Flores-Reyna, Luis Alonso¹; Martínez-Hernández, Miryam²

Language: English/Spanish [Versi?n en espa?ol]
References: 98
Page: 99-112
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ABSTRACT

Introduction: this literature review examines changes in the salivary proteome related to the presence of periodontitis that can be used for diagnosis and monitoring of the disease, this is possible because whole saliva contains a variety of local mediators derived from microbial and host responses, as well as systemic (plasma) markers that may be useful in periodontal diagnosis. Objective: to document reported changes in the salivary proteome associated with the presence of periodontitis with potential for use in the diagnosis and monitoring of the disease. Material and methods: the PubMed, SpringerLink, Google Scholar, WILEY and ScienceDirect databases were queried using the search terms "salivary proteome AND salivary diagnosis; salivary protein profile AND periodontal disease AND periodontitis" to identify publications reporting changes in the salivary protein profile of subjects diagnosed with periodontitis. Review articles, original articles published in indexed journals and consensus documents in English and Spanish were included. Results: salivary proteins such as α-amylase, cystatin-C, and mucin-5B were reported as increased by some authors, while proteins like cystatin-SN, lactoperoxidase, and mucin-7 were reported as decreased when comparing the salivary protein proteome of systemically healthy subjects with periodontitis versus subjects without periodontitis. These results confirm the existence of changes in the salivary proteome associated with the presence of periodontitis. Conclusions: the changes in the salivary proteome associated with the presence of periodontitis identified in the present literature review require further investigation because of their potential for use in the diagnosis and monitoring of periodontitis.

INTRODUCTION

Periodontal diseases are a group of inflammatory conditions that affect the dental supporting tissues - gingiva, periodontal ligament, root cementum, and alveolar bone - within this group of conditions, we find gingivitis and periodontitis.^1,2 The development of periodontitis is associated with the presence of a dysbiotic dental biofilm, which induces the deregulation of the host immune response.³ This dysregulation, together with hereditary and environmental factors⁴ such as smoking,⁴ finally leads to the destruction of periodontal tissues and consequent tooth loss.^3,5 It is estimated that 743 million people suffer from periodontitis, making it the sixth most prevalent disease in the world and the third most common oral disease.^6-8 The correct diagnosis of periodontitis is crucial for the successful treatment of the disease and is generally based on the evaluation of clinical parameters of inflammation, such as bleeding upon probing (BP),⁴ probing depth (PD),⁹ and changes in clinical insertion levels (CIN),¹⁰ as well as the extent and pattern of alveolar bone loss, the latter being evaluated radiographically. Today, dentoalveolar radiographs and periodontal probing continue to be the main tools for diagnosing and evaluating the presence and progression of periodontal disease.

The periodontal probe was described by Orban in the 1950s as the "clinician's eyes below the gingival margin"¹¹ and has been used since and has been used then to record the extent of periodontal damage.^12-14 However, its use has certain limitations. Some of these limitations may be the result of interference in the periodontal probe's path of insertion due to the presence of calculus on the tooth surfaces, overhanging restorations; incorrect pressure or angle of insertion during probing. In addition, the degree of inflammation of the periodontal tissues affects the sensitivity and reproducibility of the measurements.¹⁵ All of these factors increase the likelihood of a false positive or false negative periodontal diagnosis, which commonly results in under- or over-treatment of periodontitis.¹⁶ Additionally, changes in CINs are only detectable when an attachment loss equivalent to ≥ 2 mm has occurred, indicating that periodontal probing provides historical evidence of the presence and extent of periodontal disease, without providing real-time or predictive evidence of the course of the disease.^17,18

The limitations that currently exist to diagnose and monitor periodontitis by periodontal probing and the use of radiographic evidence have marked a challenge in biomedical research, so in recent decades more and more studies have focused on identifying quantifiable biomarkers in total saliva that can reliably reflect the pathophysiological state of the gingival sulcus, and that can complement periodontal probing to achieve early detection and correct monitoring of periodontitis.^18,19 The present review aims to describe the existence of changes in the salivary proteome associated with the presence of periodontitis with the potential to be used for disease diagnosis and monitoring. To that end, an extensive search of scientific literature in indexed journals was conducted from March 2020 to March 2021, following a previously established methodology.^20,21 The articles included in this review were consulted in electronic bibliographic information sources: PudMed, SpringerLink, Google Scholar, WILEY, and ScienceDirect, using the following keywords: "(Salivary proteome) AND (Salivary diagnosis); (Salivary protein profile) AND (Periodontal disease) AND (Periodontitis)" and that were written in English or Spanish.

