Proteomic analysis identified salivary immunoglobulin gamma-3 chain C as a potential biomarker for systemic lupus erythematosus CURRENT

Background Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies and systemic inflammatory response. We aimed to characterize the salivary protein components and find biomarkers in patients with SLE. Methods The pooled salivary proteins of patients with SLE and healthy controls were subjected to 2-dimensional gel electrophoresis. The spots exhibiting > 2-fold intensity change between SLE and healthy controls were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. The proteomic analysis using 2-dimensional gel electrophoresis and mass spectrometry revealed 10 differentially expressed protein spots, which included immunoglobulin gamma-3 chain C region (IGHG3), immunoglobulin alpha-1 chain C region (IGHA1), protein S100, lactoferrin, leukemia-associated protein 7, and 8-oxoguanine deoxyribonucleic acid glycosylase. The patients with SLE exhibited enhanced salivary IGHG3 (3.9 ± 2.15 pg/mL) and lactoferrin (4.7 ± 1.8 pg/mL) levels than patients with rheumatoid arthritis (1.8 ± 1.01 pg/mL and 3.2 ± 1.6 pg/mL, respectively, p < 0.001 for both) or healthy controls (2.2 ± 1.64 pg/mL and 2.2 ± 1.7 pg/mL, respectively, p < 0.001 for both). The salivary IGHG3 levels correlated with erythrocyte sedimentation rate (r = 0.26, p = 0.01), anti-double-strand deoxyribonucleic acid antibody levels (r = 0.25, p = 0.01), and nephritis (r = 0.28, p = 0.01). The immunoglobulin gamma-3 chain region (IGHG3), (IGHA1), protein S100-A8 (S100A8), lactoferrin, and 8-oxoguanine DNA glycosylase (OGG1) were analyzed by western blotting using the rabbit anti-human IGHG3 polyclonal (MBS248789, MyBiosource, San Diego, CA, USA), rabbit anti-human IGHA1 polyclonal (MBS9206028, MyBiosource), rabbit anti-human rat S100A8 polyclonal (MBS127619, MyBiosource), mouse anti-human lactoferrin monoclonal (ab10110, Abcam, Cambridge, UK), and rabbit anti-human OGG1 polyclonal (NB100-106, Novusbio, Centennial, CO, USA) antibodies, respectively. The proteins were subjected polyacrylamide gel electrophoresis using 10% (for IGHG3) or 15% (for IGHA1, S100/A8, lactoferrin, and OGG1) gel. The resolved proteins were transferred to a polyvinylidene fluoride membrane. The membranes were incubated with secondary antibody (goat anti-rabbit antibody A120-101P for IGHG3, IGHA1, S100A8, and OGG1, and goat anti-mouse antibody for lactoferrin, Bethyl Laboratories, Mongomery, TX, USA) diluted at 1:10,000 (IGHG3 and S100/A8), and 1:2,000 (IGHA1, lactoferrin, and OGG). All analyses were performed in triplicates. The protein concentration was determined by the optical density of specific immunoreactive bands, and the optical density of each bands was measured using image J software (NIH, Bethesda, MD, USA). assay; ESR, erythrocyte sedimentation rate; HCs, healthy controls; IGHA1, immunoglobulin chain C region; IGHG3, immunoglobulin gamma-3 chain C region; LTF, lactoferrin; LN, lupus nephritis; MS, mass spectrometry; OGG1, 8-oxoguanine DNA glycosylase; RA, rheumatoid arthritis; RBC, red blood cells; ROC, receiver operating characteristic; S100A8, protein S100-A8; SLE, systemic lupus erythematosus; SLEDAI; SLE disease activity index, SS, Sjogren’s syndrome.

biomarkers are promising candidates for the diagnosis of SS, which is an autoimmune disorder involving salivary gland and shares several common characteristics with SLE [16,17]. Proteomic analysis revealed a differential salivary protein composition between primary and secondary SS [18].
The levels of salivary α-enolase, β-2 microglobulin, and immunoglobulin kappa light chain are different between patients with SS and those with other autoimmune diseases exhibiting sicca symptoms.
Currently, there are limited studies that have analyzed the saliva samples of patients with SLE. In this study, we analyzed the composition and concentration of salivary proteins in patients with SLE by 2-DE with MS. The clinical relevance of the differentially expressed proteins was analyzed in patients with SLE.
The information on medical history and clinical manifestations was collected from a chart review and blood test results, such as complete blood count, erythrocyte sedimentation rate (ESR) and the levels of anti-nuclear antibody, complement 3 and 4, and anti-dsDNA antibody. The patients with RA were enrolled as a disease control to analyze the differential expression of specific proteins between SLE and RA, which are both chronic autoimmune diseases.
Basic characteristics of second participant group is here: the mean age of patients with SLE, patients with RA, and HCs was 39.8 ± 9.8, 41 ± 7.9, and 39.5 ± 6.9 years, respectively. The mean age was not different between the three groups. Among the patients with SLE, 41 patients (43.6%) tested positive for anti-dsDNA antibody, 28 patients (29.8%) had mucocutaneous symptoms, 31 patients (33.0%) had arthritis, and 29 patients (30.9%) had nephritis. The mean SLE disease activity index (SLEDAI) was 3.8 ± 4.2. Among the 57 patients with RA, 45 patients (77.6%) tested positive for rheumatoid factors and their mean disease activity score including 28 joints (DAS28) was 3.3 ± 1.15.

