Decreased salivary lactoferrin levels are specific to Alzheimer’s Disease

Alzheimer’s disease (AD) is one of the most devastating neurodegenerative disorders representing a major public health concern with more than 30 million people affected worldwide. The cause of the disease is still unknown but the most accepted hypothesis states that amyloid-β (Aβ) accumulation in the brain could initially trigger the pathological cascade.

Accumulated evidence suggests that bacterial and viral infections may be implicated in AD pathogenesis [, , , , ]. In the cascade of events preceding AD, oral and gastrointestinal microorganisms may play a role [,], and different types of microbes have shown to stimulate Aβ aggregation and deposition [,]. Therefore, it may exist an interplay between genetic and environmental risk factors including toxins and/or bacterial, viral and fungal pathogens in the sporadic form of late-onset AD reflecting its complex and multifactorial aetiology [].

The question of whether oral infections could be considered as a risk factor for AD has generated considerable research in recent years. Antimicrobial proteins and peptides (APPs), also called host defence agents, are the primary effector molecules of innate immunity. A novel role for APPs has been proposed in AD pathology. The emerging role of microbes and innate immune pathways in AD pathology also suggests that APPs may be considered for early therapeutic interventions in future clinical trials. Pathogen-targeting agents and brain infections markers are involved in amyloid aggregation, reinforcing the possible relationship between AD and brain infections [,].

Biomarkers reflecting innate immune system integrity could therefore be useful for both, accurate and early diagnosis, as well as disease prognosis. A promising biomarker candidate is lactoferrin (Lf), an iron-binding protein belonging to the transferrin family and expressed in all body fluids, particularly in exocrine fluids, tears or saliva []. Lf has a wide variety of physiological functions including, antioxidant activities, neuroprotective properties, regulation of the immune response, anti-inflammatory and anti-carcinogenic potential [, , , , ]. In addition, Lf has been previously detected in senile plaques, neurofibrillary tangles [] and microglia from AD brains []. Since Lf is one of the major antimicrobial peptides in saliva, it further represents an important defensive element by inducing a broad-spectrum of antimicrobial effects against bacteria, fungi, protozoa, viruses and yeasts []. The antimicrobial effects of Lf are conferred by its highly positive charged N-terminal region []. These functions are maintained by its hydrolysis products, a number of Lf-derived peptides that by retaining the cationic N-terminal region of the native protein also retain many of the Lf activities and in some cases, they can be even more potent than the parent protein [,].

We have previously shown decreased Lf levels in saliva samples from mild cognitive impairment (MCI) and AD patients compared with normal elderly subjects []. In the present study, we have evaluated the diagnostic utility of salivary Lf in a well-characterised cohort of AD and frontotemporal dementia (FTD) patients, as well as in normal elderly subjects. Results have been lately validated in an independent cohort of MCI patients and healthy controls. In order to assess the salivary Lf specificity and its relationship with the pathophysiological process of AD, only participants with known amyloid-Positron-Emission Tomography (PET) status were included.

Here we show that reduced salivary Lf levels seem to be specific to AD as such observation is not extended to healthy elderly controls or FTD, as example of different dementia, neither to advanced age healthy controls. We demonstrate that reduced Lf levels are associated to positive amyloid PET result, one of the available pathophysiological validated AD biomarkers. These findings agree with our previously reported study in which reduced salivary Lf levels were found in amnesic MCI and AD patients [].