Functions of saliva in the oral ecosystem

The human oral cavity represents a complex ecosystem where external factors and host elements interact in a dynamic equilibrium that is reflected in saliva. Saliva plays different roles in the maintenance of oral health, for example, it lubricates oral surfaces, maintains tooth integrity by reducing enamel demineralization, provides elements of innate and adaptive host immunity, and acts as the primary nutrient for the resident oral microbiota, which is mainly organized in the form of biofilms on the different oral surfaces.^22,23

It is now known that the microbiome has coevolved with humans over the centuries, and its relevance is crucial because it plays a determining role in the establishment of health and disease states.²⁴ Under equilibrium conditions, the oral microbiome maintains a symbiotic and dynamic relationship with the host, which will be a determining factor in health. However, changes in the microbial population may promote the development of a pathological inflammatory state as a consequence of microbiome dysbiosis.²⁵ Inflammatory states in the host are often favored by modifiers such as the presence of systemic diseases (e.g. diabetes mellitus type I and II), poor oral hygiene, smoking, or alterations in salivary flow.^26-28 Dysbiosis of the oral microbiome is the determinant factor in the development of periodontal disease²⁹ which induce changes in the salivary proteome.

Changes in the salivary proteome and their potential for diagnosis and monitoring of periodontal disease

Saliva is a biological fluid that is made up of a variety of functionally and structurally complex biomolecules, such as proteins, lipids, and carbohydrates,²² which is why it is currently considered one of the most valuable biofluids for biomedical research. It has a pH between 6-7, reaching daily secretion volumes of 0.5-1.5 L (0.5 mL/min).³⁰ Secretion values are influenced by factors such as age, medication, hydration, psychological conditions such as stress and depression, and circadian rhythms.^31-33 It is composed of 99-99.5% water and only 0.5-1% protein and minerals (32,34). Its average protein concentration is between 0.7-2.4 mg/mL^-1; and being an ultrafiltrate of plasma, it shares 30% of its protein content with that biofluid.³¹

The term "proteome" refers to the complete set of proteins that can be expressed by a genome, cell, tissue, or organism at any given time.³⁵ Thus, the salivary proteome associated with the presence of periodontitis refers to all the proteins present in the saliva of an individual with this disease,^36,37 Saliva is produced in the salivary glands within acinar cells where, after stimulation, it passes into a branched network of ducts to be secreted into the oral cavity. It is here that pristine saliva mixes with other biocomponents and aggregates derived from various sources such as blood, gingival crevicular fluid, food debris, oral cells, microbiota, as well as DNA and RNA; the mixture of all these components makes up what is known as total saliva.^29,38 Today, ~ 2,643 proteins have been identified in total saliva³⁹ among which, it is possible to identify 9 different families that together represent about 40% of the total salivary protein content. Table 1 describes these 9 protein families.^22,40-63

The heterogeneity and diversity of the protein content present in total saliva allow this biofluid to be considered a "reflection of oral and systemic health".^30,64,65 Thus, the identification of salivary protein expression profiles associated with the presence of periodontitis is relevant, since these changes constitute the basis for the identification of possible biomarkers of periodontal disease that may contribute to the early diagnosis and correct monitoring of periodontitis.^29,38 A biomarker refers to an objective indicator of the medical condition observed from outside the patient, that can be measured accurately and reproducibly.⁶⁶ In this regard, it has been reported that potential biomarkers of periodontal disease can be nonspecific, which correspond to changes in the concentrations of proteins synthesized by the salivary glands associated with the presence of periodontitis⁶⁵ and specific, which are a direct product of the protein synthesis derived from the inflammatory process occurring during periodontal disease.⁶⁷

The main findings derived from the search for non-specific biomarkers associated with the presence of periodontal disease are described below:

