Our study is the first to evaluate the salivary cytokine profile in patients with ischemic stroke. We have demonstrated that assessment of TNF-α, IL-6, and IL-10 levels in both NWS and SWS significantly differentiates stroke patients from healthy controls. However, particular attention should be paid to salivary TNF-α, which may be a non-invasive biomarker of cognitive function/physical status in stroke subjects.
In recent years, increasing attention has been given to the potential use of saliva in laboratory diagnostics. This biomaterial allows for repeated, non-invasive, and stress-free sampling, which is especially important in disabled and elderly patients 54. Saliva is also a non-infectious and relatively durable bioliquid 29,55. The concentration of many biomarkers in saliva highly correlates with their blood/CSF levels, allowing saliva to be used in the diagnosis of various systemic diseases 56–59. Indeed, saliva can be an alternative to blood for the determination of enzymes, hormones, immunoglobulins, and redox biomarkers 60,61. Saliva has also been shown to be valuable in assessing cytokines, chemokines, pro-inflammatory enzymes, and growth factors 62–64. Although salivary cytokines' diagnostic utility has been demonstrated in metabolic and neurodegenerative diseases 35–39, there are no data on ischemic stroke patients.
We showed that salivary TNF-α and IL-6 were significantly higher, whereas IL-10 content was statistically lower in ischemic stroke patients than healthy controls. TNF-α/IL-10 and IL-6/IL-10 ratio were also significantly higher in NWS and SWS of stroke patients, indicating an intensification of the inflammatory process and a limited anti-inflammatory role of IL-10. As we have shown by ROC analysis, salivary biomarkers are also characterized by a high diagnostic utility in stroke diagnostics. Salivary TNF-α, IL-6, and IL-10 as well as TNF-α/IL-10 and IL-6/IL-10 ratio significantly distinguish stroke patients from controls with high sensitivity and specificity. This fact should not be surprising because inflammatory and immune factors have been indicated as potential stroke biomarkers 65,66. Indeed, in stroke patients, activation of CNS cells (mainly microglia and astrocytes) and increased expression of blood cytokines, interleukins, chemokines, adhesion molecules, and pro-inflammatory enzymes is observed 67. Interestingly, the severity of inflammation correlates with brain infarct size and neurological deficits in CT and physical examination 68−70. However, cytokines' diagnostic utility has only been demonstrated in plasma/serum and CSF of ischemic stroke patients 70−72. Our study is the first to show the utility of saliva to determine cytokine levels in stroke subjects. This raises hopes for non-invasive, painless, and non-stressful stroke diagnostics. It is believed that cytokines synthesized in the stroke focus pass through the damaged BBB into the blood and then into saliva by passive transport (diffusion or ultrafiltration) or by a specific transporter (facilitated diffusion or active transport) 73,74. The BBB has a prominent role in the development of the inflammatory response by increasing the cytokine circulation between the CSF and the bloodstream 9. Unfortunately, in our research, we did not assess the cytokine content in blood, so further studies are needed to evaluate stroke patients' saliva-blood relationship. Nevertheless, it can be speculated that, as in other CNS diseases, salivary cytokine concentrations reflect their plasma/CSF levels 25,28,75.
In our patients, the content of pro-inflammatory TNF-α and IL-6 was higher, and anti-inflammatory IL-10 was lower in both NWS and SWS of stroke patients. However, it is important to note that saliva's cytokine profile depends on the type of salivary gland involved in saliva secretion. Indeed, up to 70% of non-stimulated saliva is produced in the submandibular glands, making the NWS composition very similar to blood plasma. During stimulation, the parotid gland's contribution to saliva formation increases 76. Stimulated saliva is characterized by significantly higher concentrations of Na+, HCO3− and Cl− ions and lower contents of K+ and phosphate ions and mucins compared to non-stimulated saliva 77. Cytokine production in salivary gland cells also increases, making the composition of SWS more different from that of plasma 78. Therefore, evaluation of cytokines in NWS rather than in SWS may be of greater diagnostic value. In our study, among all assessed cytokines, tumor necrosis factor α deserves special attention. Salivary TNF-α differentiates stroke patients from controls with high sensitivity and specificity and correlates with cognitive dysfunction in the ACE III scale and severity of functional impairment in the BBS and ADL scale. Therefore, in the next step, we examined how this parameter distinguishes stroke patients according to cognitive impairment. For this purpose, stroke subjects were divided into three groups: normal cognition (100 − 89 in the ACE III scale), mild dementia (88 − 61 in the ACE III scale), and moderate dementia (< 61 in the ACE III scale) 52,53. Salivary TNF-α differentiates (with high sensitivity and specificity) stroke patients with normal cognitive function from patients with mild to moderate dementia (Fig. 1). Although further studies in a larger patient population are needed, TNF-α may be a potential biomarker of cognitive impairment/functional status in stroke cases. As shown in previous studies, elevated TNF-α serum level was noted already in the first few hours after the ischemic stroke 10,16. Serum TNF-α also correlates with brain infarct volume and severity of neurological symptoms as well as reflects the degree of patients' functional disability 79,80. Indeed, TNF-α has a unique role in stroke pathogenesis. Primarily, it is the first inflammatory mediator produced in the ischemic area 6,80. Secondly, it stimulates the synthesis/secretion of chemotactic factors that stimulate peripheral blood leukocytes and activates other cytokines like IL-1, IL-6, and interferon-gamma (IFN-γ) 10. TNF-α also enhances MMPs activity 81, thereby increasing endothelial cell permeability and BBB dysfunction 82. Our study is the first to demonstrate that salivary TNF-α can be a potential biomarker of stroke patient’s functioning. Interestingly, the level of TNF-α in NWS correlates more strongly with the cognitive and physical functional status of stroke patients. Thus, non-stimulated saliva has a better diagnostic potential than SWS.
Despite the unquestionable advantages, the use of saliva in diagnostics also has some drawbacks. The biomarker concentration in saliva may be highly dependent on the degree of salivary gland secretion 83. Because salivary hypofunction may occur in stroke patients 84,85, in our study, the concentration of inflammatory biomarkers was standardized to total protein content and salivary flow rate. Nevertheless, both the concentration (pg/mL), salivary output (pg/min), and specific content (per mg protein) of TNF-α and IL-6 were significantly higher, whereas IL-10 was statistically lower in NWS and SWS of stroke patients compared to controls. Importantly, we did not observe a relationship between salivary gland function and the assessed cytokines. This suggests a negligible effect of salivary hypofunction on salivary TNF-α, IL-6, and IL-10 levels. Nevertheless, aging, medications, diet supplements, smoking, as well as oral and systemic diseases can also modify the cytokine profile of saliva 86–88. At this time, it is important to emphasize the careful selection of the control group in the presented study. Control individuals are matched not only by age and sex to the study group but also by other clinical features, including concomitant diseases, medications, oral hygiene status, etc. This significantly reduces the impact of confounding factors on the obtained results.
In summary, stroke patients have increased salivary secretion of pro-inflammatory cytokines and decreased anti-inflammatory mediators. Salivary TNF-α, IL-6, and IL-10 may be potential non-invasive biomarkers that distinguish stroke patients from controls with high sensitivity and specificity. Of particular note is salivary TNF-α, which may indicate cognitive/physical impairment in post-stroke individuals. Further studies are needed in a larger stroke population to assess cytokines at both the salivary and central (plasma/serum) levels. Evaluation of other inflammatory mediators for their potential diagnostic significance also requires research.