Introduction
Synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome was first proposed by Chamot et al. [
1] in 1987 as a rare disease that occurs in 30- to 50-year-old individuals. The predominance of individual clinical manifestations varies among patients with SAPHO syndrome [
2]. In 1988, 4 diagnostic criteria for SAPHO syndrome were first proposed by Benhamou [
3,
4], including (1) osteoarticular manifestations with polymeric acne and fulminant acne or septic hidradenitis, (2) osteoarticular manifestations with palmar pustulosis, (3) hyperosteogeny (upper chest wall, extremities, or spine) with or without skin lesions, and (4) chronic multifocal recurrent osteomyelitis (CMRO) involving the axial or peripheral bones with or without skin lesions. Patients with one of the 4 conditions listed above can be diagnosed with SAPHO syndrome. SAPHO syndrome is often unrecognized or misdiagnosed due to its challenging diagnosis caused by the wide variability in musculoskeletal and cutaneous manifestations [
5,
6].
Bone pain caused by severe osteoarticular lesions is one of the most common symptoms prompting SAPHO patients to visit hospitals. Therefore, in some clinical studies, the visual analogue scale (VAS) score, which evaluated the degree of pain, was used to measure the disease activity of SAPHO [
7]. However, pain, especially chronic pain, is influenced by and interacts with physical, psychological, social, and contextual factors. The VAS score does not accurately represent the disease severity. Moreover, the levels of C-reactive protein (CRP) and the erythrocyte sedimentation rate (ESR) are also elevated in most patients, but not in complete accordance with the activity of SAPHO syndrome [
8]. Thus, no specific markers have been identified for the diagnosis or monitoring of the disease status of patients with SAPHO syndrome.
Currently, standardized treatment protocols are not available for patients with SAPHO syndrome. Most treatments are empirical and mainly attenuate the pain associated with SAPHO syndrome. Nonsteroidal anti-inflammatory drugs and analgesics are applied as first-line agents. Anti-rheumatic drugs have also exerted beneficial effects on some patients [
3,
9]. Because of the inhibition of bone resorption and the anti-inflammatory effect, bisphosphonates are clinically used for palliative treatment of patients with SAPHO syndrome. Osteocalcin and β-crosslaps (β-CTx), which can reflect the bone metabolism, have been suggested as an ideal prognostic marker for bisphosphonates treatments. However, they could not reflect the therapeutic effect of the treatment methods other than bisphosphonates. More predictors of the efficacy of treatment with antibiotics, bisphosphonates, or immunosuppressive drugs are available.
The pathogenesis and etiology of SAPHO syndrome are not yet clear; they may be related to heredity, infection, and immunity [
10]. Previous studies have reported multiple dysfunctions of the immune system in SAPHO. For example, according to Nedelec et al. [
11], an infectious state contributing to strong humoral and cellular proinflammatory responses may trigger SAPHO syndrome, and the cellular immune response may also be abnormal. Activation of the Th17 axis, but not the Th1 or Th2 axis, has been observed [
12]. From the perspective of autoimmunity, several previous studies have assessed anti-thyroid peroxidase (TPO), anti-thyroglobulin (Tg), and anti-nuclear antibodies in patients with SAPHO syndrome [
13]. Regardless, these autoantibodies are not specific to SAPHO syndrome and have little significance. Thus, the identification of target autoantigens is important for understanding the etiology of SAPHO syndrome and for the diagnosis and/or monitoring of the disease status.
Multiplex assays have emerged for autoantibody high-throughput screening, enabling the rapid identification of subsets of patients to facilitate diagnostic and predictive medicine [
14]. In this study, a 17K whole-genomic protein microarray was applied to screen the profile of serum autoantibodies in patients with SAPHO syndrome to identify specific biomarkers for the diagnosis or disease status monitoring.