• 1. α-amylase: it is the most abundant protein in total saliva. It has been reported that several isoforms of this protein are increased in subjects with periodontal disease, reaching levels higher than 600 μg/mL in total saliva of subjects with severe periodontitis, and decreasing to values close to 300 μg/mL after periodontal treatment.^38,40,56
• 2. Cystatins: it has been suggested that cystatins act as modulators of the enzymatic activity of the periodontium during the development of periodontal disease.⁶⁸ Within this family of proteins, cystatin SN has inhibitory effects in vitro on cathepsins B, H, and L, involved in the catabolism of structural proteins of periodontal tissues,^38,42,68 while cystatins C and S have been shown to inhibit the growth of Porphyromonas gingivalis.⁶⁹ Hartenbach et al.⁷⁰ reported an increase in cystatin SA levels in subjects with periodontal disease, probably due to an attempt to slow down the proteolytic activity triggered by periodontitis. Gonçalves et al.³⁸ reported a decrease in the concentrations of cystatin SN, so values higher than 280 μg/mL and lower than 240 μg/mL of cystatin SA and SN, respectively, could be expected in subjects with periodontitis.⁴⁰
• 3. Defensins: based on their cysteine pairing pattern, two subfamilies, called α-defensins and β-defensins, can be distinguished.⁴¹ The former are produced and stored by neutrophils, while the latter are synthesized mainly by keratinocytes.^60,71 Both have antimicrobial properties attributed to their positive charge. In addition to their antimicrobial properties, β-defensins also exhibit antifungal properties, in particular against Candida albicans.^44,72 Although the increased concentration of defensins in the total saliva of subjects with periodontal disease has not been reported, it was recently demonstrated that β-defensin levels increase in the gingival crevicular fluid of subjects with periodontitis.⁴⁵
• 4. Staterins: staterins are peptide precursors of the acquired salivary film (ASF)^50,73 that allow the interaction of Fusobacterium nucleatum with primary colonizers of dental biofilm.⁷³ It has previously been reported that the concentration of staterin-derived peptides in subjects with periodontitis is five times lower compared to the levels quantified in total saliva of periodontally healthy subjects, i.e., values lower than 2.4 μg/mL of staterin-derived peptides could be found, compared to periodontally healthy subjects where values close to 12 μg/mL can be expected.⁴⁰ Due to this negative correlation between the concentration of this family of proteins in total saliva and the presence of periodontitis, authors such as Inzitari et al.⁴⁹ express their interest in the potential of peptides belonging to this family for monitoring the development of periodontitis.
• 5. Histatins: these are basic peptides rich in histidine residues.^51,74 Histatin 5 has the ability to inhibit arginine-specific gingipains (Arg-gingipains or Rgp) and lysine-specific gingipains (Lys-gingipains or Kgp), produced by P. gingivalis;^75,76 it also inhibits the gelatinolytic activity of matrix metalloproteinases (MMPs) -2 and -9 by up to 99%.^58,75 In addition, histatins 1, 2, and 3 play a role in wound healing, participating in angiogenic processes, and promoting wound re-epithelialization and fibroblastic proliferation.^53,77 It has been reported that histatin 1 could have diagnostic applications since the increase of its concentration in total saliva has been related to the presence of periodontal disease.^58,70 It has been found that in subjects with periodontitis, the concentration in total saliva of histatin 1 could reach values higher than 27 μg/mL.⁵²
