**2. Inflammation in amyloid diseases**

Inflammation is the term given to a form of immune defense that is widespread and requires a complex network of immune effector mechanisms to address tissue dysfunction or injury. The notion that inflammation is involved in amyloid pathogenesis was first suggested in the 1970s in two reports on serum amyloid A (SAA) [11, 12]. Nowadays, it is known that this particular amyloidogenic protein expression is regulated by IL-6, an inflammatory cytokine, which results in increased levels of SAA [13]. Hyperexpression of SAA for long periods of time results in amyloid formation and deposition in tissue, characterizing amyloid A (AA) amyloidosis [13]. This particular type of amyloidosis occurs in chronic inflammation conditions, such as autoimmune diseases or cancer [13]. With the exception of inflammation-induced amyloidosis, such as AA amyloidosis, the role of inflammation in most amyloid diseases is a more recent concept that emerged in the late 1980s with the observation of microglia, a brain immune cell, found near amyloid plaques in postmortem brain tissue from Alzheimer's disease (AD) patients [14–16]. After these first observations, more evidence on the involvement of inflammatory mechanism in amyloid pathogenesis has emerged.

### **2.1 Alzheimer's disease (AD)**

Alzheimer's disease (AD) is known as the most common neurodegenerative disease worldwide. It afflicts over 40 million people in the world, and because aging is the major risk factor for developing AD, its incidence will likely increase in the future as medical advances lead to increasing life span [17]. Although there is no definite known causative agent, most scientists agree that amyloid-β peptide (Aβ) is an important factor leading to AD [18]. Aβ is highly amyloidogenic, meaning that it has great potential to aggregate in solution [18]. Aβ is a small peptide ranging from 25 to 42 amino acids [18]. Extracellular aggregates of Aβ are the main protein present in amyloid plaques, a hallmark of AD [18]. Moreover, a small fraction of AD patients is diagnosed with familial AD, which is known to be the result of an infrequently inherited autosomal dominant mutation in one of the three genes

**43**

*The Role of Inflammation in Amyloid Diseases DOI: http://dx.doi.org/ 10.5772/intechopen.81888*

*2.1.1 Role of microglia in AD*

to brain diseases, such as AD.

role of Aβ in AD is very complex.

involved with the production of Aβ: the amyloid precursor protein (APP) gene, presenilin 1 (PSEN1), and presenilin 2 (PSEN2) [19]. The first findings pointing to a possible involvement of inflammatory mechanisms emerged around 1980 when scientists reported reactive microglia and astrocytes surrounding amyloid plaques of AD patients [14–16]. Nowadays, after years of research, inflammation is known to be implicated in AD by having a role in neuronal damage [20], Aβ generation [21], increased hyperphosphorylation of tau protein [22] (another hallmark of AD), and cognitive impairment [23]. In this part of the chapter, we will be focusing on

Microglia are the immune cells of the brain. They derive from myeloid precursors which migrate into the brain during early embryonic development and play a major role in maintaining a healthy environment in the brain [24, 25]. Microglia use their surface receptors to constantly scan the central nervous system (CNS) for microbes or other damaging molecules [26]. When activated by a stimulus, microglia mediate innate and adaptive immune responses or perform various functions in response to CNS disease or injury [26]. Microglia are of great importance for brain homeostasis, but uncontrolled or overactivated microglia can also contribute

Years of research is allowing deeper understanding of how microglia contribute to AD. Microglia produce inflammatory mediators, such as TNF-α [26], that can have a detrimental role when overproduced for long periods of time. Microglia are able to produce large quantities of TNF-α upon exposure to fibrillar and oligomeric Aβ [23, 27]. TNF-α is increased in the serum and CSF of AD patients and has additionally been detected in amyloid plaques [28–32]. Inhibiting TNF-α production with the use of unspecific anti-inflammatory compounds, such as minocycline, or a specific neutralizing TNF-α antibody, (infliximab) results in downregulated inflammatory pathways (e.g., MAPK, AKT, and NF-κB) and abrogates cognitive deficits in mice [23, 33]. It has been shown that fibrillar and oligomeric Aβ can induce production of not only TNF-α but also other important inflammatory cytokines in microglia by binding and activating several receptors [23, 34]. This suggests the existence of a universal epitope found in aggregated material and a nonspecific response to amyloids. Also supporting this idea is the fact that two generic, widely used, conformation-specific antibodies have been generated (A11 and OC antibodies) that recognize mutually exclusive structural epitopes in a range of amyloid-forming proteins, including Aβ, independently of any primary amino acid sequence similarities. A11 antibodies recognize anti-parallel β-sheet structures found in intermediate states, and OC antibodies detect parallel β-sheets found in mature amyloid fibrils [35]. Inflammation is a downstream consequence of aggregated Aβ binding to receptors such as TLR-4 (toll-like receptor 4), RAGE (receptor for advanced glycation end products), CD36 [23, 36, 37], etc. It is important to note that some of these receptors are also able to recognize other aggregated non-Aβ materials [38]. Interestingly, these receptors are not only present in microglia, but some are also present in endothelial cells and neurons [39]. This suggests that the

