Major sources of iron dysregulation stem from externally induced stressors (Kell and Pretorius, 2018). This form of iron dysregulation can be initiated by several factors that contribute to or cause cell death, such as mechanical damage (Zhang et al., 2013), nutritional stress (Schaffer, 2016), pharmacological stress (Primohamed et al., 2004), and of course oxidative stress (Kerley et al., 2018). Another source of free iron is via heme metabolism, due to the functioning of heme oxygenase-1 (HO-1), which catalyzes the degradation of heme (Pretorius and Kell, 2014). Since upregulation of HO-1 activity occurs in systemic inflammatory disorders in which erythrocytes are lysed, it may also be an important marker of inflammation and iron dysregulation.

Additionally, hepcidin, produced by the liver, is a key regulator of iron metabolism (Michels et al., 2015; Reichert et al., 2017). Decreases in hepcidin levels enhance surface exposure of ferroportin (Ganz and Nemeth, 2012) on enterocytes, macrophages and hepatocytes to increase serum ferritin levels (illustrated by Figure 2). Hepcidin expression is induced by inflammatory markers such as LPS, IL-1β, and IL-6, while increases in 1,25(OH)₂D₃ (calcitriol) levels cause hepcidin levels to decrease (Kell and Pretorius, 2018). According to a report by Bacchetta et al. (2014), decreases in hepcidin levels by 1,25(OH)₂D₃ are due to suppression of the HAMP gene by the vitamin D receptor (VDR). Chromatin immunoprecipitation assays confirmed the binding of VDR to the vitamin D response element within the proximal promotor region of the HAMP gene (Bacchetta et al., 2014). While this process is intricate, it appears that alterations in vitamin D metabolism could potentially instigate iron dysregulation.

Intestinal inflammation caused by gut dysbiosis can impact iron homeostasis within the GI tract (Cherayil et al., 2011), however, whether these findings have been extrapolated to serum iron homeostasis has not yet been elucidated. While iron dysregulation within the GI tract and gut dysbiosis potentially exacerbate one another, Constante et al. (2017) concluded that luminal heme originating from gastrointestinal bleeding or dietary components more likely contributes to dysbiosis of the gut microbiota in mice than vice versa. However, dietary non-heme iron intake from food has been associated with a 30% increased risk of Parkinson’s disease (p = 0.02) (Logroscino et al., 2008). In the same study authors also observed that supplemental iron intake was associated with a borderline increase in Parkinson’s disease among men (Logroscino et al., 2008). Nonetheless, the most prominent cause of iron dysregulation in the form of elevated serum ferritin levels is cell death (Kell and Pretorius, 2018).

In AD, iron dysregulation and the benefits of its chelation have been known for decades (Crapper McLachlan et al., 1991), and many reviews point to the role of poorly liganded iron in AD development (Exley, 2006; Castellani et al., 2007, 2012; Kell, 2009, 2010; Sandro and Muckenthaler, 2009; Grunblatt et al., 2011; Weinreb et al., 2011; Peters et al., 2015; Wood, 2015; Belaidi and Bush, 2016). Unliganded iron and the accompanying oxidative damage is causatively related to neuro-inflammation (Bester et al., 2013; Hongkuan et al., 2015). According to Nunomura et al. (2001), neurons with neurofibrillary tangles show a 40–56% decrease in relative 8-hydroxyguanosine (8-OHG) levels when compared to neurons without neurofibrillary tangles in AD patients. 8-OHG is a marker of hydroxyl radical formation (Kell, 2009, 2010), so this finding indicates that oxidative damage is an early event in AD progression, which dissipates with lesion formation (Nunomura et al., 2001). This is further supported by (Moreira et al., 2005) who found one of the earliest pathological events in AD to be an imbalance between free radical scavenging and generation. The relationship between poorly liganded iron and oxidative stress involves iron’s catalytic contribution to the Fenton and Haber-Weiss reactions. These reactions are involved in the formation of the highly reactive hydroxyl radical (OH^-), the most biologically detrimental free radical species, specifically with regards to macromolecules such as lipids, proteins and nucleic acids (Moreira et al., 2005; Kell, 2009, 2010; Lipinski and Pretorius, 2013; Pretorius et al., 2016a). Notably, iron chelation can reverse tau protein hyperphosphorylation in mice brains (Guo et al., 2013), thereby reducing the formation of intracellular neurofibrillary tangles.

