Protein Composition And Function Of Serum Extracellular Vesicles In SAPHO Syndrome

BackgroundSynovitis,acne,pustulosis,hyperosteogeny and osteoarthritis syndrome(SAPHO syndrome)is a rare chronic inflammatory disease.It has osteoarthritis,synovitis,osteoporosis and hyperosteogeny which often involve the anterior chest wall and axial bone,and skin manifestations such as palmoplantar pustulosis and acne.Bone and joint manifestations are the characteristics of SAPHO syndrome,which causes pain,limited activity and stiffness of joints in the later stage of the course,the morphological changes and even collapses seriously affect the quality of life of patients.The pathogenesis of SAPHO syndrome is still unclear.The chronic inflammatory manifestations and bone and joint symptoms of SAPHO syndrome suggest that immune abnormalities and bone metabolic imbalance play an important role in the occurrence and development of SAPHO syndrome.In recent years,with the more and more in-depth study of extracellular vesicles,its role in the regulation of immune and bone metabolism is gradually revealed.Studies have shown that extracellular vesicles are involved in the regulation of bone metabolism and inflammation in physiological and disease conditions.Many inflammatory cytokines are also important regulators of bone metabolism.Extracellular vesicles,which contain various protein components and shoulder the function of cell communication,may play a role as a "bridge" between immunity and bone metabolism in SAPHO syndrome.At present,the research on serum-derived extracellular vesicles(SEVs)of SAPHO syndrome is still blank.MethodsIn this study,SEVs were isolated by ultracentrifugation.After the successful isolation of SEVs was proved by electron microscopy,the quantitative proteomics method was used to analyze the SEVs of SAPHO patients and healthy volunteers,so as to clarify the protein expression profile and differential expression proteins(DEPs)of SEVs in SAPHO patients.Bioinformatics analysis identified DEPs and potential SAPHO disease biomarkers involved in bone metabolism.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression of potential biomarkers in serum and SEVs isolated by reagents of patients and healthy volunteers,and to verify its ability to assist the diagnosis of SAPHO.Further,the levels of SAA1 in SEVs of SAPHO,rheumatoid arthritis(RA)and ankylost spondylitis(AS)patients were compared by ELISA to determine whether SAA1 can be used for the differential diagnosis of SAPHO syndrome,RA and AS.In order to verify the regulatory effect of SEVs on bone metabolism in SAPHO patients,SEVs were extracted by reagent.Western Blot(WB)and Nanoparticle Tracking Analysis(NTA)confirmed the successful isolation of SEVs.The osteoclastogenesis and osteogenesis experiments were conducted with human and mouse cells stimulated by SEVs respectively.The cells with phenotypic changes under the stimulation of different SEVs were analyzed by quantitative proteomics and bioinformatics to find the DEPs,that is,the possible reasons for the phenotypic differences.ResultsThe results showed that there were significant differences in SEVs protein expression profiles between SAPHO patients and healthy volunteers,and 17(35%)of the DEPs were involved in inflammation and bone metabolism.SEVs were extracted from 54 SAPHO patients and 84 healthy volunteers by reagents.Serum Amyloid A-1(SAA1)was found to be highly expressed in the serum and SEVs of SAPHO patients.It has the potential to assist in the diagnosis of SAPHO syndrome.However,there was no significant difference in the expression of SAA1 in SEVs among 54 SAPHO patients,22 RA patients and 29 AS patients.Bone formation experiments showed that SEVs in SAPHO patients had no significant effect on the osteogenesis and osteoclastogenesis processes of cells derived from mice and healthy volunteers.However,in the experiment of inducing differentiation of peripheral blood mononuclear cells(PBMCs)from patients with SAPHO into OCs,the differentiation process was inhibited by SAPHO SEVs in 24 out of 36 cases.Statistical analysis indicated that the occurrence of inhibition was related to the active stage of the patients.Furthermore,quantitative proteomics was used to detect the protein composition changes of OCs stimulated by SEVs from SAPHO pateints and healthy volunteers.It was found that the expression of Nucleolin(NCL)was upregulated in OCs stimulated by SAPHO SEVs,and it was the key node of the interaction network of DEPs.SignificanceIn this study,we reported the protein expression profile and DEPs of SEVs in SAPHO patients for the first time.Among the DEPs,the up-regulated protein SAA1 is an inflammatory marker which is helpful for the diagnosis of SAPHO syndrome.SEVs in SAPHO patients could inhibit the formation and function of OCs in patients in active stage,which may have a negative role in regulating the immune response.NCL is a potential key regulator of OCs generation in SAPHO patients in active stage.