Identification of potential saliva and tear biomarkers in primary Sjögren's syndrome, utilising the extraction of extracellular vesicles and proteomics analysis

Abstract

Background: There is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sjögren's syndrome (pSS) patients. Incorporation of additional diagnostics involving screening for disease-specific biomarkers in biological fluid is a promising concept that requires further investigation. In the current study we aimed to explore novel disease biomarkers in saliva and tears from pSS patients.

Methods: Liquid chromatography-mass spectrometry (LC-MS) was performed on stimulated whole saliva and tears from 27 pSS patients and 32 healthy controls, and salivary and tear proteomic biomarker profiles were generated. LC-MS was also combined with size exclusion chromatography to isolate extracellular vesicles (EVs) from both fluids. Nanoparticle tracking analysis was conducted on joint fractions from the saliva and tears to determine size distribution and concentration of EVs. Further EV characterisation was performed by immunoaffinity capture of CD9-positive EVs using magnetic beads, detected by flow cytometry. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold, and the proteins were further analysed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), for gene ontology overrepresentation, and the Search Tool for the Retrieval of Interacting Genes/Proteins for protein-protein interaction network analysis.

Results: Upregulation of proteins involved in innate immunity (LCN2), cell signalling (CALM) and wound repair (GRN and CALML5) were detected in saliva in pSS. Saliva EVs also displayed biomarkers critical for activation of the innate immune system (SIRPA and LSP1) and adipocyte differentiation (APMAP). Tear analysis indicated overexpression of proteins involved in TNF-α signalling (CPNE1) and B cell survival (PRDX3). Moreover, neutrophil gelatinase-associated lipocalin was upregulated in saliva and tears in pSS. Consistently, DAVID analysis demonstrated pathways of the adaptive immune response in saliva, of cellular component assembly for saliva EVs, and of metabolism and protein folding in tears in pSS patients.

Conclusions: LC-MS of saliva and tears from pSS patients, solely and in combination with size-exclusion chromatography allowed screening for possible novel biomarkers encompassing both salivary and lacrimal disease target organs. This approach could provide additional diagnostic accuracy in pSS, and could possibly also be applied for staging and monitoring the disease.

Keywords: Adaptive immunity; Autoimmunity; Biomarkers; Extracellular vesicles; Inflammation; Innate immunity; Proteomics; Saliva; Sjögren’s syndrome; Tears.

Fig. 1

Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis delineating cellular pathways that involve proteins identified in whole saliva, tear fluid, and extracellular vesicles (EVs). Cellular pathways involving innate and adaptive immune responses, cellular component assembly, metabolism and protein folding were identified using DAVID (v 6.7, https://david.ncifcrf.gov) analysis for each sample of whole saliva, tear fluid and EVs

Fig. 2

Protein-protein interaction networks of upregulated proteins associated with primary Sjögren’s syndrome identified in stimulated whole saliva. Two distinct protein-protein interaction networks are visualised. One is involved in metabolism and redox reactions, while the other plays a central role in both innate and adaptive immunity and contains the most upregulated protein in the patient group, namely neutrophil gelatinase-associated lipocalin (LCN2). The five most upregulated proteins in the patient group (Table 3) are indicated with red circles. The Search Tool for the Retrieval of Interacting Genes/Proteins (http://string-db.org/) was used to generate the networks, where potential interactions of proteins with medium confidence are shown. The different clusters are indicated by the same colour. The colour of the connecting lines indicates the type of evidence used in predicting the associations (red gene fusion, yellow text-mining extracted from literature, purple protein-protein interaction datasets, light blue protein interaction groups, black linked across species). CALM calmodulin, CALML5 calmodulin-like protein 5, GRN granulin adipocyte plasma, APMAP membrane-associated protein, GNA13 guanine nucleotide-binding protein subunit alpha-13, WDR1 WD repeat-containing protein 1, SIRPA tyrosine-protein phosphatase non-receptor type substrate 1, LSP1 lymphocyte-specific protein 1

Fig. 3

Protein-protein interaction networks of upregulated proteins associated with primary Sjögren’s syndrome detected in extracellular vesicles from whole saliva. One major protein-protein interaction network is visualised. The proteins identified are involved in the cytoskeleton, in addition to cell migration and cell junction. Out of the five most upregulated proteins in pSS (Table 4), indicated with red circles, both guanine nucleotide-binding protein subunit alpha-13 (GNA13) and WD repeat-containing protein 1 (WDR1) are present within this protein network. The Search Tool for the Retrieval of Interacting Genes/Proteins (http://string-db.org/) was used to generate the networks, where potential interactions of proteins with medium confidence are shown. The different clusters are indicated by the same colour. The colour of the connecting lines indicates the type of evidence used in predicting the associations (red gene fusion, yellow text-mining extracted from literature, purple protein-protein interaction datasets, light blue protein interaction groups, black linked across species). APMAP membrane-associated protein, SIRPA tyrosine-protein phosphatase non-receptor type substrate 1, LSP1 lymphocyte-specific protein 1

Fig. 4

Protein-protein interaction networks of upregulated proteins associated with primary Sjögren’s syndrome detected in tear fluid. Two protein-protein interaction networks are visualised. One is central in the formation of the cytoskeleton and cell migration, while the other is involved in redox reactions and oxidative stress and contains one out of the five most upregulated proteins, as indicated with red circles (Table 5), namely thioredoxin-dependent peroxidase reductase (PRDX3). The Search Tool for the Retrieval of Interacting Genes/Proteins (http://string-db.org/) was used to generate the networks, where potential interactions of proteins with medium confidence are shown. The different clusters are indicated by the same colour. The colour of the connecting lines indicates the type of evidence used in predicting the associations (red gene fusion, yellow text-mining extracted from literature, purple protein-protein interaction datasets, light blue protein interaction groups, black linked across species). APEX1 DNA (apurinic or apyrimidinic site) lyase, CPNE1 copine, ACO2 aconitate hydratase, LMO7 LIM domain only protein 7