Some receptors that primarily bind monomeric Aβ are not involved in pathological, inflammatory processes. Receptors such as LRP1 (low-density lipoprotein receptor-related protein 1), PrPc (cellular prion protein), and PICALM (phosphatidylinositol-binding clathrin assembly protein) are able to bind monomeric Aβ and are thought to be involved in Aβ clearance, decreasing the Aβ burden and plaque formation in the brain [40]. Moreover, mutations in PICALM, which is a gene

summarizing the role of microglia in the progression of AD.

#### *The Role of Inflammation in Amyloid Diseases DOI: http://dx.doi.org/ 10.5772/intechopen.81888*

involved with the production of Aβ: the amyloid precursor protein (APP) gene, presenilin 1 (PSEN1), and presenilin 2 (PSEN2) [19]. The first findings pointing to a possible involvement of inflammatory mechanisms emerged around 1980 when scientists reported reactive microglia and astrocytes surrounding amyloid plaques of AD patients [14–16]. Nowadays, after years of research, inflammation is known to be implicated in AD by having a role in neuronal damage [20], Aβ generation [21], increased hyperphosphorylation of tau protein [22] (another hallmark of AD), and cognitive impairment [23]. In this part of the chapter, we will be focusing on summarizing the role of microglia in the progression of AD.

### *2.1.1 Role of microglia in AD*

*Amyloid Diseases*

for these diseases.

**2. Inflammation in amyloid diseases**

mechanism in amyloid pathogenesis has emerged.

**2.1 Alzheimer's disease (AD)**

For decades, scientists believed that amyloid fibrils were stable and inert, and the end result of a nontoxic natural pathway that serves to scavenge and store highly reactive and toxic oligomeric intermediates formed during the process of protein folding [3–5]. The reactive hydrophobic residues found exposed in oligomeric intermediates are hidden in mature amyloid fibrils, thus unable to react with important cellular components [6]. Although the latter is true about amyloid fibrils, research in the last two decades shows that these structures are neither inert nor nontoxic [5, 7]. When extracellular, amyloid fibrils are readily identified by the host immune system, mainly by macrophages or neutrophils [8–10]. These immune cells have receptors that recognize and bind to the amyloid fibrils and intermediates, activating a signaling cascade that results in the production of proinflammatory molecules [8–10]. In this chapter, we will review the inflammatory component of some clinically important amyloid diseases, such as Alzheimer's disease (AD) and familial amyloid pathologies, such as hereditary TTR amyloidosis (hATTR or familial amyloid polyneuropathy—FAP). We will also discuss how inflammatory cells contribute to disease progression by causing bystander damage to tissues or by enhancing protein aggregation, in the case of AA amyloidosis. Finally, we will provide the recent therapeutic approaches based on immune regulatory strategies

Inflammation is the term given to a form of immune defense that is widespread and requires a complex network of immune effector mechanisms to address tissue dysfunction or injury. The notion that inflammation is involved in amyloid pathogenesis was first suggested in the 1970s in two reports on serum amyloid A (SAA) [11, 12]. Nowadays, it is known that this particular amyloidogenic protein expression is regulated by IL-6, an inflammatory cytokine, which results in increased levels of SAA [13]. Hyperexpression of SAA for long periods of time results in amyloid formation and deposition in tissue, characterizing amyloid A (AA) amyloidosis [13]. This particular type of amyloidosis occurs in chronic inflammation conditions, such as autoimmune diseases or cancer [13]. With the exception of inflammation-induced amyloidosis, such as AA amyloidosis, the role of inflammation in most amyloid diseases is a more recent concept that emerged in the late 1980s with the observation of microglia, a brain immune cell, found near amyloid plaques in postmortem brain tissue from Alzheimer's disease (AD) patients [14–16]. After these first observations, more evidence on the involvement of inflammatory