Materials and Methods
Patients and Healthy Controls
Healthy controls (HC), patients with SAPHO syndrome, patients with systemic lupus erythematosus (SLE), and patients with rheumatoid arthritis (RA) were recruited from the Peking Union Medical College Hospital (PUMCH). The committees of both the PUMCH and Chinese Academy of Basic Medical Science approved the use of clinical samples for this project (identifier: ZS-944). The subjects meeting the diagnostic criteria proposed by Benhamou [
4] for SAPHO syndrome were included in this study. The exclusion criteria are as follows: (1) women in pregnancy or lactation, (2) septic osteomyelitis, (3) infectious chest wall arthritis, (4) infections PPP, (5) palmoplantar keratodermia, (6) DISH except for fortuitous association, (7) osteoarticular manifestations of retinoid therapy. The patients with SLE and RA fulfilled the American College of Rheumatology (ACR) criteria for SLE [
15] or RA [
16], respectively, but did not meet the criteria for SAPHO syndrome. The demographic characteristics, osteoarticular symptoms, skin manifestations, and lesion sites of patients with SAPHO syndrome on bone scintigraphy were recorded. Laboratory evaluation included erythrocyte sedimentation rate (ESR, 0–15 mm/h for male and 0–20 mm/h for female), hypersensitive C-reaction protein (hsCRP, 0–3 mg/L), β-CTx (0.21–0.44 ng/mL), and osteocalcin (1.8–8.4 ng/mL) that were also collected. Serum samples were collected, centrifuged at 1000×
g for 10 min, aliquoted, and stored at − 80 °C until use.
Protein Microarray Profiling
17K HuProt™ human whole-proteome microarray slides (CDI, USA) were initially blocked with 3% BSA-TBST buffer at room temperature (RT) for 1 h. The sera from every 5 patients or HCs were mixed as the primary antibody and incubated with the arrays for 1 h at RT. TBST buffer was used to wash the arrays five times. The secondary antibody, a Cy3-conjugated goat anti-human IgG antibody (Biolegend, USA), was diluted and incubated with the arrays for 1 h at RT. The arrays were washed as described above. The primary antibody against the positive control, i.e., a rabbit anti-GST monoclonal antibody (CST, USA), was diluted and incubated with the same microarray slides at RT for 1 h. The slides were washed as described above. The secondary antibody for the positive control, Cy5-labeled goat anti-rabbit IgG (Biolegend, USA), was diluted 1:500 and incubated with the array for 1 h at RT, followed by washing as described above. Three independent protein chip tests were performed, and the chips were scanned using a GenePix 4000B fluorescence microarray scanner (CapitalBio, China). The protein chip data were processed by GenePixPro 5.1 software. The mean value of duplicates was used for data analysis. The signal-to-noise ratio at a wavelength of 532 nm (SNR532) and the ratio of 532 nm to 635 nm (see below) were used for the quantitative analysis of protein spots. The specific equations used for the calculations are as follows: SNR532 = (mean foreground at 532 nm − mean background at 532 nm)/(standard deviation of the background at 532 nm); ratio = (mean foreground at 532 nm − mean background at 532 nm)/(mean foreground at 635 nm − mean background at 635 nm).
The criteria for choosing candidates were as follows: points with SNR532 values over 2, which were found in SAPHO patients but not in HCs, were considered positive points.
Plasmids, Recombinant Proteins, and Gene Cloning
The plasmid used for Sp17 overexpression in eukaryotic cells was constructed with the Gateway Cloning System (Invitrogen, USA) according to the manufacturer’s instructions. An alternate Sp17 plasmid was constructed for Escherichia coli expression by cloning the full-length sequence of the Sp17 gene into the PET-30a easy vector and transforming it into E. coli BL-21(DE3) (TransGen, China). Correct construction of the plasmid was confirmed by DNA sequencing. BL-21 cells were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG; 1 mmol/L; Sigma) at 37 °C for 6 h, and the recombinant Sp17 protein was purified using prepacked HisTrap high-performance columns (GE Health, USA).
