Porphyromonas gingivalis, a periodontitis causing bacterium, induces memory impairment and age-dependent neuroinflammation in mice

Periodontium is composed of gingiva, periodontal ligament, alveolar bone and cementum. Infection of the periodontium, known as periodontitis, is a chronic peripheral inflammatory disease, initiated by microbes residing in the oral cavity. It is commonly caused by specific bacteria, such as P. gingivalis, a Gram-negative bacterium, which is a key periodontal pathogen. P. gingivalis and its toxic components, including fimbria, gingipains, and lipopolysaccharide (LPS), are closely related to periodontitis. Clinically, chronic periodontitis is characterized by the presence of gingival erythema, edema, periodontal pockets, and destruction of the tissue supporting the teeth [1‐3]. Periodontal microorganisms and their products may enter into the circulation leading to bacteremia and systemic dissemination of bacterial products [4]. Moreover, periodontitis can induce systemic effects by promoting the expression of inflammatory mediators such as pro-inflammatory cytokines. Thus, periodontitis has been confirmed to be associated with systemic diseases including cardiovascular disease, diabetes, atherosclerotic, and respiratory diseases [5].

In recent years, AD, well known as a progressive neurodegenerative disease, has been recognized as the leading cause of cognitive and behavioral damage [6]. It has been increasingly claimed that peripheral infections could activate already primed microglial cells within the central nervous system (CNS) which promotes the development of neurodegeneration in AD [7]. The mechanism by which peripheral pro-inflammatory molecules might increase the brain’s molecular inflammatory pool involves at least two pathways, the systemic circulation and/or the neural pathways. Once in the brain, pro-inflammatory molecules might directly elevate the expression levels of the pro-inflammatory cytokine pool locally, or indirectly activate glial cells by regulating the secretion of additional pro-inflammatory cytokines. Therefore, Kamer et al. first proposed that the pool of the inflammatory molecules in the brain could be enhanced by periodontitis, which is characterized by elevated inflammatory levels, and consequently promoting the development of AD [5]. Recently, there are increasing studies supporting this hypothesis. A close relationship between immunological mediators, such as TNF-α and antibodies against periodontal pathogens, and AD, has been reported. Furthermore, these mediators can be used as AD diagnostic factors [8]. In addition, a report stated that poor dentition is associated with cognitive impairment [9]. A positive correlation between cognitive decline in AD patients and both acute and chronic inflammation was revealed by a human trial [10]. Moreover, a study conducted by Sophie et al. reported that LPS from periodontal pathogens could gain access to the brain tissue of AD patients during life, demonstrating the vital role of inflammatory factors in the pathology of AD [11]. Therefore, these reports suggest a possible connection between periodontitis and AD. However, most of the previous studies have not demonstrated a clear causative relationship between periodontitis and cognitive impairment. The experimental model used in this study was more representative of periodontitis infection as it utilizes the entire P. gingivalis in order to take into consideration all the bacteria components and secreted compounds which might be contributing to the development of periodontitis and might also affect cognitive memory. Therefore, in this study we test our hypothesis that periodontitis may cause cognitive impairment via age-dependent neuroinflammation in the P. gingivalis infection animal model.

Periodontitis is a chronic inflammatory disease, which could induce systemic host responses. Numerous reports have shown that periodontitis could raise the serum pro-inflammatory state, characterized by increased levels of C Reactive Protein (CRP) and pro-inflammatory cytokines (e.g. TNF-α), and decreased levels of anti-inflammatory markers (e.g. IL-10) [17]. Several studies have reported that peripheral inflammation could activate microglia cells and promote the generation of pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α, in the brain, resulting in neuroinflammation [18]. Neuroinflammation, including activation of microglia cells, participation of astrocytes, and involvement of neurons, has been suggested to contribute to the development of neurodegenerative diseases such as AD, Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis [14, 15, 19, 20]. Therefore, many studies focus on the correlation between periodontitis and AD. In addition, several reports support the hypothesis that periodontal inflammation can affect cognition. Kamer et al. found that older Danish adults with periodontitis or severe tooth loss exhibit impaired cognition in comparison to healthy subjects [4]. Animal studies have also provided evidence on the impact of tooth loss on neuroinflammation and cognition. Female transgenic mice after tooth extraction were significantly impaired in learning and memory abilities which confirmed that neuronal cell loss in the hippocampus could be triggered by tooth loss causing memory impairment [21]. Although tooth loss can occur for several reasons, periodontitis is one of the major causative factors [22]. Additionally, P. gingivalis has been shown to secrete other components associated with periodontitis, which might also affect cognitive function [23]. These results indicate that periodontitis may impair cognition. However, there is limited information on the association between P. gingivalis infection and cognition. In the present study, we have observed that P. gingivalis infection elevated the expression levels of the pro-inflammatory cytokines TNF- α, IL-6, and IL-1β in the brains of middle-aged mice. It was also noted that middle-aged P. gingivalis infected mice displayed impaired learning and memory abilities. These findings provided further evidence for supporting the association between P. gingivalis periodontal infection and cognitive impairment.

