Expression of SALL4 in the synovial tissue of refractory rheumatoid arthritis patients

Rheumatoid arthritis (RA) synovitis is characterized by synovial inflammation, characterized by synovial proliferation, neovascularization, and leukocyte infiltration into the joint space,1 leading to irreversible cartilage and bone damage.2 This synovial inflammation stimulates the formation of pannus, which is an invasive tumor-like mass, ultimately contributing to the pathogenesis of RA. RA shares several similarities with cancer, including joint pain, swelling, and stiffness, which are accompanied by hypoxia in the synovial tissue and tumor environment.3 Spalt-like transcription factor 4 (SALL4) plays an essential role in the occurrence and development of tumors.4 However, SALL4 expression in synovial tissue of RA is yet to be investigated. This study aimed to investigate SALL4 expression in synovial tissue derived from patients with RA, using immunohistochemical techniques. Nineteen patients with RA consented to enrollment to donate synovial tissue for the experiment. All the patients (5 males, 14 females) with an average age of 53 (29–66) years met the 2010 standards of the American College of Rheumatology (Supporting information: Table S1). The disease activity score 28 (DAS28) scores of all the patients were higher than 2.6, including one patient with low disease activity (2.6–3.2), eight patients with moderate disease activity (3.3–5.1) and ten patients with high disease activity (>5.1) (Supporting information: Table S2). Refractory RA is the disease that has not been relieved after combined treatment with multiple immunosuppression and the disease is still in high activity after treatment. The detailed clinical features and treatment background of the patients are shown in Supporting information: Tables S1–S3. SALL4 expression in the synovium was detected by immunohistochemical methods. We divided 19 patients into SALL4-positive and SALL4-negative groups according to SALL4 expression or not. The clinical characteristics of patients were compared based on SALL4 expression in synovial tissue. Synovial tissue was procured from patients with RA and preserved in a 10% formalin solution. Following this collection, the synovial tissue underwent a routine protocol of tissue grossing, processing, and sectioning. The slides were stained with hematoxylin and eosin, and two slides of each case were stained for immunohistochemical markers of SALL4. To perform immunohistochemistry, blocks were stained with a mouse monoclonal antibody kit (Bio-SB Company, CA, USA) using the Dako Agilent Autostainer Link 48 slide stainer (Agilent Technologies, Beijing, China). The slides were examined for histopathological features of the biopsy and immunohistochemical characteristics of intraepithelial lymphocytes. Data were statistically analyzed using SPSS 27.0 software (IBM Corp, NY, USA). Data with a normal distribution and homogeneity of variance are expressed as the mean ± standard deviation, and an independent sample t-test was used for comparison between the two groups. A value of p < 0.05 indicates a statistically significant difference. There were 19 patients in this study, three of whom had positive SALL4 expression. The overall positivity rate of SALL4 was 15.8%. Remarkably, among the treatment-resistant patients with RA, three individuals showed SALL4 expression in synovial tissue (Figure 1A–C). These three individuals did not respond to multiple disease-modifying antirheumatic drug treatments, which ultimately led to knee arthroplasty as a definitive intervention. In contrast, the remaining patients with RA showed no SALL4 expression in synovial tissue (Figure 1D–F). SALL4 is expressed in tumor cells, and tumor is a proliferative disease, so SALL4 is associated with proliferation. RA is essentially a proliferative disease, which is mainly characterized by synovial hyperplasia, so we believe that SALL4 is expressed in the synovial tissue of RA patients. Our experiment provided preliminary results. However, the mechanism of how SALL4 affects the abnormal proliferation of RA synovial tissue is unknown. SALL4 expression in the intracellular matrix is not consistent with previous studies. Two previous studies examined SALL4 expression in breast and lung cancer and showed that SALL4 was mainly found in the cytoplasm with a diffused distribution in the nucleus.5, 6 We speculate that SALL4, as a transcription factor, may be able to shuttle between the nucleus and cytoplasm. Previous studies have suggested that SALL4 is expressed in the nucleus and cytoplasm, but our study found that SALL4 only exists in the cytoplasm. For this difference, we speculate that the expression of SALL4 in the nucleus interferes with cell division and proliferation at the genetic level, making the tumor cells proliferate indefinitely. In RA patients, SALL4 is only expressed in the cytoplasm, so synovial proliferation is different from the unlimited proliferation of tumor cells. Interestingly, in our study, patients with SALL4 showed higher levels of erythrocyte sedimentation rate, rheumatoid factors-IgA, rheumatoid factors-IgG, and DAS28 than those with negative SALL4 expression (all p < 0.05). These findings suggest an association between SALL4 expression and the production of RA antibodies and disease activity. In summary, our study provides evidence that SALL4 expression is present in the synovial tissue of certain patients with refractory RA, suggesting a role of SALL4 in the development of refractory RA. These findings offer a new perspective for the diagnostic evaluation of refractory RA. Further validation using a larger sample size and additional experiments, such as immunohistochemistry, are necessary to confirm the clinical significance of SALL4 levels in refractory RA. All authors were involved in the drafting of the article or critical revision of the important intellectual content and approved the final version to be published. We thank the medical staff of the Rheumatology and Immunology Department of the Second Affiliated Hospital of Shanxi Medical University for their support and help, the Science and Technology Department of Shanxi Province for their support, the patients who participated in the study, and the anonymous reviewers for their valuable suggestions. This study was funded by the Shanxi Provincial Department of Science and Technology (Grant/Award Number: 201803D421072). The authors declare no conflict of interest. The authors certify that they have obtained all appropriate patient consent forms. The study was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.