Brain Aβ aggregation is thought to play a major role in AD aetiology, but the amyloid cascade hypothesis cannot explain the pathological process of AD by itself. In 2016, a hypothesis postulated that brain infections might play an initial role in amyloid plaque formation and the development of AD [], proposing a potential novel role for APPs in AD pathophysiology []. These infections lead to persistent inflammatory stimuli altering the immune response []. Different lines of evidence suggest that AD-affected brains host virus, including herpes virus and hepatitis virus [, , , , ], and bacteria, including Chlamydia, Borrelia spirochetes, or Helicobacter Pylori []. Those bacterial and viral populations in the brain might induce Aβ aggregation, triggering the AD pathophysiological process [, , , , , , . However, it remains unclear whether the entry of pathogens occurred at the onset of AD or it may be the consequence of a leaky blood brain barrier (BBB) induced by chronic infections-derived inflammatory processes or, ultimately, if it is secondary to AD itself. It is important to recognise that infections can occur decades before evident signs of dementia. In agreement with this hypothesis, a number of risk factors, including chronic infections, obesity, diabetes, depression and stress among others, are known to contribute down-regulating the innate immune system and thus increasing the risk for infections. On this regard, is interesting to mention the recent work carried out by Ilievski and colleagues in which they have found a connection between oral infection and the development of AD-like pathology []. The authors orally administered Porphyromonas gingivalis (Pg) and/or its product gingipain to 8-month-old C57/BL6 mice for 22 weeks. Then, they found hippocampal and cortical neuropathology including Aβ plaques, neurofibrillary tangle formation, astrogliosis and microgliosis in the treated mice, but not in the control group []. More recently, Dominy and colleagues demonstrated the presence of Pg DNA and gingipain antigens in brains from AD patients [].

Oral homoeostasis is dependant upon saliva and its protein content. An alternative approach to understand the AD aetiopathogenesis has been recently reported: a potential microbial infection and innate immune origins for the disease []. This approach opened the way for a paradigm shift in studying a new “infection hypothesis,” or perhaps more broadly, “innate immune system dysregulation hypothesis”. It suggests the role of chronic infections in the development of AD, as well as a potential deregulation of the human innate immune response to these infections, as a plausible route for new investigations of the aetiology and pathophysiology of AD []. Although AD is a complex disease and microbial infection may not be the sole cause, this new paradigm may provide novel targets for the prevention and treatment of this devastating disease.

The dissemination of oral microorganisms in the brain is controlled by APPs, as part of the innate immune system. In the last decade, several studies have explored the role of these APPs as potential biomarkers for AD []. In 2017, we showed that salivary Lf levels, one of the major antimicrobial proteins in saliva, discriminates between patients with MCI and AD and control subjects []. Although Lf cleavage generates antimicrobial peptides, sometimes with higher antimicrobial capacity, the protein itself has antimicrobial effects as these properties are conferred by its highly positive charged N-terminal domain and this domain is retained by its generated peptides. These differences come from the observation of Lf-derived peptides exerted higher antibacterial capacity compared to native Lf. However, that was not the case of the antiviral capacity as several works demonstrated that mature protein has much more effect []. APPs are defensive agents constitutively expressed, but they can be modulated during inflammation or a microbial challenge, including chronic bacterial or viral infections. APPs have been studied for decades for their action against pathogens, and their immunostimulatory effects []. As part of the innate immune system, APPs are defensive peptides that enter the brain and efficiently target pathogens through synergistic pathways []. The normal production of APPs may be reduced because of a number of factors such as a weakened immune system or damaged defence cells. We then hypothesise that reduced levels of salivary Lf could reflect compromised immunity resulting in an enhanced risk of brain infections. Lf is known to be transferred from the sublingual mucosa to the brain []. In this context, favourable Lf effects on brain are expected, and they could be related to its antimicrobial capacity but also they may stimulate the non-amyloidogenic processing of APP and the α-secretase catalytic activity and expression in a mouse model of AD [].

