Oral Infections and Cytokine Levels in Patients with Alzheimer’s Disease and Mild Cognitive Impairment Compared with Controls

Abstract

Background: Oral infections are prevalent in the adult population. Their impact includes the implication as a risk factor for Alzheimer’s disease (AD), altering its progression. One of the potential mechanisms involves immune mediators such as circulating cytokines.

Objective: The goal of the present study was to investigate the prevalence of oral infections and blood levels of IL-1β, TNF-α, and IL-6 in patients with AD, mild cognitive impairment (MCI), and controls.

Methods: Sixty-five elderly were evaluated (25 AD, 19 MCI, and 21 controls) by the following methods: Mini Mental State Exam, Questionnaire of Functional Activities, periodontal and oral evaluation, and blood concentrations of IL-1β, TNF-α and IL-6.

Results: Patients with AD had high serum IL-6 levels (p = 0.029), and patients with periodontitis had high serum TNF-α levels (p = 0.005). There was an association between IL-6 and TNF-α in patients with AD/MCI and periodontitis (p = 0.023).

Conclusion: The increased levels of TNF-α and IL-6 in this study suggests their implication in the overlapping mechanisms between oral infections and AD. Longitudinal studies are necessary for further investigation.

Keywords

Alzheimer’s disease IL-6 inflammation mild cognitive impairment oral infection periodontitis

INTRODUCTION

Alzheimer’s disease (AD) is the most common acquired cerebral degeneration and the main cause of dementia in Western countries (50% – 66%). It interferes in personal, social, and work activities. Its pathophysiology is based on molecular and cellular neuronal abnormalities that include senile plaques and neurofibrillary tangles with a chronic inflammatory background [1]. One of the main factors involved in the development and progression of AD is a cerebral inflammatory process. Some inflammatory biochemical markers seem to be elevated in patients with AD when compared with healthy subjects of the same age. The plasmatic levels of interleukin 6 (IL-6) and the cerebral levels of IL-6, interleukin 1 Beta (IL-1β) and tumor necrosis factor alpha (TNF-α), were increased in these patients [2].

Dental infections, especially periodontitis, are associated with dementia and are risk factors for the onset or the aggravation of AD [3 –6]. Chronic periodontitis is an infectious-inflammatory disease caused by bacteria that destroy the connective and bone tissue of the periodontium [7]. This chronic infection generates chronic inflammation with the release of local and systemic inflammatory and immune mediators such as cytokines, which are elevated in the serum of patients [8]. Besides, oral infections at the mucosa, such as candidiasis, are more frequent in AD. They are also able to increase serum IL-6 and TNF-α levels [9]. Thus, the objective of this study was to investigate the prevalence of oral infections and serum levels of IL-6 and TNF-α in patients with AD, mild cognitive impairment (MCI), and non-demented elderly.

MATERIALS AND METHODS

Subjects

Twenty-five patients with AD and 19 patients with MCI diagnosed according to the criteria of the National Institute for Communicative Disorders and Stroke (Alzheimer’s Disease and Related Disorders Association) (NINCDS-ADRDA) [10] were evaluated. The diagnosis was performed by an experienced neurologist of the Group of Behavior and Cognitive Neurology of the Hospital das Clinicas (Medical School of the University of Sao Paulo). The patients were compared with 21 healthy subjects. They were non-demented elderly that had been followed at the Group of Multidisciplinary Assistance of Elderly of the Geriatric Clinic at the same hospital. All patients, controls, relatives and/or guardians were informed about the purposes of the study and all signed the informed consent. The local Ethics Committee approved the protocol.

Patients with AD, MCI, and controls were matched according to educational status, age, gender and the prevalence of hypertension and diabetes (p > 0.05; SPSS 17.0; SPSS Inc., Illinois, USA). A single trained neurologist who was blinded to the dentist evaluation performed the neurological evaluation of all subjects.

Exclusion criteria were other neurodegenerative or neurological conditions (e.g., Parkinson’s disease, multiple sclerosis, other causes of dementia, neural infections such as Hansen’s disease, stroke) and cerebral, facial, or cervical tumors.

Protocol of evaluation

A single trained dentist performed the complete oral exam and clinical questionnaire to identify absences of teeth and aspects of the oral mucosa, tongue, and orofacial pain [11]. The decayed, missing, and filled tooth index was determined as well as the O’Leary plaque index (PI). The periodontal probing was performed to determine the gingival bleeding index (BI), probing pocket depth (PPD), the clinical attachment level (CAL), and cement-enamel junction distance (CEJ) [12, 13].

