Patterns of inflammation and dysfunction in pSS: identification of proteomic salivary biomarkers correlated with different disease phenotypes.

Background: Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease mainly characterized by dysfunction and inflammation of the exocrine glands. A great deal of variation in the intensity of the infiltrate in minor salivary gland biopsies (MSGBs) and in the salivary flow production has been described in pSS, originating different phenotypes of the disease. Recently, a growing interest has arisen for the discovery of novel diagnostic salivary biomarkers for pSS and its subsets by using 2-DE and MALDI-TOF/MS. However, results from 2-DE analysis are often limited to highly abundant proteins while many proteins at low levels of expressions often elude detection. Moreover, limited data are available regarding the identification of salivary biomarkers that might be potentially associated with different disease phenotypes shedding new light on the mechanisms involved in pSS pathogenesis. Aims of the study: 1) to characterize salivary proteomic biomarkers in pSS by using LC-MS/MS after the removal of the high-abundance proteins in order to broaden the panel of candidate biomarkers for SS. 2) to search for specific candidate biomarkers associated with the phenotypes of the patients, defined on the basis of the degree of lymphocytic infiltration in MSGBs and on the variation of the salivary flow rate. Methods: Unstimulated salivary flow (USF) was collected in standard conditions from 18 patients with pSS (AECG criteria, 2002). Six patients presented a high focus score (FS≥3) and normal salivary flow rate (group A), six patients presented a FS≥3 and an USF rate <1.5 ml/15 (Group B), and 6 patients were characterized by a low focus score (FS<3) and USF rate <1.5 ml/15 (group C). Six healthy volunteers represented the controls. High-abundance proteins were depleted using affinity and immunodepletion methodologies. A high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for the proteomic analysis. Principal component analysis (PCA) was utilized for statistical analysis. Results: We identified a number of differently expressed proteins in the saliva of pSS patients vs controls, including proline-rich proteins, calcium-binding proteins, profilin and other cell motion-related proteins, proteins involved in apoptosis, defence-response proteins and inflammatory-response proteins. More specifically, when compared to healthy volunteers, 7 proteins were significantly decreased and 66 proteins were significantly increased in patients included in the group A. Similarly, when compared to controls, we found that 18 proteins were significantly decreased and 159 significantly increased in the saliva of patients included in the group B whereas, 16 proteins were significantly decreased and 164 significantly increased in patients of the group C. When we used PCA, differentially expressed proteins distinctively discriminate between the four sample groups and especially between healthy volunteers and group B and group C samples. Conclusions: Depletion techniques of high-abundance proteins seem to increase visualization of low-abundance proteins unrevealing novel biomarkers potentially related to specific phenotypes of pSS disease. Once validated, these candidate biomarkers might be useful to better clarify pSS pathogenesis and, ultimately, treatment.