Dormancy is extremely common in microbiology, even among non-sporulating bacteria (Kaprelyants et al., 1993; Lewis, 2007; Kell et al., 2015; Kell and Kenny, 2016), for sound evolutionary reasons (Mukamolova et al., 2003). Our major hypothesis involves shared symptoms across numerous chronic inflammatory diseases caused by dormant microbes (Pretorius et al., 2016a). As reviewed in 2016 (Pretorius et al., 2016a), microbial reactivation or resuscitation, which may be autocatalytic (Mukamolova et al., 1998), indicates that these so-called dormant microbes which are non-growing and appear operationally dead (Kell et al., 1998), can recover culturability. Metals, although explicitly involved in production of free radicals and potentially oxidative stress in AD, are also involved in the process of dormant microbe reactivation (Bester et al., 2015; Kell et al., 2015). All microbes (with the possible exception of Borrelia) require free available iron to grow. This follows from the in vivo limitation of microbial growth in the absence or decreased availability of free iron (Markel et al., 2007; Reid et al., 2009; Chu et al., 2010; Nairz et al., 2010, 2014; Skaar, 2010; Haley and Skaar, 2012; Armitage and Drakesmith, 2014; Subashchandrabose and Mobley, 2015; Damron et al., 2016; Carver, 2018). It is its absence in normal metabolism that causes dormant bacteria to remain in that state. With this in mind, iron dysregulation specifically in the form of high SF levels (Kell and Pretorius, 2014), accelerates AD pathology in two ways (Pretorius et al., 2016a) which suggests the opportunity of its use as a biomarker for early diagnosis and/or a target for slowing down disease progression (Bester et al., 2013).

Dysbiosis refers to alteration in the composition of the microbiota and has been implicated in the etiology of various disease states (de Oliveira et al., 2017; Kho and Lal, 2018). Perturbations of the gut microbiota can occur as consequences of antibiotic treatment (especially during infancy), dietary changes, sedentary behavior, food additives, non-steroidal anti-inflammatory and other drugs (Wallace, 2013; Ticinesi et al., 2017; Maier et al., 2018) and various other conditions (Pistollato et al., 2016). Dietary changes, for example, may explain up to 57% of the total structural variation the gut microbiome (Brown et al., 2012). Using a humanized mouse model in which adult human fecal microbiota were transplanted into germ-free mice, Brown et al. (2012) reported a shift in the composition of microbiota after switching the mice from a low-fat, plant polysaccharide-rich diet to a Western diet. While diets rich in refined sugars result in different changes to the microbiota from diets lacking fiber, due to the intricate balance that exists between microbial species, alterations in one species can disrupt the entire microbial community (Brown et al., 2012). Thus, alterations in diet, although modifiable, could potentially be a major hurdle in treatment dysbiosis-related conditions if not adequately addressed. Furthermore, physical activity and its relative intensity can induce or prevent gut dysbiosis by influencing splanchnic blood flow, intestinal permeability and inflammatory cytokine expression (Monda et al., 2017). For example, in mice, even in combination with a high-fat diet, exercise may reduce inflammatory infiltrate into epithelial cells, protecting the integrity of the GI wall (Campbell et al., 2016).

Broad-scale fluctuations in the gut microbiota play significant roles in disease advancement through immune and systemic activation (Vogt et al., 2017). There is considerable evidence for the existence of the gut-brain axis which allows bi-directional communication through various pathways such as neural, endocrine and immune mechanisms (Gareau, 2014; Lyte, 2014; Bienenstock et al., 2015; Köhler et al., 2016; Fung et al., 2017; Sandhu et al., 2017). Within this context, changes in microbial communities could lead to pathophysiological changes in the brain of individuals with AD. Dysbiosis is often associated with increased anxiety and memory impairment due to decreased secretion of neurotrophic factors such as brain-derived neurotrophic factor (Stilling et al., 2014).