• 6. Mucins: they are the major protein component secreted by the submandibular and sublingual glands. It has been reported that the concentration of MUC-1 in total saliva increases in subjects with periodontal disease, especially when the clinical parameters of PD, CIN, and BP are increased, which could be a defense mechanism of the salivary glands and epithelia against the development of periodontitis, through the agglutination of microorganisms and their subsequent swallowing.⁵⁶ In addition, MUC-1, -4, and -16 play an important role in the distant metastasis of certain oral carcinomas, as they act as mediators between leukocytes and cancer cells in the tumor microenvironment and facilitate the colonization of distant disseminated cells.⁷⁸ Taking the above into consideration, these proteins have been the subject of study for alternatives in the treatment of various carcinomas such as pancreatic, ovarian, breast, and head and neck carcinomas, through mucin-based radioimmunotherapy (RIT) and vaccination, focusing on proteins such as MUC1, -4, -5AC, -5B, -16 and -17.⁷⁸
• 7. P-B peptides: they are proline-rich peptides secreted by all salivary glands, which, contrary to what was believed, are not the product of the degradation of other proteins but mature peptides by themselves, whose functions in total saliva have not yet been fully elucidated.⁴⁹ However, they have been reported to possess antimicrobial properties.⁵⁸ Within their possible applications in periodontal diagnosis, it has been suggested that the concentration of fragments from these peptides in total saliva is doubled in subjects with periodontal disease compared to periodontally healthy subjects.^58,79
• 8. Peroxidases: there are three main subgroups of peroxidases: lactoperoxidase (hLPO), which is secreted by the salivary glands, myeloperoxidase (hMPO).^60,61 which comes from neutrophil granules and catalase, which comes from erythrocytes and can catalyze the conversion of hydrogen peroxide (H₂O₂) into water (H₂O).⁶⁰ These enzymes have been shown to play an important role in what would be the onset of dysbiosis in the dental biofilm.⁸⁰ Unfortunately, there are insufficient studies that provide quantitative data on the changes in the concentrations of peroxidases in total saliva associated with the presence of periodontitis.
• 9. Proline-rich proteins (PRPs): this family of proteins, which is divided into 3 subtypes: acidic PRPs (PRPs-a), basic PRPs (PRPs-b) and glycosylated PRPs (PRPs-g), have several functions such as preventing the overgrowth of hydroxyapatite crystals on enamel surfaces, in addition to participating in the formation of ASF.^40,57 They also possess antimicrobial activity, so it has been suggested that an increase in their secretion in total saliva could represent a defense mechanism against an increase in the bacterial load in the oral cavity.⁵⁸ Its possible diagnostic role is not entirely clear, since while it has been reported that the concentration of PRPs-a increases in subjects with periodontal disease.⁷⁰ Trindade et al.⁵⁸ report that PRPs-a and PRP-b isoforms do not present statistically significant quantitative changes when comparing their concentration in total saliva of subjects with periodontitis vs. periodontally healthy subjects.