To construct a UACA overexpression plasmid, the full-length gene sequence was purchased (Sino Biological, China) and cloned into the cFUGW vector using Phusion DNA polymerase (Biolabs, USA). The experimental procedures were performed according to the manufacturer’s instructions. The correct plasmid was transfected into 293 T cells using Lipofectamine 2000 (Invitrogen, USA).
Western Blotting Analysis
In western blotting studies, 20 μg per lane whole-cell lysate was separated by SDS-PAGE, and the proteins were transferred to NC membranes. The NC membranes were cut into different strips for incubation with sera from different HCs or SAPHO syndrome patients. The primary antibody, sera from HCs or SAPHO syndrome patients, was diluted at 1:100 and incubated at 4 °C overnight. The secondary antibody, goat anti-human IgG (Thermo Fisher, USA), was diluted 1:5000 and incubated with the membrane for 2 h at RT. The NC membranes were combined in imaging steps. Chemiluminescent horseradish peroxidase (HRP) substrate (Pierce, USA) was added to the spliced NC membrane, followed by detection using a chemiluminescence imaging analysis instrument (Clinx, China).
Enzyme-Linked Immunosorbent Assay
Serum total IgG (Abcam, USA) and UACA autoantibody levels (CUSABIO, China) were assessed according to the manufacturer’s instructions.
For the Sp17 autoantibody test, His-tagged recombinant Sp17 (0.2 μg/mL) was coated onto 96-well test plates. Wells without antigen coating were used as blank controls. The plates were washed and blocked with 5% BSA-PBST. Serum samples, as the first antibody, were diluted at 1:300. Wells with anti-His antibody were used as a positive control. Blank wells did not include any reagents, and negative control wells contained phosphate-buffered saline (PBS). The plates were incubated at 37 °C for 1 h and then washed 5 times. HRP-labeled anti-human IgG (Thermo Fisher, USA) was diluted and incubated at 37 °C for 1 h. TMB was added after 5 washes, and the reaction was terminated by adding H2SO4 (0.2 mol/L). Absorbance at OD450 was measured using a microplate reader (Thermo Fisher, USA).
Statistical Analysis
We performed statistical analysis using SPSS 19.0 software (IBM Corp., USA). Independent samples t tests were employed to compare means between the two groups. Analysis of variance (ANOVA) was used for three or more sets of data. Spearman correlation was applied to analyze correlations. The chi-squared test (or Fisher’s exact test if required) was used for categorical variables. Means between different treatment cycles were compared with paired t tests.
Discussion
The major finding of this study is the identification of an anti-Sp17 autoantibody as a potential biomarker for the diagnosis and monitoring of disease activity in patients with SAPHO syndrome. Although the pathogenesis of SAPHO syndrome is multifactorial, immunological dysfunction plays a crucial role in its development [
19]. Total IgG levels in the sera from patients with SAPHO syndrome were elevated compared to HCs, indicating the excessive activation of humoral immunity in the patients with SAPHO syndrome. This finding is consistent with previous findings that SAPHO syndrome is accompanied by remarkably elevated serum IgG4 levels [
20]. We then screened the profile of autoantibodies in the sera of SAPHO patients by using protein chips containing 17,000 human proteins, and two specific autoantibodies against Sp17 and UACA in the sera of SAPHO patients were detected. However, only the anti-Sp17 autoantibody was confirmed in sera from a larger group of SAPHO patients by using ELISA and western blot assays; in contrast, levels of anti-UACA autoantibodies were undetectable by both ELISA and western blot assays. Thus, anti-UACA autoantibodies are not suitable for development as markers.
Sp17 autoantibody levels in the sera from patients with SAPHO syndrome are associated with disease activity, which was confirmed by a correlation analysis of Sp17 autoantibodies with two inflammatory markers: hsCRP and ESR. Indeed, serum levels of Sp17 autoantibodies exhibited a significant positive correlation with serum levels of hsCRP and ESR in patients with active SAPHO syndrome. Importantly, the serum anti-Sp17 autoantibody level may be a better specific marker for the early diagnosis or monitoring of disease activity in patients with SAPHO syndrome than serum hsCRP and ESR levels. In fact, serum hsCRP and ESR levels are elevated in patients with many inflammatory diseases and immunological disorders [
21,
22], but elevated serum levels of anti-Sp17 autoantibodies have not been reported in patients with any other autoimmune diseases.