Alzheimer's disease (AD) is known as the most common neurodegenerative disease worldwide. It afflicts over 40 million people in the world, and because aging is the major risk factor for developing AD, its incidence will likely increase in the future as medical advances lead to increasing life span [17]. Although there is no definite known causative agent, most scientists agree that amyloid-β peptide (Aβ) is an important factor leading to AD [18]. Aβ is highly amyloidogenic, meaning that it has great potential to aggregate in solution [18]. Aβ is a small peptide ranging from 25 to 42 amino acids [18]. Extracellular aggregates of Aβ are the main protein present in amyloid plaques, a hallmark of AD [18]. Moreover, a small fraction of AD patients is diagnosed with familial AD, which is known to be the result of an infrequently inherited autosomal dominant mutation in one of the three genes

**42**

Microglia are the immune cells of the brain. They derive from myeloid precursors which migrate into the brain during early embryonic development and play a major role in maintaining a healthy environment in the brain [24, 25]. Microglia use their surface receptors to constantly scan the central nervous system (CNS) for microbes or other damaging molecules [26]. When activated by a stimulus, microglia mediate innate and adaptive immune responses or perform various functions in response to CNS disease or injury [26]. Microglia are of great importance for brain homeostasis, but uncontrolled or overactivated microglia can also contribute to brain diseases, such as AD.

Years of research is allowing deeper understanding of how microglia contribute to AD. Microglia produce inflammatory mediators, such as TNF-α [26], that can have a detrimental role when overproduced for long periods of time. Microglia are able to produce large quantities of TNF-α upon exposure to fibrillar and oligomeric Aβ [23, 27]. TNF-α is increased in the serum and CSF of AD patients and has additionally been detected in amyloid plaques [28–32]. Inhibiting TNF-α production with the use of unspecific anti-inflammatory compounds, such as minocycline, or a specific neutralizing TNF-α antibody, (infliximab) results in downregulated inflammatory pathways (e.g., MAPK, AKT, and NF-κB) and abrogates cognitive deficits in mice [23, 33]. It has been shown that fibrillar and oligomeric Aβ can induce production of not only TNF-α but also other important inflammatory cytokines in microglia by binding and activating several receptors [23, 34]. This suggests the existence of a universal epitope found in aggregated material and a nonspecific response to amyloids. Also supporting this idea is the fact that two generic, widely used, conformation-specific antibodies have been generated (A11 and OC antibodies) that recognize mutually exclusive structural epitopes in a range of amyloid-forming proteins, including Aβ, independently of any primary amino acid sequence similarities. A11 antibodies recognize anti-parallel β-sheet structures found in intermediate states, and OC antibodies detect parallel β-sheets found in mature amyloid fibrils [35]. Inflammation is a downstream consequence of aggregated Aβ binding to receptors such as TLR-4 (toll-like receptor 4), RAGE (receptor for advanced glycation end products), CD36 [23, 36, 37], etc. It is important to note that some of these receptors are also able to recognize other aggregated non-Aβ materials [38]. Interestingly, these receptors are not only present in microglia, but some are also present in endothelial cells and neurons [39]. This suggests that the role of Aβ in AD is very complex.

Some receptors that primarily bind monomeric Aβ are not involved in pathological, inflammatory processes. Receptors such as LRP1 (low-density lipoprotein receptor-related protein 1), PrPc (cellular prion protein), and PICALM (phosphatidylinositol-binding clathrin assembly protein) are able to bind monomeric Aβ and are thought to be involved in Aβ clearance, decreasing the Aβ burden and plaque formation in the brain [40]. Moreover, mutations in PICALM, which is a gene

#### *Amyloid Diseases*

that encodes a clathrin assembly protein and thus is involved in endocytosis, have been shown to be a risk factor for developing late-onset AD [41]. More convincing evidence of the significant role of neuroinflammation in AD is found in recent genome-wide association studies (GWAS). These studies have identified more than 20 gene variants as risk factors for developing late-onset AD. These diseasemodifying genes include genes involved in both innate and adaptive immune system responses: *CR1*, *CLU*, *CD33*, *MS4A, ABCA7 EPHA1, TREM2,* and *HLA*-*DRB5/ HLA*-*DRB1* [41, 42]. It is interesting to note that all of these genes are present in microglia cells as well [41]. It is thought that these genes can change microglia function and increase the risk of AD.

