One of the main characteristics of primary Sjögren’s syndrome (pSS) is chronic dysfunction and destruction of the salivary and lacrimal glands; their secretory biofluids should reflect the glandular biological status. Saliva is a heterogeneous biofluid comprised of biomolecules and omics constituents that are altered in response to various diseases. Scientific effort has evaluated saliva proteome to diagnose, monitor, and prognosticate pSS. This article reviews the recent advances in salivary proteomics in the context of pSS, highlighting the most significant and promising findings. Determining saliva as a credible means of early disease detection could lead to translational advantages and significant clinical opportunities for pSS.
Key points
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Sjögren’s syndrome (SS) saliva proteome analysis reveals a unique expression profile.
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Major classification biomarkers of SS are present in saliva, including anti-Ro/SSA and anti-La/SSB.
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Prevalidation studies suggest salivary autoantibodies as strong biomarker candidates in SS.
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Further clinical validation of the verified and prevalidated proteomic signatures is needed in independent multicenter validation cohorts.
Introduction
Primary SS (pSS) is characterized by chronic dysfunction and destruction of exocrine glands, in particular the salivary and lacrimal glands, leading to persistent dryness of the mouth and eyes. Due to the direct involvement of those glands in the pathophysiology of the disease, saliva and tears are thought to reflect the glandular dysfunction and destruction. Saliva and tears drain the main targets of autoimmune response, which in the case of pSS are the salivary and lacrimal glands.
Saliva presents obvious advantages in terms of accessibility and unstimulated secretion, hence, ease of collection. Although primarily considered an indispensable element of early digestion, saliva is a heterogeneous biofluid comprised of biomolecules and omics constituents, which may become altered in response to various diseases, including pSS. The advancements of high-throughput technologies and analytical techniques have made saliva proteomics an ideal tool to study the underlying pSS glandular autoimmune exocrinopathy. In recent years, scientific effort has been undertaken for the purpose of evaluating saliva proteome to diagnose, monitor, or prognosticate pSS. The results from these studies are encouraging; thus, saliva has been emerged as a novel and promising biofluid for the discovery of definitive disease-specific biomarkers for pSS with potential clinical and translational impact.
This discussion begins by exploring the production, secretion and composition of saliva. Next, the proteomics of saliva in the context of pSS are delved into by describing several studies conducted from different groups including the authors’. Last, potential pitfalls are addressed and promising topics highlighted for the near future of saliva-proteome research in pSS.
Introduction
Primary SS (pSS) is characterized by chronic dysfunction and destruction of exocrine glands, in particular the salivary and lacrimal glands, leading to persistent dryness of the mouth and eyes. Due to the direct involvement of those glands in the pathophysiology of the disease, saliva and tears are thought to reflect the glandular dysfunction and destruction. Saliva and tears drain the main targets of autoimmune response, which in the case of pSS are the salivary and lacrimal glands.
Saliva presents obvious advantages in terms of accessibility and unstimulated secretion, hence, ease of collection. Although primarily considered an indispensable element of early digestion, saliva is a heterogeneous biofluid comprised of biomolecules and omics constituents, which may become altered in response to various diseases, including pSS. The advancements of high-throughput technologies and analytical techniques have made saliva proteomics an ideal tool to study the underlying pSS glandular autoimmune exocrinopathy. In recent years, scientific effort has been undertaken for the purpose of evaluating saliva proteome to diagnose, monitor, or prognosticate pSS. The results from these studies are encouraging; thus, saliva has been emerged as a novel and promising biofluid for the discovery of definitive disease-specific biomarkers for pSS with potential clinical and translational impact.
This discussion begins by exploring the production, secretion and composition of saliva. Next, the proteomics of saliva in the context of pSS are delved into by describing several studies conducted from different groups including the authors’. Last, potential pitfalls are addressed and promising topics highlighted for the near future of saliva-proteome research in pSS.
Saliva: production and secretion
Saliva is produced by several salivary glands located around the oral cavity, including the parotid, submandibular, sublingual, and minor salivary glands and the posterior deep lingual glands (von Ebner glands). Salivary glands are comprised of clustered acinar cells called acini, which concertedly produce approximately 500 mL to 1500 mL of saliva daily.
Two categories of acinar cells are found in the salivary glands: (1) serous cells (most commonly found in the parotid gland), which secrete a nonviscous watery product, and (2) mucous cells (predominant in the sublingual gland), which secrete a mucous-like product of high viscosity. The solution produced by these cells contains electrolytes, mucins, and enzymes, which subsequently flow into collecting tubes, where their composition can be further altered by the reabsorption of specific molecules before release into the mouth as saliva.