In order to assess the learning and memory abilities of mice, behavioral tests are required. The MWM test was used to assess learning and memory [24]. It is a maze where the animals must search for a hidden platform which is submerged under the water surface and placed in a fixed location [25]. Our setup is free from motivational stimuli, like deprived food and water, electrical stimuli, and buzzer sounds, which are likely to impact the normal process of memory. Thus, this task is more precise than active/passive avoidance tasks as it eliminates factors which may interfere with the learning and memory like visual acuity and motor function [12, 26]. In general, the MWM is an effective and accurate test for assessing learning and memory abilities. The escape latency data could be interpreted as spatial learning. During the training days, mice spent less time finding the platform, indicating that they have learned and memorized the location of the platform [26]. The number of times of crossing the platform is regarded as the evaluation outcome of the memory assessment of the mice, and a reduction of the crossing times through the platform indicates memory impairment. In our present study, the results showed that the escape latency and the crossing times were statistically significant different between middle-aged mice with P. gingivalis periodontal infection and control mice. Therefore, it is possible that P. gingivalis periodontal infection promotes age-dependent neuroinflammatory responses though pro-inflammatory cytokines release.

It has been demonstrated that inflammation induces alterations in neurovascular functions, resulting in an increase in the blood–brain barrier permeability, reduction of nutrient supplements, and aggregation of toxins. Elevated levels of pro-inflammatory mediators in the blood, such as IL-β and TNF-α, can lead to their direct or indirect transport to the brain, which might accelerate the development of brain impairment [27]. During the process of neuroinflammation, pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and others) are essential neuroinflammation mediating signaling molecules [28]. TNF-α has been reported to play a pivotal role in the development and functions of the CNS, including neuron compliance, cognition, and behavior [29]. In the brain, trauma, infection, or the presence of endogenic abnormal protein aggregates, such as amyloid-β (Aβ) peptides in AD, can activate the secretion of TNF-α, primarily produced by glial cells. In addition, TNF-α has been proven to activate immune/glial cells leading to the augment of amyloid-β precursor protein and Aβ deposits in vitro [20]. One study demonstrated that a single intraperitoneal LPS injection in mice caused peripheral TNF-α expression, both at the mRNA and the protein levels, in the brain [30]. As a key molecule, IL-1β is capable of triggering the production of various inflammatory mediators via activating microglia cells. Thus, there is a close correlation between IL-1β levels and neuroinflammation in AD [31]. Some reports have deduced that afferent neurons can respond directly to peripheral cytokines, like IL-1, for vagal sensory nerve activation. IL-1β has been proven to promote the transformation of Aβ from its non-fibrillar form to insoluble Aβ fibers, leading to increase in plaque formation [32]. Concerning IL-6, its up-regulation in the brain of transgenic mice has been shown to be associated with severe neurological dysfunction. Another study using radial arm maze test to examine the spatial learning of mice showed that IL-6 deficient mice exhibit better and faster acquisition of learning and memory abilities [14].

Our approach to study the association between periodontitis and cognition differed from previous studies, as we have used the P. gingivalis periodontal infection animal model. We have assessed the effect of the entire P. gingivalis on healthy mice, rather than assessing the individual components of P. gingivalis. In addition, we directly measured the mRNA and protein levels of TNF-α, IL-1β, and IL-6, in the brains of mice by qRT-PCR and ELISA. We found that the expression of these cytokines showed differences between young and middle-aged mice. Therefore, the effects of aging on neuroinflammation need to be considered. Most studies have used middle-aged or older mice for research purposes. In our study we chose both young and middle-aged mice and found that, unlike the middle-aged animals, the young mice with P. gingivalis periodontal infection neither displayed impaired cognition nor overexpressed pro-inflammatory factors. These differences between the young and middle-aged mice might be attributed to chronic inflammation due to aging, which exerts additional stress to the brain nerve cells of older mice and makes them more vulnerable to infection [33]. Moreover, aging-related alterations, like decreased density and plasticity of synapses, and the amount of the pathological neurofibrillary tangle formation required to cause dementia, increase with age [34, 35]. Additionally, during systemic inflammation, the functions of the blood-cerebrospinal fluid barrier (BCSFB) were significantly affected by the differential responses of glial cells to age-dependent cytokines [36]. Recent reports showed that chronic systemic inflammatory processes promoted the transformation of microglia and astrocytes into anti-inflammatory cell types in young rats, while a pro-inflammatory cell phenotype was detected in middle-aged rats [37]. Furthermore, aging is the major risk factor of AD and is correlated with elevated glial responsiveness, which might increase the brain’s susceptibility to injury and disease [21, 38]. One study supports this view by showing that the age-associated progression of the AD-like phenotype in WT mice could be initiated by chronic inflammatory conditions with enhanced accumulations of APP [39].

The present study has some limitations. First, although over-activation of microglia has been reported to be a hallmark of neuroinflammation [40], we did not investigate the activation of microglia in our study. However, the objective of this paper was to determine whether P. gingivalis periodontal infection can promote cognitive impairment via inducing neuroinflammation, which was assessed by measuring the levels of the main pro-inflammatory cytokines. Second, the brain inflammation induced by P. gingivalis periodontal infection may be mediated via the systemic circulation and/or direct neural pathways. In this study we did not access which mediating pathway is involved, which needs to be considered in the future. In addition, although TNF-α, IL-6, and IL-1β are the most reported molecules to be implicated in neuroinflammation, they cannot represent the whole range of inflammatory cytokines. Therefore, other inflammatory cytokines associated with neuroinflammation remain to be considered.

We have shown that P. gingivalis periodontal infection may induce an age-dependent brain inflammation, which increases the TNF- α, IL-6, and IL-1 β levels in middle -aged mice. Moreover, the middle-aged P. gingivalis infected mice displayed impaired cognition. Although we have not demonstrated that periodontitis is a risk factor for AD, these findings showed that P. gingivalis periodontal infection can cause memory impairment, which suggests that periodontitis might have a similar effect on the development of AD.