Sp17 autoantibody levels in the sera from patients with SAPHO syndrome are associated with bone metabolism status, which was confirmed by a correlation analysis of Sp17 autoantibodies with two bone metabolism markers: osteocalcin and β-CTx. Importantly, osteocalcin is a bone-specific calcium-binding protein released during bone formation and resorption by osteoblasts. β-CTx is the main fragment of the type I collagen degradation by osteoclasts. An elevated level means that there is osteolysis and strong bone resorption. Indeed, serum levels of Sp17 autoantibodies exhibited a significant positive correlation with serum levels of osteocalcin and β-CTx in patients with active SAPHO syndrome, which suggested serum levels of Sp17 autoantibodies are associated with osteoarthritis and osteolytic lesions.
Sp17 is a highly conserved mammalian protein, and based on early studies, it is widely believed to be a testis-specific protein that is expressed at high levels during the sperm acrosome reaction. Jong et al. [
23] further validated the distribution of Sp17 isoforms in various tissues using RT-PCR and detected the
Sp17-1a mRNA in the human adrenal glands, lymph nodes, skeletal muscle, spine, ovary, and adult testis, whereas esophageal
Sp17-1a and
Sp17-1b mRNAs have both been detected in PBMCs, the parathyroid gland, and the synovium. Sp17 was recently reported to be a highly immunogenic protein, and Sp17 autoantibodies have been detected in vasectomized men [
24] and patients with periampullary carcinoma [
25]. The findings of the present study provide new insights into the potential pathogenesis of SAPHO. Therefore, further studies should be performed to explore the mechanisms underlying Sp17 targeting by the immune system and its roles in the pathogenesis of SAPHO syndrome.
The key future characteristics of patients with SAPHO syndrome are inflammatory skin and osteoarticular manifestations. Multiple immunosuppressive drugs aiming to alleviate inflammatory symptoms have been used. Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to relieve pain and skeletal injuries. However, in most cases, the effects are transient and the disease relapses after NSAID withdrawal [
26]. As a therapeutic option for SAPHO cases that are unresponsive or refractory to conventional drugs, biological inhibitors targeting the inflammatory mediators IL-1 and TNF-α are effective at improving bone, skin, and joint manifestations. However, populations benefiting from this treatment regimen have not been clearly identified. Patients with a worsening disease or who are unresponsive to anti-TNF-α drugs have been reported [
5]. Because of the inhibition of bone resorption and the anti-inflammatory effect, some studies have used bisphosphonates to control inflammation and pain related to bone resorption. Bisphosphonates significantly and rapidly relieve symptoms in patients with SAPHO syndrome and exert a long-term effect on inflammation and spinal bone marrow edema, which is strongly correlated with musculoskeletal pain [
7]. Similar results were obtained in our study, in which VAS, hsCRP, and ESR levels were markedly decreased after pamidronate treatments. β-CTx showed a significant decrease after the first treatments, while the osteocalcin declined until the second treatment cycle compared with the baseline. Notably, during the treatment period, serum levels of Sp17 autoantibodies decreased continuously in patients with SAPHO syndrome. Serum Sp17 autoantibody was more sensitive for the efficacy of bisphosphonate treatments in SAPHO syndrome than β-CTx and osteocalcin, which further confirmed that the level correlated strongly with the disease activity and inflammatory status of patients with SAPHO syndrome.
In summary, our major finding in this study is the identification of the anti-Sp17 autoantibody as a potential biomarker for patients with SAPHO syndrome. This finding may also provide us with a novel clue for exploring the pathogenesis of SAPHO syndrome.
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