The serum level of cytokines (IL-6, IL-1β, and TNF-α) were investigated by venous blood samples of the subjects, acquired concomitant to clinical exam. The blood samples were obtained in dry tubes of 9 mL (Vacuette^®) and immediately centrifuged, and the serum was immediately stored in a –80°C freezer. After the storage of all samples, a multiplex panel (MILLIPLEX map High Sensitivity Human Cytokine Panel^®) was used to perform the cytokine analysis.

Statistical analysis

Data were tabulated and analyzed according to means, standard deviations, frequencies, and normality tests for the distribution of quantitative variables (Shapiro-Wilk test and Q-Q plot). Variables with normal distribution were analyzed with one-way ANOVA, MANOVA and Pearson’s correlation coefficient. Non-parametric chi square test was also performed. The level of significance was 5%.

RESULTS

The demographic characteristics and the results of cognitive and functional evaluation are illustrated in Table 1. Patients with AD had higher impairment of cognition and function than the other groups (p < 0.001).

AD patients showed higher prevalence of candidiasis as well as coated tongue (Table 2). There were no differences in periodontal indexes. Patients with AD and MCI had more absent teeth (p = 0.032) and a longer duration of edentulism (p = 0.032) than healthy elderly (Table 3). The main cause for tooth loss in AD patients was periodontal disease (P < 0.001); in opposition, decay was the main cause of tooth loss of the MCI group and the control group (Table 3).

The cytokine levels (IL-1β, TNF-α, and IL-6) can be observed in Fig. 1. Patients with AD had significantly higher IL-6 levels than controls (p = 0.029), and patients with periodontal disease had significantly higher TNF-α levels than patients with healthy periodontal tissues (p = 0.005). The multivariate analysis showed an association between IL-6 and TNF-α in patients with AD or MCI and periodontal disease (p = 0.023).

The concentration of IL-6 was negatively correlated with the MMSE score (p = 0.018; Pearson) and positively correlated with the Pfeffer’s Functional Assessment Questionnaire (FAQ) (p < 0.001; Pearson), which means that the high serum IL-6 level was observed in subjects with low performance in cognitive tests. The concentration of TNF-α was positively correlated with the periodontal indexes PPD, CAL, BI (p < 0.001; Pearson), and PI (p = 0.008; Pearson), which means that patients with worse periodontal condition had higher serum TNF-α level. Finally, IL-6 and TNF-α were positively correlated in all studied groups (p < 0.001; Pearson).

DISCUSSION

This study shows that the elevated concentration of IL-6 in patients with AD was associated with high serum TNF-α level of subjects with periodontal disease. This finding suggests that immune-inflammatory mechanisms of periodontitis may underlie its role in the onset, progression or aggravation of AD. IL-6 is a biomarker for cognitive impairment [14], and TNF-α might be a biomarker of the association of periodontitis with AD by these results.

The etiology of AD is not yet completely elucidated, and the involvement of chronic inflammation in its pathophysiology is still controversial. The cerebral inflammatory process could be a consequence of the deposits of senile plaques and neurofibrillary tangles, and its immune-inflammatory mediators might accelerate the neurodegenerative process [15, 16]. This hypothesis is supported by the evidence of IL-6 and C reactive protein as biomarkers of cognitive impairment [14 , 18].

Common immune-inflammatory backgrounds of neurodegeneration in AD and bone destruction in periodontitis supports their overlapping. Oral infections are frequent in the adult population and their systemic impact is important, supporting prevention and early treatment to avoid complications, such as AD, at old age [3 , 19–21]. Systemic inflammation of periodontitis is implicated in the progression and aggravation of several chronic illnesses [5], and it includes dementia.

The present results are in agreement with previous observations showing the association of early tooth loss due to periodontal disease with dementia [22]. The main cause of tooth loss was periodontitis, which corroborates to the association of cytokine expression and AD. The impact of tooth loss on nutrition, especially when occurring under the age of 35 is another risk factor of AD [23]. Oral rehabilitation with prostheses is compulsory to improve nutritional status that have an implication in general health and in prevention of cognitive deficit. Thus, oral diseases may play a role in AD by immune-inflammatory and indirect nutritional consequences [24]. Besides systemic inflammatory mechanisms, oral bacteria can reach the cerebral tissue and induce chronic local infection and inflammation in the brain [25].