Saliva sample collection
As salivary proteins exhibit diurnal variations, the saliva samples were collected from all participants between 9:00 and 11:00 am. The subjects were not allowed to eat, drink, smoke, or perform oral hygiene procedures for at least 1 h prior to the sample collection. The saliva samples were collected for 5 min after the subjects rinsed their mouth with water [20]. The saliva secretion was not stimulated in the study subjects. The subjects were asked to keep their mouths closed and expectorate the saliva into a tube once per minute. Each saliva sample was immediately treated with the protease inhibitors to preserve the integrity of the protein constituents. The saliva samples were centrifuged at 3,000 rpm for 15 min at 4°C. After removing the clear supernatant, the samples were aliquoted and stored at -20°C until further use.

Two-dimensional gel electrophoresis (2-DE)
The samples from 11 patients with SLE or 11 HCs were pooled equally to avoid intra-class variations that were detected between the patients in 2-DE analyses. A 1 mL aliquot of the sample was concentrated 10 times using the Amicon-3K centrifugal filters at 14,000 g and 4°C for 20 min. The proteins in the salivary samples were precipitated using 500 μL of trichloroacetic acid/acetone (90%; v/v)-dithiothreitol mixture overnight at -20°C. The samples were centrifuged at 10,000 rpm and 10°C for 10 min. The supernatant was collected and the samples were pretreated with 250 μL of rehydration buffer. Next, the samples were centrifuged at 10,000 rpm and 10°C for 10 min to remove any insoluble material. The protein concentration of the samples was estimated by Bradford protein assay (Bio-Rad, Hercules, CA, USA).

Liquid chromatography tandem mass spectrometry (LC-MS)
With 2-DE proteomic analysis of the saliva samples of 11 patients with SLE and 11 HCs, the proteins separated into numerous spots with different concentrations. The proteins in 20 spots were subjected to liquid chromatography tandem-mass spectrometry (LC-MS) to analyze the proteins with high specificity [21]. The gel pieces containing the protein spots were destained, reduced, alkylated, and digested with modified sequencing grade trypsin (Sigma, MO, US A), as previously described [22].
Peptide mixtures were lyophilized and stored at -80°C for further LC-MS analysis. The data were manually examined, and the overlapping peaks were discarded (additional file 2). The threshold level for differentially expressed proteins was defined as at least 2-fold increase or decrease in spot intensity that was statistically significant. The MS spectra of the peptide peaks were searched against the Uniprot Human database using the Mascot version 2.3 (Matrix Science, London, UK). For quantitative protein profiling, only the proteins identified by multiple peptides with significant MASCOT score (p < 0.05) were considered.

Western blotting analysis
and clinical features in patients with SLE were determined using the Spearman's rank correlation coefficient. On the receiver operating characteristic (ROC) curve analysis of salivary proteins, area under curve (AUC), sensitivity, and specificity were calculated. The difference was considered statistically significant when the p value was less than 0.05. All statistical analyses were performed in the Statistical Package for the Social Sciences version 22.0 (IBS Corp, Armonk, NY, USA) and SAS9.4 (SAS institute Inc, Cary, NC, USA).

Salivary protein identification
The 2-DE analysis revealed a differential salivary protein expression pattern between patients with SLE and HCs ( Fig. 1). The protein identity, fold change, and peptide sequence between the two groups were determined by LC-MS analysis and quantitative protein profiling. Among the 10 spots exhibiting fold change values higher than 2, two spots were identified as alpha-amylases, protein S100, and OGG1 ( Table 2). The other spots were identified as IGHG3, IGHA1, lactoferrin, and leukemiaassociated protein 7.