Human Lf exerts multifunctional effects displaying extensive antimicrobial, anti-inflammatory and immunomodulatory activities. Lf may deprive microorganisms such as bacteria, yeasts and parasites binding and sequestering iron, which is essential for their growth []. Lf has different iron binding statuses: the iron-free (apo) form, the native form, and the iron-saturated (holo) form. It has been shown that both, the apo-Lf form as well as the holo-Lf form mediate agglutination of microorganisms indicating that Lf possess strong iron-binding independent bacteriolytic properties []. Moreover, previous studies also demonstrated that Lf showed antioxidant effects depending on its iron binding status []. Oxidative stress or damage such as protein oxidation, lipid oxidation, DNA oxidation, and glycoxidation is closely associated with the AD development []. The antioxidant effect of Lf could be explained as Lf restrains membrane lipid peroxidation by scavenging free iron radicals. These iron radicals are cytotoxic activators of lipid peroxidation and oxidative stress. It has been shown that Lf simultaneously enhances antioxidant activity by reducing ROS and enhancing SOD1 and reducing inflammatory markers in AD mice []. In a recent study with AD patients, Lf treatment showed significant improvements in antioxidant and anti-inflammatory markers in serum, and reduced Aβ42 and phosphorylated tau levels, as well as improved cognitive function []. Lf antioxidant activities have also been described in PD experimental models in which this protein was able to prevent oxidative stress and to upregulate the expression of antioxidant enzymes. Simultaneously, the expression of glial markers and proinflammatory cytokines were found reduced [, ]. Therefore, a boosting effect of Lf may be potentially observed when this protein exerts its antioxidant properties through its iron scavenging abilities and this effect would potentiate the immune response [].

In the present study, we report that salivary Lf levels are significantly reduced in AD patients but not in patients with other dementia such as FTD, suggesting a specific Aβ-related link. To our knowledge, this is the first study at examining the relationship between salivary Lf and brain Aβ plaques measured in vivo by amyloid-PET neuroimaging. Amyloid-PET has emerged as a tool for the diagnostic accuracy of prodromal AD, as amyloid deposition occurs years before symptoms onset. MCI encompasses a heterogeneous syndrome with several aetiologies including neurodegenerative diseases, vascular, metabolic or psychiatric illness [, . Inaccuracy of clinicopathological correlation and common coexistence of different pathophysiological processes make difficult in most cases to establish a suitable diagnosis []. Furthermore, MCI without evidence of AD pathophysiology continues to be a mixed and frequently poorly defined group. In our study, Lf showed high variability levels in this group of patients, likely reflecting their biological diversity.

We show that the presence of a positive amyloid-PET signal, that indicates high amyloid plaque load, is highly associated with reduced levels of salivary Lf. These findings support the hypothesis that AD pathophysiology may be related to salivary levels of Lf and the innate immune response to brain infections. Innate immune pathways are commonly activated in other neurodegenerative disorders [, , , ]. Therefore, it is remarkable that salivary Lf levels will not decrease in other dementia such as FTD. A possible explanation could be that the hypothalamic region, that controls body innate immunity, [] is affected in AD [, , , ], but not in FTD. In addition to amyloid plaques and neurofibrillary tangles, functional studies suggest that hypothalamic dysfunction is a common event in AD, often early in the course of disease []. Although there are evidences indicating that certain hypothalamic regions are also affected in FTD, specifically those that correlate with abnormal eating behaviours [], they are different to those affected in AD. The apparently AD-specific salivary Lf reduction may thus not only be useful in the differential diagnosis but could also provide important insights into selective immune vulnerability in neurodegenerative diseases. The secretion of salivary proteins is controlled by cholinergic parasympathetic nerves that release acetylcholine, evoking the secretion of saliva by acinar cells in the salivary gland []. These parasympathetic nerves are connected with the hypothalamus [, , , ]. We propose that early hypothalamic Aβ accumulation may disrupt hypothalamic function affecting salivary gland regulation that ultimately results in reduced salivary Lf secretion.

