP antibody in RA in future investigations.
However, there were obvious limitations in this study. First, the number of RA patients recruited from our clinical center was limited in this study, and it might restrain the demonstration of clinical correlation of anti-cLBP antibodies due to lack of statistical power. It will be important to recruit more RA patients from multiple clinical centers in our future studies. Another weakness of this study is that we did not reveal the exact pathogenesis of anti-cLBP in RA, especially how it affected immune responses during infection, which should also be revealed in the future investigations.
In conclusion, this study has successfully set up the protein analysis method for RA associated autoantigens by high resolution mass spectrometry, and identified 11 citrulline-containing proteins in the serum of RA patients. Eight of the proteins are novel antigens in RA, which have not been reported before. Two (Apo B-100p and C3) of 11 citrulline-containing proteins appeared to be citrulline on at least 1 position. The identified epitopes of citrulline-containing proteins in RA will be helpful for the development of assays for ACPA profiling. Such profiles may benefit the ACPApositive patients with RA concerning disease responsiveness to some kinds of treatments. The biologic function of many proteins will be affected because of citrullination, and it will be promising to investigate to what extent this contributes to pannus formation and cartilage and bone damage.
And we also found that anti-cLBP antibody was elevated in RA, especially in seronegative patients, and correlated with disease activity and infection, which implicated anti-cLBP might be involved in the infection of RA.

Conclusions
We had set up the protein analysis method for RA associated autoantigens by high resolution mass spectrometry, and identified 11 citrulline-containing proteins in the serum of RA patients. And we revealed that anti-cLBP autoantibody is a novel biomarker in RA, especially in seronegative RA, and associated with disease severity.  For normally distributed data, results were expressed as mean ±SD; differences between groups were analyzed with the t test. Data not distributed normally expressed as median (range), differences were analyzed with the Mann-Whitney U-test. Classified data were presented as n (%) and analyzed by chi-square (χ 2 ) test. P < 0.05 were considered statistically significant. ERY: urine erythrocyte; PRO: urine protein.