Amyloid by definition is protein that is deposited as an insoluble fibril as a result of successive alterations in the protein folding process, known as amyloidosis. Similarly to prions, there is no change in the primary amino acid sequence of the proteins when they adopt an insoluble amyloid form (Kell and Pretorius, 2018). Interestingly, self-associating amyloidogenic lipoproteins are secreted by most microbial species (Schwartz et al., 2016), and amyloid is one of the main secretory products of the gut microbiome (Zhao et al., 2017). The role of the gut in dissemination of amyloid proteins has been reviewed by Pistollato et al. (2016), in which the authors adopted the unifying prion concept to explain transmission of these prion-like proteins. The cumulative life-long contribution of microbial amyloid to neurodegenerative pathophysiology is little recognized. Progressive production and aggregation of amyloids contributes to the pathogenesis of diseases in which amyloids accumulate, which is often mediated by microglial cells (Zhao et al., 2017). Thus, bacterial amyloids originating from the gut microbiome may enhance inflammatory responses to cerebral accumulation of Aβ and play a role in the pathogenesis of AD (Pistollato et al., 2016).

For example, functional amyloid from Klebsiella pneumoniae can depolymerise from its fibrillary state to release oligomers which induce cytotoxicity equal to that caused by pathological Aβ in AD (Shahnawaz and Soto, 2012). The molecular mimicry theory (Ebringer and Rashid, 2009; Proal et al., 2013, 2017) includes microbial curli fibers² and Aβ as a protein–protein interaction which could result in cross-seeding, even if these proteins are essentially unalike (Friedland, 2015; Zhao et al., 2015). These extrinsic curli proteins act as pathogen-associated molecular patterns with increased β-pleated sheet structures, and can cross-react with antibodies to Aβ plaques (Miklossy, 2016). It is plausible that extracellular senile plaques, due to their morphological appearance and density, may in fact be constituted of curli-like Aβ compositions. In theory, this gives bacterial phylotypes a more blatant role in AD causality (Pritchard et al., 2017). The influence of gut microbiota on amyloid formation and propagation is more significant throughout aging as both the epithelium of the GI tract and the blood-brain barrier (BBB) increase in permeability to smaller molecules (Tran and Greenwood-Van Meerveld, 2013; Pistollato et al., 2016).

Some of the foremost bacterial species of the GI tract, such as the Gram-negative bacilli Bacteroides fragilis and Escherichia coli, secrete a multifarious selection of pro-inflammatory neurotoxins which are not only pathogenic but also detrimental to the homeostatic functioning of neurons (Zhao et al., 2017). The shedding of inflammagens such as LPS from gut-microbiota-related dysbiosis causes systemic and neuro-inflammation and promotes gut permeability. Shedding can occur in response to varying physiological and environmental signals with the most extreme example of microbial shedding known as the Jarisch-Herxheimer reaction. This is basically an uninhibited cytokine storm, which is caused by prompt release of inflammagenic material, often following antibiotic treatment of syphilis (Kell and Pretorius, 2018), or other diseases caused by spirochetes. As mentioned earlier, this may promote neuronal dysfunction and apoptosis, impairment of synaptic plasticity and induced vulnerability to cognitive decline (Daulatzai, 2014). Bacterial LPS has also been found in AD hippocampal brain lysates where the mean LPS levels were three-fold higher in the hippocampus of AD patients than in age-matched controls. This was said to increase to 26-fold in advanced AD hippocampal cases (Zhao et al., 2017).

LPSs are sparingly soluble but over a period of time characteristically form large heterogenous aggregates which are exceptionally immunogenic (Zhao et al., 2015). Interestingly the glycosylphosphatidyl-inositol-anchored LPS and microbe-detecting CD41 receptor, which is crucial in the neutralization of invading microbes, is similarly, stimulated by Aβ fibrils (Dowhan, 2014), relating innate-immune signaling with amyloidogenesis in AD. It has also been noted that previous bacterial infections which resulted in the formation of antibodies to amyloids or bacterial inflammagens may predispose central nervous system amyloids to ensuing attack by antibodies, which results in upregulation of neuro-inflammation (Via et al., 2015; Zhao et al., 2015). The inflammagenic potency of LPS is so pronounced that it is frequently used to stimulate in vivo AD models (Lee et al., 2008; Catorce and Gevorkian, 2016; Zakaria et al., 2017).