Composition
Saliva is a clear, slightly acidic, hypotonic fluid, which is continuously secreted and is predominantly composed of water (99.5%). The remaining 0.5% is comprised of inorganic ions, including sodium, chloride, potassium, and calcium along with organic components, such as proteins, amino acids, antibodies, hormones, enzymes, lipids and cytokines, among many others. In addition, recent studies have shown that saliva actually contains a variety of genomic, transcriptomic, proteomic, microbiologic, and immunologic analytes that may be capable of identifying both local and systemic disorders in afflicted individuals. Therefore, saliva is now the focal point of multiple investigations aimed at establishing oral fluids as the preferred diagnostic medium.
In 2004 the National Institute of Dental and Craniofacial Research provided funding to 3 research groups comprising the Saliva Proteome Consortium, in an effort to identify and catalog the human saliva proteome, including saliva proteins as well as their structurally modified forms (eg, glycosylated and phosphorylated). These studies revealed the salivary proteome as a sizeable collection of up to 1166 protein molecules—914 in parotid saliva and 917 in submandibular/sublingual saliva. A majority of these proteins are synthesized and secreted into the oral cavity by the acinar cells of the salivary glands. In consideration, a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes, ranging from structural functions to enzymatic and catalytic activities, with a majority of them mapped to the extracellular and secretory compartments.
Function
Saliva plays a key role in maintaining the oral health and homeostasis, by lubricating and moistening the oral tissues to aid in swallowing, chewing, speech, and taste. Saliva also has a critical role in initiating and facilitating digestion. In addition, maintenance of oral health largely depends on saliva’s cleansing actions and intrinsic antipathogenic characteristics.
Saliva for biomarker discovery in primary Sjögren syndrome
An Attractive Biofluid
The revelation that saliva is comprised of analytes capable of reflecting health status presents a significant translation potential. In considering the simplicity of saliva collection and its potential as a diagnostic medium, oral fluids have rapidly become the focus of investigation for several disease biomarkers. In that sense, saliva is widely recognized as an attractive biofluid for study of pSS, featuring several undisputable advantages over blood, the most important that it can be obtained using noninvasive techniques. As a clinical tool, saliva can be easily collected, stored, and shipped compared with serum. More importantly, for patients, the noninvasive collection techniques significantly reduce anxiety and discomfort and make procurement of repeated collections a cost-effective approach for longitudinal monitoring.
Challenges
Although saliva exerts several compelling advantages over serum and other invasively collected biofluids, there are challenges that need to be overcome. Unlike blood, saliva proteome seems sensitive to degradation and research has been carried out to minimize those processes. Hence, a major challenge is to collect and store saliva under conditions that prevent proteolysis, degradation, or dephosphorylation. The authors have shown that collection of saliva into ice-cold tubes, addition of protease inhibitors, and storage immediately at −80°C result in minimal proteolysis. Another critical step for proper saliva proteomic analysis is the removal of the mucins. These proteins are responsible for the “sticky” appearance of saliva and might interact with standard immunologic assays. Mucins removal can be achieved either by centrifugation or by using special filter-containing collection devices.
Immunologic Proteins in Saliva
One of the current criteria used for diagnosis of pSS is serum positivity for anti-Ro/SSA and/or anti-La/SSB. Studies from different groups have demonstrated the presence of these 2 autoantibodies in whole and parotid saliva collected from pSS patients. This finding is of fundamental importance for pSS because it provides evidence that oral fluids are capable of reflecting the autoantibody load, thus presenting an alternative, noninvasive procedure for the diagnosis of the disease. Other autoantibodies that have been described in saliva are anti-mAChR, anti-spectrin, and rheumatoid factor. In an attempt not only to discover but also validate potential autoantibody biomarkers in saliva, the authors’ laboratory has identified 24 potential autoantibodies that can discriminate patients with SS from both patients with systemic lupus erythematosus and healthy individuals. Four of these saliva autoantibodies, namely anti-transglutaminase, anti-histone, anti-Ro/SSA, and anti-La/SSB, were further successfully validated in independent SS, systemic lupus erythematosus, and healthy control subjects. Hence, saliva autoantibodies seem promising biomarkers to be used in a clinical setting.