Salivary IGHG3 and lactoferrin in SLE
The salivary levels of IGHG3 and lactoferrin were measured using ELISA (Fig. 3). The ROC curve analysis of salivary IGHG3 and lactoferrin revealed that the AUC was 0.78 (95% confidence interval 0.72-0.84) and 0.79 (95% confidence interval 0.73-0.85), respectively (Fig. 4). The sensitivity and specificity of salivary IGHG3 were 67.3% and 76.8% with a cut-off value of 2.37 pg/mL for the diagnosis of SLE, respectively. The sensitivity and specificity of salivary lactoferrin were 77.3% and 73.9% with a cut-off value of 4.36 pg/mL for the diagnosis of SLE, respectively.

Discussion
The 2-DE with MS proteomic analysis of saliva revealed that the densities of ten spots were significantly different between patients with SLE and HCs. These spots were identified as IGHG3, IGHA1, protein S100, lactoferrin, OGG1, and leukemia-associated protein 7. Immunoblotting analysis revealed that the expression levels of salivary IGHG3 and lactoferrin in patients with SLE were significantly higher than those in patients with RA and HCs. Additionally, the salivary IGHG3 levels correlated with the ESR, anti-dsDNA antibody, and nephritis in patients with SLE.
The role of IgG in the pathogenesis of SLE is well known. The interaction between anti-dsDNA IgG antibody and pleural mesothelial cells induces the synthesis of proinflammatory cytokines, which is the pathogenic mechanism underlying serositis in SLE [23]. Elevated level of serum IgG is a poor prognostic factor for autoimmune hepatitis associated with SLE. Additionally, the deposition of IgG is reported to cause tissue damage [24,25]. The serum IgG levels correlated with the serologic activity and can predict disease flares in patients with LN. Additionally, IgG induces the expression of calcium/calmodulin-dependent protein kinase IV, which is highly expressed in the podocytes of patients with LN causing renal damage [26,27]. IGHG3 is a constant region of immunoglobulin heavy chains. IGHG3 enables the binding of IgG to the Fcγ receptor (FcγR) of neutrophilic granulocyte and macrophages. FcγRI on the surface of dendritic cells promotes the antigen presentation of dendritic cells to the T cells [28]. Aberrant expression of FcγR for IgG was observed in patients with arthritis and SLE. FcγR is involved in the antigen presentation and immune-complex-mediated maturation of dendritic cells, regulation of B-cell activation, and plasma cell survival in SLE [29]. The role of FcγR and the effect of its gene polymorphisms on the susceptibility or manifestations of SLE has been previously studied [30][31][32]. A study on patients with autoimmune hemolytic anemia demonstrated that the levels of IGHG3 in the red blood cells (RBC) were associated with the frequency of RBC transfusion after diagnosis [33]. The enhanced expression of IGHG3, an immunoprotein, in RBC may result in severe hemolysis. Our ROC analysis suggested that the salivary IGHG3 had reliable specificity and sensitivity to differentiate the patients with SLE from patients with RA and HCs. The mechanism underlying elevated salivary IGHG3 levels in patients with SLE must be investigated further. However, our results suggest that salivary IGHG3 may be a differential diagnostic biomarker for SLE. Moreover, salivary IGHG3 levels correlated with the disease activity markers, such as the ESR and anti-dsDNA antibody levels. Additionally, the levels of IGHG3 were significantly elevated in patients with LN.
Lactoferrin, also known as lactotransferrin, is a multifunctional glycoprotein that belongs to the transferrin family. Lactoferrin expression is detected in the mucosal secretions and secondary granules of polymorphonuclear leukocytes [34]. Lactoferrin not only plays an important role in protection against microorganisms, but also in immunomodulation, inflammation, and anticancer activity through its interactions with the host immune system [35,36]. Lactoferrin-specific IgG autoantibodies were detected in the serum of patients with SLE or RA [37]. The release of surfaceexpressed lactoferrin from the polymorphonuclear neutrophils modulates the cytokine production in the T helper cell type 1 (Th1). The decreased expression of lactoferrin in patients with SLE is associated with abnormal Th1/Th2 production [38].

Conclusions
This work revealed the differential salivary protein composition in patients with SLE and RA and HCs.
The levels of salivary IGHG3 and lactoferrin in patients with SLE were significantly higher than those in patients with RA and HCs.

Ethics approval and consent to participate
This study was conducted according to the Declaration of Helsinki and Good Clinical Practice guidelines. All subjects provided their informed consent for participating in the study. The study protocol was approved by the institutional review of board of our hospital (BMR-SMP- .

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Additional Files
Additional file 1. Supplementary Fig. 1       Concentrations of salivary immunoglobulin gamma-3 chain C region in patients with lupus nephritis The levels of salivary immunoglobulin gamma-3 chain C region (sIGHG3) patients with lupus nephritis was significantly higher than that in patients without lupus nephritis. All measurements were performed in duplicates.

Supplementary Files
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