LTA is the cell wall equivalent of LPS in Gram-positive bacteria and is equally capable of inducing an inflammatory response through its interactions with toll-like receptor (TLR) 2 (Kell and Pretorius, 2018). LTA has not been as well studied as LPS with regards to inflammagenesis, however, recent work suggests that LTAs may in fact be more potent than LPSs (Pretorius et al., 2018). The stimulation of inflammatory cytokine production by both LPS and LTA are mediated through TLR binding (reviewed by Kell and Pretorius, 2018). In response to inflammagens, concentrations of acute phase biomarkers and inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8, C-reactive protein (CRP), serum amyloid A (SAA), and fibrinogen increase significantly (deRosset and Strutz, 2015; De Buck et al., 2016; Pindjakova et al., 2017).

Additionally, increased BBB permeability typically observed in individuals with AD can be caused by shed bacterial inflammagens. The BBB plays a fundamental role in the initiation and continuance of chronic inflammation during AD (Zenaro et al., 2017). While Aβ accumulation in vasculature results in inflammatory events that increase BBB permeability in AD (Erickson and Banks, 2013), proteolytic enzymes such as carbonic anhydrases, peptidyl deiminases and gingipains, and appendages such as curli fibers, fimbriae and other amyloid-like proteins, which are carried with LPS (Pritchard et al., 2017) and contribute significantly to BBB permeabilization. BBB dysfunction during AD effects Aβ clearance, endothelial cell function, tight junction integrity and may activate glial cells which accelerates migration of leukocytes to the brain (Zenaro et al., 2017). This could stimulate the beginning of chronic neuro-inflammation in AD.

Atopobiosis refers to the appearance of some of the gut microbes in the wrong place (Potgieter et al., 2015). The sterility of blood is seemingly a controversial topic. The presence of a “physiological” blood microbiota has been reported by several studies using 16S ribosomal DNA quantitative polymerase chain reactions (Amar et al., 2013; Lelouvier et al., 2016; Martel et al., 2017; Proal and Marshall, 2018). Yet, in these studies the plausible source of these microbes from gut permeability was not investigated. Authors of the same studies have reported that changes in the composition of this blood microbiota can be associated with disease. However, based on immunological and clinical understanding (Ochei and Kolhatkar, 2000) the detection of bacteria in blood is always abnormal (Kinnby and Chavez de Paz, 2016; Andreadou et al., 2017) and thus referred to as atopobiosis (Potgieter et al., 2015). As seen in Figure 3, atopobiosis is caused by bacterial translocation in which bacteria move from the GI tract to normally sterile blood and tissues (Potgieter et al., 2015). Once translocation has taken place, these bacteria can cause direct infection and inflammation. The process of bacterial translocation has been reviewed by Potgieter et al. (2015) but can briefly be described by the simultaneous dysfunction of three role players: (1) dendritic cells (2) GI epithelium and, (3) M cells which overlay Peyer’s patches. It is a combination of various compromised mucosal defenses that leads to bacterial translocation. When the influx of gut microbes and toxins is particularly abundant, it is often referred to as a “leaky gut” (Maes, 2009; Fasano, 2012; Quigley, 2016; Mu et al., 2017; Kell and Pretorius, 2018) and is due to severely compromised epithelial tight junctions (Fasano, 2012).

Bacteria that have successfully translocated to the blood have the ability to become dormant and reactivate upon appropriate stimuli, such as iron dysregulation in the form of high SF levels as discussed in section Causes of Iron Dysregulation. In contrast to conventional infections, these bacteria do not multiply but enter dormant states (Kell and Kenny, 2016; Pretorius et al., 2016a; Kell and Pretorius, 2018). This is especially relevant in clinical settings when toxic concentrations of antibiotics promote bacterial persistence or adoption of a dormant state that permits their survival (Kell and Pretorius, 2018). Even though the major source of this dormant blood microbiome has been reviewed here as a dysbiotic and permeabilized GI tract, we also recognize periodontitis, gingivitis, urinary tract infections and even insemination as possible points of microbial infection and subsequent translocation (Kamer et al., 2008; Flores-Mireles et al., 2015; Kenny and Kell, 2017; Pretorius et al., 2017; Pritchard et al., 2017). Current research has drifted toward the correlation between an individual’s infectious burden and risk of developing AD, with particular emphasis on herpes simplex virus 1 (HSV1) and human herpesvirus 6 and 7 infections (Honjo et al., 2009; Itzhaki, 2014, 2017; Bu et al., 2015; Itzhaki et al., 2016; Itzhaki and Tabet, 2017; Eimer et al., 2018; Itzhaki and Lathe, 2018; Readhead et al., 2018; Tzeng et al., 2018). Dormant bacteria can survive in leukocytes (Thwaites and Gant, 2011; Liehl et al., 2015) and erythrocytes (Potgieter et al., 2015) to establish the dormant blood microbiome. In Potgieter et al. (2015) transmission electron micrographs of cellular inclusions thought to be L-forms of bacteria inside erythrocytes of individuals diagnosed with AD can be seen (see also Mattman, 2001). Figure 4 illustrates the association of bacteria with pathological coagulations within the blood of individuals diagnosed with AD. This dormant blood microbiome affects the integrity of the hematological system, promoting pathological coagulation.