In addition to the changes in protein concentrations produced by salivary glands associated with the presence of periodontal disease described above, a large number of cytokines, pro-inflammatory mediators, and matrix metalloproteases (MMPs) are produced during the inflammatory process characteristic of periodontitis. It has been reported that particularly MMP-8, -9 and -13 are involved in the cascade of events leading to the degradation of gingival tissues and alveolar bone tissue 81, so that elevated proteolytic activity is expected to be reflected in total saliva of subjects with periodontal disease. As previously mentioned, changes in the salivary proteome derived from the protein synthesis associated with the inflammatory process occurring during periodontal disease constitute the basis for the identification of periodontitis-specific biomarkers.⁶⁵ The most relevant ones are described below:

• 1. Immunoglobulin (Ig) A: the main immunoglobulin isotype found in saliva.⁸² It constitutes the main mechanism of the specific immunologic response against pathogenic microorganisms.^83,84 As part of the defense mechanisms within the oral cavity, it is expected that its levels are increased in subjects with periodontal disease.^83,84 Additionally, it has been reported that their levels in saliva decrease significantly after effective periodontal therapy.⁸⁴
• 2. Interleukin (IL)-1β: a proinflammatory cytokine that plays a key role in the pathogenesis of periodontitis.⁸⁵ It is mainly secreted by neutrophils, dendritic cells, macrophages, and fibroblasts^84,86 as is its counterpart IL-1α.⁸⁷ It has been reported that its concentration in total saliva is increased in subjects with periodontal disease.^86,88 Together with MMP8, IL-1β has been the most investigated salivary biomarker in the field of diagnostic accuracy, both with a clinically acceptable efficacy for the diagnosis of periodontitis.⁸⁹
• 3. Interleukin (IL)-17a: this is a proinflammatory cytokine produced by activated Th17 lymphocytes that induces inflammation and bone resorption, stimulating the release of chemokines and the expression of matrix metalloproteases (MMPs), such as MMP-1 and -8.⁹⁰ Some of its effects are produced by its synergy with IL-1β and tumor necrosis factor (TNF)-α.⁸⁸ Liukkonnen et al.⁸⁸ reported an increase in IL-17a levels in the total saliva of subjects with localized periodontal disease, so this cytokine has been considered as a possible biomarker of periodontitis in its early stages.
• 4. Prostaglandin (PG) E₂: it is an inflammatory mediator product of the metabolism of arachidonic acid, released mainly by neutrophils, macrophages, and fibroblasts.⁸⁶ This inflammatory mediator increases osteoclastic activity and bone resorption, which translates clinically into a loss of periodontal attachment levels.^91,92 Nowadays, there is not enough data to define the changes in total saliva concentrations of PGE₂ alone, however, an increase in its concentration in conjunction with IL-1β has been reported as the severity of periodontal disease increases, reaching increases of up to 194%.⁸⁶ Other authors⁹² have also reported increases in PGE₂ associated with other biomolecules such as TNF-α or nitrous oxide.
• 5. Tumor necrosis factor-α (TNF-α): like PGE₂, TNF-α is an inflammatory mediator that has a strong positive influence on osteoclastic activity and bone resorption.⁹³ Frodge et al.⁹⁴ reported an increase in the concentration of this cytokine in the total saliva of subjects with periodontal disease.

In addition to the changes in the concentrations of various proteins in total saliva described above, Table 2 shows a summary of salivary proteins that are also under study for their possible potential to be used as biomarkers of periodontal disease.^{37,38,40,58,70,95}

Insights from biomarkers identified in the salivary proteome for the diagnosis and monitoring of periodontal disease

As we have seen, saliva contains an abundance of proteins and other biomolecules that may reflect the pathophysiological state of periodontal tissues during periodontitis, so it has become increasingly clear that salivary diagnostics could offer a safe and noninvasive approach to disease detection and monitoring, and that it also has a high potential to revolutionize the diagnosis of periodontitis.⁹⁶

The scientific community remains focused on generating the highest quality evidence on the diagnostic accuracy of salivary biomarkers so that they can be used routinely in the near future.^89,97 Currently available evidence indicates that MMP-8, MMP-9, IL-1β, IL-6, and hemoglobin (Hb) are the salivary biomarkers with the highest ability to detect periodontitis in systemically healthy subjects; MMP-9 and IL-1β also show a good ability to distinguish between cases without periodontitis. Other molecules such as cysteine, macrophage inflammatory protein 1α (MIP-1α), and nitric oxide (and its related metabolites), have also been identified as promising salivary biomarkers as reported in a recent meta-analysis.⁸⁹ However, further studies are required to confirm these findings.

The proteomic era has made the identification of multiple potential biomarkers in total saliva an achievable goal, coupled with the recent creation of the International Consortium for Salivary Biomarkers of Periodontitis, which aims to ensure the implementation of standardized protocols for clinical research.⁶⁷ Likewise, advancements in diagnostic technologies suggest that the diagnosis and monitoring of periodontal disease using molecular tools is a realistic proposal, today closer than ever before.⁹⁸

CONCLUSIONS

Increased understanding of the components of the salivary proteome, its modifications, and dynamics in health and disease will allow saliva to enter clinical practice as an alternative biological fluid for the diagnosis and monitoring of periodontal disease, serving as a complement to periodontal probing, which currently remains the fundamental component of a comprehensive dental examination.

ACKNOWLEDGMENTS

The authors would like to thank the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT) of the UNAM, Code: IA208220, for the support provided for this work.

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AFFILIATIONS

¹ Alumno Especialidad en Periodoncia e Implantología. División de Estudios de Posgrado e Investigación. Facultad de Odontología. Universidad Nacional Autónoma de México.

² Laboratorio de Biointerfases. Facultad de Odontología. División de Estudios de Posgrado e Investigación. Facultad de Odontología. Universidad Nacional Autónoma de México.

CORRESPONDENCE

Dra. Miryam Martínez-Hernández. E-mail: miryam_mh@comunidad.unam.mx

Received: Abril 2021. Accepted: Agosto 2021.