In 2011, the NIA-AA guidelines proposed a framework for the use of imaging and CSF biomarkers in the preclinical, MCI and dementia stages of AD [,,,]. However, these current biomarkers used in AD research are expensive or invasive, making difficult or impossible to include them in the routine clinical use in non-specialised health settings. Thus, the present research framework concludes the urgent need for the development of early diagnostic methods, incorporating new biomarkers that can accurately detect and diagnose AD. In recent years, there have been intensified efforts in searching minimum or non-invasive peripheral biomarkers in blood or saliva, for the early diagnosis of AD. Our present work show that salivary Lf levels are decreased in AD and are associated with amyloid-PET imaging profile even in prodromal disease, assuming the multifactorial and heterogeneous aetiology of dementia. The good correlation found in our study between amyloid-PET status and saliva Lf levels suggest that this inexpensive and easy-to-access measurement could be a potential candidate-screening tool in patients with cognitive impairment or suspicion of AD dementia.

In this study, we analysed Lf levels in cohort 1 where patients and control subjects were recruited from regions in Central and South of Spain. Second, the analysis was validated using the cohort 2 composed of donors recruited from Central Spanish region. Our present data demonstrates that the accuracy of salivary Lf as AD biomarker remains in these two separated geographic areas, suggesting that salivary Lf levels can be considered as reproducible biomarker and they may not be affected by other possible influences, including lifestyle and exposure to environmental factors. Other works showed on the contrary, that salivary Lf concentrations might be affected by circadian rhythms and physical activity [, , ], or environmental and lifestyle factors, including habits like smoking and medication-intake [, , ]. Therefore, it will be important to replicate these findings in larger-scale prospective and longitudinal studies with other different cohorts even with much more geographic separation. This will allow to accurately analysing the influence of other potential confounding variables.

In conclusion, to our knowledge, this is the first study that assesses the diagnostic performance and specificity of a salivary-based single biomarker to detect prodromal AD and AD dementia based on their amyloid-PET biomarker profile. Importantly, our findings have been replicated in an independent cross-sectional study able to replicate the diagnostic performance of saliva Lf concentration. Finally, this study also proposes that salivary Lf represents one of the main/first lines of defence against pathogens, and that low Lf levels in saliva may represent a reduced protection exacerbating the risk of AD.

We are grateful to the patients and donors without whom these works would not have been possible. We thank also Dr. Alberto Lleo and Dr. Pablo Martinez-Lage for their scientific discussion.

This study was supported by Dr. Carro grants from Instituto de Salud Carlos III (FIS15/00780, FIS18/00118), FEDER, Comunidad de Madrid (S2017/BMD-3700; NEUROMETAB-CM), and CIBERNED (PI2016/01). This study was also supported by research grants from the Spanish Ministry of Economy and Competitiveness (SAF2017-85310-R to Dr. Cantero, PSI2017-85311-P to Dr. Atienza); International Centre on ageing CENIE-POCTEP (0348_CIE_6_E to Dr. Atienza); and CIBERNED (CB06/05/1111 to Dr. Cantero). Dr. Bueno receives research funding from the Instituto de Salud Carlos III, Spain (PIE16/00021, PI17/01799). The H2H-Spain Study was supported in Spain by grant PIE16/00021 from Instituto Carlos III, Ministry of Science, Innovation and Universities, and additional funds from the Centro Nacional de Investigaciones Cardiovasculares (CNIC). The CNIC is supported by the Ministry of Economy, Industry and Competitiveness and the Pro CNIC Foundation, and is a Severo Ochoa Centre of Excellence (SEV-2015–0505). The funders had no role in the conceptualisation, study design, data collection analysis and preparation of this manuscript.

Dr. Perry has served as a consultant for Neurtrope, Neurotez, Investiture. Dr. Bueno receives research funding from Astra-Zeneca, BMS, Janssen and Novartis; has received consulting fees from Astra-Zeneca, Bayer, BMS-Pfizer, Novartis; and speaking fees or support for attending scientific meetings from Astra-Zeneca, Bayer, BMS-Pfizer, Ferrer, Novartis, and MEDSCAPE-the heart.og. Dr. Carro and Dr. Orive are co-founders of GEROA Diagnostics. No other disclosures were reported.