Fibrinogen is the precursor of fibrin and thus plays a major role in thrombosis and hemostasis (Pretorius and Kell, 2014). During inflammation, circulating fibrinogen levels are increased, while fibrinolysis is impaired. Amyloidogenesis of the fibrin fibers occurs, resulting in the formation of dense matted deposits which trap other blood cells and distort their shape. The combination of the increased propensity to form a stiffer fibrin network and reduced fibrinolysis is a feature of numerous inflammatory diseases such as AD (Ahn et al., 2010; Zamolodchikov and Strickland, 2012). Figure 5 illustrates some of the morphological changes of fibrin fibers due to the addition of LPS.

The presence of excessive (unliganded) iron and the consequential production of hydroxyl radicals is one of the leading causes of altered fibrin cross-linking (Pretorius and Kell, 2014; Kell and Pretorius, 2015b). Dense matted deposits produced in the presence of ferric ions are abnormally resistant to chemical and proteolytic degradation, hypothesized to be due to the existence of intermolecular hydrophobic bonds (Lipinski and Pretorius, 2012). As reviewed, it seems that iron dysregulation may serve as a causative factor for multiple pathologies involved in AD progression. This altered fibrinogen structure, with excessive cross-linking and increased β-pleated sheet structure, has been implicated in the development of neuro-inflammation and memory impairments. Increased fibrinogen levels are a strong indicator of cerebrovascular risk since fibrinogen binds to Aβ which further delays clot degradation (Bester et al., 2013; Pretorius et al., 2016a). In addition, pharmacological inhibition of the fibrinogen–Aβ interaction with Ru-505 altered thrombus structure and paused cognitive decline in mice (Ahn et al., 2014), providing a strong association between pathological clotting and neurodegenerative diseases.

The dynamics of erythrocytes represent an important but understudied feature of the cardiovascular system. Central to this is their rheology, viscosity, aggregation and deformability (Baskurt et al., 2011; Pretorius and Kell, 2014). It is thought that alterations in erythrocytes and their rheology can contribute to the pathogenesis of AD by hindering oxygen delivery (Tripathy et al., 2013). Dense matted deposits may also trap erythrocytes, impairing their effective delivery of oxygen to the brain. Proficient oxygen delivery is vital for normal functioning of the brain, underpinned by that fact that 20% of an individual’s total oxygen intake is used by the brain. Neurons are particularly susceptible to hypoxic periods, which can cause irreversible neurological consequences (Lipinski and Pretorius, 2013) after only a few minutes. Altered functioning of erythrocytes is also strongly suggested by changes in their size distribution (Ozturk et al., 2013; Weuve et al., 2014; Pilling et al., 2017; Winchester et al., 2018).

As reviewed by Pretorius and Kell (2014), erythrocyte deformability is a complex process which is significantly altered by pathophysiological conditions. Briefly, reduced erythrocyte deformability is an important feature of inflammation in which reactive oxygen species cause degradation of spectrin and band 3, for example, which are important membrane proteins. Since the plasma membrane and the cytoskeleton are responsible for the preservation of erythrocyte shape and stability, modifications of the phospholipid bilayer can affect deformability. In 2010, Mohanty et al. (2010) reported that 15% of erythrocytes in AD were elongated and suggested that a potential link between changes in the erythrocyte proteome could contribute to AD pathology. Deformability and impaired reformability, particularly after erythrocytes have moved through capillaries, reduces their oxygen carrying capacity, potentiating transient states of hypoxia. Figure 6 illustrates erythrocyte deformability as well as loss of membrane integrity in typical blood samples from individual’s diagnosed with AD. It is clear to see how the loss of the typical bi-concave structure of these erythrocytes would impair their physiological functioning.