In the present population studied, there were no statistical differences in the prevalence of dental diseases and periodontal parameters, except infections of the oral mucosa, a finding similar to earlier observations, which was the subject of a previous publication [11]. In that previous publication, the severity of periodontitis was not different but its prevalence was higher in patients with AD. Although the current evaluation showed similar prevalence of periodontitis between studied groups, the previous history of causal factors for dental extractions showed periodontitis as the reason for teeth loss only in AD patients. However, candidiasis can also increase the evaluated cytokines in blood serum [9]. We suggest monitoring blood serum levels of IL-6 and TNF-α and evaluating the clinical signs of periodontitis and other oral infections in AD is essential.

TNF-α, IL-1β, and IL-6 are capable of stimulating the synthesis of Aβ-amyloid 1-42 peptide (Aβ₄₂) and the phosphorylation of tau protein (P-tau), and Aβ₄₂ and P-Tau can stimulate the production of TNFα, IL-1β, and IL-6 by glial cells [26]. Thus, periodontitis and oral mucosa infections, as potential causes of TNF-α increase in the blood serum, is a candidate for a modifying factor for the development of AD [19, 21].

The world’s population got older in the last century, and as dementia is becoming more prevalent, it is becoming a public health problem. Co-morbidities are frequent and may play a role in the aggravation of symptoms. Non-modifiable risk factors include age, presence of APOE ɛ4, and family history [27]. Oral infections can be assessed, treated, and prevented [11 , 29], and their blood serum biomarkers (IL-6 and TNF-α) may help in the analysis of their risks in dementia.

In conclusion, this case-control transversal study shows evidence of the association between cytokines expression in the blood serum with oral infections and AD. The next step is to investigate the role of periodontitis and oral mucosa infections in the incidence of dementia in longitudinal studies, to elucidate the mechanisms underlying the overlap between TNF-α and IL-6 in neurodegeneration. Examining clinical implications is necessary to prevention, and the treatment of oral infections in adults and the elderly will improve the quality of aging because these infections potentially have a role as risk factors for AD.

Footnotes

ACKNOWLEDGMENTS

This study was supported by FAPESP (Foundation for Research of the State of Sao Paulo – 2011/18756-9).

Authors’ disclosures available online ().

References

Querfurth

, LaFerla

(2010) Alzheimer’s disease. N Engl J Med 28, 329–344.

Rogers

(2008) The inflammatory response in Alzheimer’s disease. J Periodontol 79, 1535–1543.

Kamer

(2010) Systemic inflammation and disease progression in Alzheimer disease. Neurology 74, 1157–1158.

Rethman

(2010) Inflammation in chronic periodontitis and significant systemic diseases. J Calif Dent Assoc 38, 247–257.

Hatipoglu

, Kabay

, Güven

(2011) The clinical evaluation of the oral status in Alzheimer-type dementia patients. Gerodontology 28, 302–306.

Mancini

, Grappasonni

, Scuri

, Amenta

(2010) Oral health in Alzheimer’s disease: A review. Curr Alzheimer Res 7, 368–373.

Tatakis

, Kumar

(2005) Etiology and pathogenesis of periodontal diseases. Dent Clin North Am 49, 491–516.

Bretz

, Weyant

, Corby

, Ren

, Weissfeld

, Kritchevsky

, Harris

, Kurella

, Satterfield

, Visser

, Newman

(2005) Systemic inflammatory markers, periodontal disease, and periodontal infections in an elderly population. J Am Geriatr Soc 53, 1532–1537.

Pietruski

JK1

, Pietruska

, Jabłońska

, Sacha

, Zaremba

, Stokowska

(2000) Interleukin 6, tumor necrosis factor alpha and their soluble receptors in the blood serum of patients with denture stomatitis and fungal infection. Arch Immunol Ther Exp 48, 101–105.

10.

Mckhann

, Drachman

, Folstein

, Katzman

, Price

, Stadlan

(1984) Clinical diagnosis of Alzheimer’s disease: Report of the NINCDS-ADRDA work group under the auspices of department of health and human services task force on Alzheimer’s disease. Neurology 34, 939–944.

11.

