Proteomic and histopathological characterisation of sicca subjects and primary Sjögren's syndrome patients reveals promising tear, saliva and extracellular vesicle disease biomarkers

Abstract

Background: Mononuclear cell infiltration of exocrine glands, production of Ro/SSA and La/SSB autoantibodies, along with oral and ocular dryness, are characteristic features of primary Sjögren's syndrome (pSS). Non-SS sicca subjects, an underexplored group in relation to pSS, display similar sicca symptoms, with possible mild signs of inflammation in their salivary glands, yet with no serological detection of autoantibody production. In this study, we investigated inflammatory manifestations in the salivary gland tissue, tear fluid and saliva of non-SS subjects, as compared to pSS patients and healthy individuals.

Methods: Fifteen non-SS, 10 pSS and 10 healthy subjects were included in the analyses. Histological evaluation of salivary gland biopsies was performed. Liquid chromatography-mass spectrometry (LC-MS) was conducted on tear fluid and stimulated whole saliva, and proteomic biomarker profiles were generated. Extracellular vesicle (EVs) isolation and characterisation from both fluids were also combined with LC-MS. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold. Database for Annotation, Visualization and Integrated Discovery (DAVID) and Functional Enrichment Analysis Tool (FunRich) were applied for functional analyses.

Results: Histopathological evaluation of salivary gland biopsies showed implications of milder inflammation in non-SS subjects through mononuclear cell infiltration, fibrosis and fatty replacement, as compared to pSS patients. Although unaffected in the non-SS group, upregulation of proinflammatory pathways and proteins involved in ubiquitination (LMO7 and HUWE1) and B cell differentiation (TPD52) were detected in tear fluid of pSS patients. Moreover, overexpression of proteins STOM, ANXA4 and ANXA1, regulating cellular innate and adaptive immunological pathways, were further identified in EVs from tear fluid of pSS patients. Finally, whole saliva and EVs isolated from whole saliva of pSS patients expressed proteins vital for innate MHC class I cellular regulation (NGAL) and T cell activation (CD44).

Conclusions: Non-SS sicca subjects may show implications of mild inflammation in their glandular tissue, while their protein profile was strikingly more similar to healthy controls than to pSS patients. Hence, the tear and salivary biomarkers identified could be implemented as potential non-invasive diagnostic tools that may aid in increasing diagnostic accuracy when evaluating non-SS subjects and pSS patients and monitoring disease progression.

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

Fig. 1

Histopathological evaluation of minor salivary gland biopsies shows implications of inflammation in the target organ of non-SS sicca subjects. Haematoxylin and eosin staining of minor salivary gland biopsies taken from the non-SS and pSS subjects included in the study allowed the evaluation for mononuclear cell infiltration, fibrosis, and the presence of fatty infiltration in their salivary gland tissue. a Non-SS subject with normal salivary gland morphology. b Non-SS individual with fibrosis in the salivary gland tissue. c Non-SS participant with mild focal inflammation in the salivary gland and a focus score < 1. d Salivary gland biopsy of a pSS patient with a focus score value of 3 and GC-like structure within the focal infiltrate. Areas of inflammation are indicated by black arrow

Fig. 2

Upregulation of proinflammatory pathways detected in tear fluid of pSS patients. For functional analysis of the proteomics data, DAVID (v 6.7, https://david.ncifcrf.gov) was applied using a FDR with a maximum 5% cut-off, and cellular processes for the upregulated proteins in the pSS patients were identified. FunRich (http://www.funrich.org/) was then used to visualise the fraction of proteins involved in each of these upregulated signalling pathways. a Upregulated signalling pathways identified in the pSS patients, as compared to non-SS sicca controls. b Comparing pSS patients to healthy controls helped detect similar cellular processes as with the non-SS subjects, affecting both innate and adaptive immunological processes. Percentage values indicate the amount of overexpressed proteins involved in upregulating each of the cellular processes identified

Fig. 3

Overexpression of proteins regulating cellular innate and adaptive immunological pathways detected in EVs from tear fluid of pSS patients. Following LC-MS of EVs extracted from tear fluid, DAVID analysis (v 6.7, https://david.ncifcrf.gov) was applied using a FDR with a maximum cut-off of 5%. Cellular processes for the upregulated proteins in the pSS patients were identified, and FunRich (http://www.funrich.org/) was then used to visualise the segment of proteins involved. a Upregulated signalling pathways distinguished in EVs isolated from tears of pSS patients, as compared to non-SS subjects. b Comparing pSS patients to healthy controls, the most upregulated of cellular processes in the pSS patient group was again retina homeostasis, followed by other central innate and adaptive immune responses. Percentage values represent the fraction of overexpressed proteins contributing to the upregulation of each cellular process