A few studies have reported saliva cytokines in the context of pSS. Data from these reports congruently show significantly higher levels of T H 1, T H 2, and T H 17 cytokines in saliva of pSS patients. The lack of appropriate controls in a majority of these studies, non-SS sicca patients, however, does not allow a definitive conclusion regarding the specificity of these salivary cytokines to discriminate pSS from non-SS sicca subjects. Kang and colleagues found that salivary T H 1/T H 2 ratios, represented by interferon-γ/interleukin (IL)-4 and by tumor necrosis factor α/IL-4 ratios, were features that most differentiated SS and non-SS sicca and were correlated with the clinical parameters of SS.
To conclude, the available data indicate that immunologic proteins, including the major pSS-related autoantibodies and cytokines, can be detected in saliva and their levels are significantly increased in patients suffering from pSS. These findings, although not independently validated in large clinical cohorts, confirm that saliva is capable of reflecting the autoimmune exocrinopathy in pSS.
Salivary Proteomics for Primary Sjögren Syndrome Diagnosis
Because saliva is the product of salivary glands, the primary targets of the autoimmune response in pSS, it is believed that this secreted fluid can directly mirror the glands’ pathophysiology. To analyze the proteomic content of saliva, scientists often use traditional techniques, including liquid chromatography, gel and capillary electrophoresis, nuclear magnetic resonance, mass spectrography, and immunoassays. More contemporary methods, however, including immune-response protoarrays and 2-D electrophoresis (2DE) coupled with mass spectrography, are also used and have allowed investigators to analyze several salivary analytes. The development of emerging high-throughput proteomic approaches allows the investigation of the whole and gland-specific protein composition.
The main goal of these studies has been the discovery, verification, and validation of a panel of protein biomarkers so that they can be used in early detection of pSS. Such approaches have highlighted distinct protein patterns characteristic of pSS. These protein signatures mostly comprise secretory proteins, enzymes, calcium-binding proteins, and abundantly expressed immune-related molecules, such as β 2 -microglobulin. Other protein molecules that have received particular attention include cathepsin-D, α-enolase, cystatins, defensins, and Ig γ light chain. Collectively, these studies have presented evidence that inflammatory phase proteins are elevated in saliva from pSS patients and this finding correlates with the chronic autoimmune inflammation of the salivary glands in pSS. Similarly, the increased expression of salivary β 2 -microglobulin, Ig κ light chain, and Ig γ light chain was attributed to B-cell activation in the periphery. In an effort to catalog salivary biomarkers according to their biological pathways, a recent study showed that SS-associated salivary proteome seems profoundly altered with respect to several aspects of immunity, immune cell differentiation, and tissue homeostasis. The congruency between the proteomic signatures identified in this study and the hallmarks of salivary gland pathology in pSS greatly supports that salivary proteome reflects the biologic state of the glands. Collectively, these data indicate that saliva has the capability of revealing changes in the biologic state of the salivary glands and proteomic approaches seem a promising tool for improving early diagnosis of pSS.
Prognostic Biomarkers of Lymphoma in Primary Sjögren Syndrome
Considering the lack of well-validated prediction biomarkers, saliva might be a significant pool of candidate molecules for early identification of pSS patients at higher risk for developing mucosa-associated lymphoid tissue (MALT) lymphoma. The authors’ group has performed a proteomic and transcriptomic analysis of human parotid glands from patients with pSS and patients with pSS and MALT lymphoma. This study revealed that 70 proteins were up-regulated in SS/MALT lymphoma samples compared with both non–SS control and pSS samples. Intriguingly, 45% of the up-regulated proteins had an mRNA transcript (gene-expression level) that was concordantly differentially expressed. Most of the proteins with up-regulated levels in pSS/MALT lymphoma were related to signal transduction, gene regulation, apoptosis, and the immune response. Among these targets, a few have previously been linked to lymphoma, and, in particular, 2 cancer-related proteins, Rho-GDP dissociation inhibitor and cyclophilin A, are of biologic significance.
In another proteomic approach to analyze whole saliva from pSS and pSS/MALT lymphoma patients, Baldini and colleagues showed several qualitative and quantitative modifications in the expression of putative albumin, immunoglobulin J chain, Ig kappa chain C region, α 1 -antitrypsin, haptoglobin, and Ig α 1 -chain C region. These studies suggest that clinical and functional changes of the salivary glands driven by lymphoproliferative processes might be reflected in patients’ whole saliva, providing further insights into the molecular mechanisms of pSS and pSS/MALT lymphoma. Reasonably, once validated and confirmed, the identified protein candidates could be translated into early prognostic biomarkers for non-Hodgkin lymphoma–susceptible pSS patients.
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