Rolim

, Fabri

, Nitrini

, Anghinah

, Teixeira

, de Siqueira

, Cestari

, de Siqueira

(2014) Oral infections and orofacial pain in Alzheimer’s disease: A case-control study. J Alzheimers Dis 38, 823–829.

12.

American Academy of Periodontology (2000) Parameter on chronic periodontitis with slight to moderate loss of periodontal support. J Periodontol 71, 853–855.

13.

American Academy of Periodontology (2000) Parameter on chronic periodontitis with advanced loss of periodontal support. J Periodontol 71, 856–858.

14.

Engelhart

, Geerlings

, Meijer

, Kiliaan

, Ruitenberg

, van Swieten

, Hofman

, Witteman

, Breteler

(2004) Inflammatory proteins in plasma and the risk of dementia. Arch Neurol 61, 668–672.

15.

Rosenberg

(2005) Clinical aspects of inflammation in Alzheimer’s disease. Int Rev Psychiatry 17, 503–514.

16.

Tan

, Beiser

, Vasan

, Roubenoff

, Dinarello

, Harris

, Benjamin

, Au

, Kiel

, Wolf

, Seshadri

(2007) Inflammatory markers and the risk of Alzheimer disease: The Framingham Study. Neurology 68, 1902–1908.

17.

Dimopoulos

, Piperi

, Salonicioti

, Mitropoulos

, Kallai

, Liappas

, Lea

, Kalofoutis

(2006) Indices of low-grade chronic inflammation correlate with early cognitive deterioration in an elderly Greek population. Neurosci Lett 398, 118–123.

18.

Kravitz

, Corrada

, Kawas

(2009) Elevated C-reactive protein levels are associated with prevalent dementia in the oldest-old. Alzheimers Dement 5, 318–323.

19.

Kamer

, Craig

, Dasanayake

, Brys

, Sobanska

, Leon

(2008) Inflammation and Alzheimer’s disease: Possible role of periodontal diseases. Alzheimers Dement 4, 242–250.

20.

Kamer

, Craig

, Pirraglia

, Dasanayake

, Norman

, Boylan

, Nehorayoff

, Glodzik

, Brys

, de Leon

(2009) TNF-α and antibodies to periodontal bacteria discriminate between Alzheimer’s disease patients and normal subjects. J Neuroimmunol 216, 92–97.

21.

Watts

, Crimmins

, Gatz

(2008) Inflammation as a potential mediator for the association between periodontal disease and Alzheimer’s disease. Neuropsychiatr Dis Treat 4, 865–876.

22.

Okamoto

, Morikawa

, Okamoto

, Habu

, Iwamoto

, Tomioka

, Saeki

, Yanagi

, Amano

, Kurumatani

(2010) Relationship of tooth loss to mild memory impairment and cognitive impairment: Findings from the Fujiwara-kyo study. Behav Brain Funct 6, 77–84.

23.

Stewart

, Sabbah

, Tsakos

, D’Aiuto

, Watt

(2008) Oral health and cognitive function in the Third National Health and Nutrition Examination Survey (NHANES III). Psychosom Med 70, 936–941.

24.

Sparks Stein

, Steffen

, Smith

, Jicha

, Ebersole

, Abner

, Dawson

3rd (2012) Serum antibodies to periodontal pathogens are a risk factor for Alzheimer’s disease. Alzheimers Dement 8, 196–203.

25.

Riviere

, Riviere

, Smith

(2002) Molecular and immunological evidence of oral Treponema in the human brain and their association with Alzheimer’s disease. Oral Microbiol Immunol 17, 113–118.

26.

Gosselin

, Rivest

(2007)Brain Behav Immun 21, 281–289 Role of IL-1 and TNF in the bra, Twenty years ofrogress on a Dr Jekyll/Mr Hyde duality of the innate immune system. .

27.

Bird

(2005) Genetic factors in Alzheimer’s disease. N Engl J Med 352, 862–864.

28.

Avlund

, Holm-Pedersen

, Morse

, Viitanen

, Winblad

(2004) Tooth loss and caries prevalence in very old Swedish people: The relationship to cognitive function and functional ability. Gerodontology 21, 17–26.

29.

Syrjälä

, Ylöstalo

, Sulkava

, Knuuttila

(2007) Relationship between cognitive impairment and oral health: Results of the Health 2000 Health Examination Survey in Finland. Acta Odontol Scand 65, 103–108.