#### *2.1.2 Role of peripheral inflammation in AD*

An emerging concept based on recent work is that peripheral inflammation, in addition to local, brain inflammation, also affects AD pathogenesis. Studies suggest that myeloid cells, such as neutrophils, can enter the brain and may also involve in Aβ clearance [10, 43]. Neutrophils can recognize fibrillar Aβ and produce *in vitro* and *in vivo* extracellular traps (NETs; a defense mechanism that results in neutrophil cell death) [10, 43]. Extracellular traps are protein and DNA-made meshes that can immobilize Aβ particles, degrade fibrillar amyloids, but are known to modulate other immune system effector mechanisms as well [44].

Acute systemic inflammation, caused by bacterial infection, exacerbates AD pathology [45], and chronic systemic inflammation, occurring in diseases such as rheumatoid arthritis (RA), depression, and obesity, has also been reported to modify the amyloid phenotype of AD mice and is considered common co-morbid states of AD patients. In autoimmune disease, chronic inflammation can increase the risk of developing AD. Patients with the autoimmune disease Sjögren's syndrome (SS) are twice as likely to develop AD [46]. In RA, it has been shown that anti-TNF-α therapy has a protective effect on dementia [47]. In the case of depression, Aβ accumulation in AD mouse models induces depressive-like behavior, which is dependent on inflammation [23]. Inflammatory cytokine production reduces serotonin levels and contributes to behavioral changes in mice with AD [23]. Again, this can be prevented by anti-TNF-α therapy [23]. Obesity is a known comorbidity of AD and a low-grade chronic inflammatory disease. In humans and AD mouse models, cafeteria diet consumption and a higher BMI are known to accelerate AD pathology [48, 49]. For example, in humans, for every 1.0 increase in BMI at age 70 years, AD risk increased by 36% in female patients [49].

In an attempt to cure AD, active immunization against Aβ was performed in mice and humans [50, 51]. Studies reported that immunization had a therapeutic effect on mouse models of AD [50–52]. Unfortunately, clinical Aβ vaccination trials have been interrupted due to the development of meningoencephalitis in 6% of the patients, likely involving the appearance of pro-inflammatory macrophages, CD4+ and CD8+ T cells [50]. As well as myeloid cells, T cells can enter the brain. There are myriads of T-cell subtypes surveilling the CSF and the meningeal membranes [53], and they can enter the brain parenchyma upon cell injury [53]. It is not only brain-local T cells that react to Aβ but also blood T cells have been shown to have hyperreactivity to the Aβ peptide [54], specifically to epitopes within the residues 15–42 [54]. This evidence together suggests that the immune system and inflammation play significant roles in AD: not only helping with homeostatic Aβ clearance and preventing AD plaque formation but also by contributing to cell injury. Unfortunately, to date, tackling the immune system to prevent AD has yet to prove clinically effective.

**45**

*The Role of Inflammation in Amyloid Diseases DOI: http://dx.doi.org/ 10.5772/intechopen.81888*

incurable and results in death [59].

**2.2 Hereditary ATTR (hATTR) amyloidosis**

Transthyretin (TTR) is a 55-kDa tetrameric protein expressed and secreted mainly not only by the liver, but also by the choroid plexus in the brain [55]. This protein received this specific name due to its function: once in the plasma or in the cerebrospinal fluid, TTR acts as a retinol-binding protein and thyroxine transporter across the body [55]. More than 100 point mutations in the TTR gene have been described worldwide and most of them culminate in the production of abnormal protein with a high thermodynamic instability compared to its wild-type counterpart [55]. Only a handful of mutations are not pathogenic, such as the T119M mutation [56]. The pathogenic V30M variant is the most common mutation affecting a large population of people worldwide and results in the accumulation of TTR in various tissues, such as cardiac and nervous tissue [57]. Most TTR mutations have a high propensity to aggregate under denaturing and even physiological conditions [58], forming amyloid fibrils that deposit in various tissues and organs [58]. For decades, most physicians and pathologists still regard hATTR amyloidosis as a disease without an inflammatory component, since most biopsies and *ex vivo* analysis showed no leukocyte infiltration [59]. However, with the appearance of new data in the last decade, hATTR amyloidosis is now being recognized as a disease with an important inflammatory component. Moreover, TTR amyloid fibrils are similar in structure to other amyloid fibrils and thus should induce similar inflammatory responses. One of the most common types of hATTR amyloidosis is known as familial amyloid polyneuropathy (FAP). FAP is an autosomal dominant hereditary disease characterized by the accumulation of amyloid fibrils in peripheral nerves, the gastrointestinal tract, and the heart [59]. This disease has three discernable stages: FAP 1 = unimpaired ambulation; mostly mild sensory, motor, and autonomic neuropathy in the lower limbs; FAP 2 = assistance with ambulation required; mostly moderate impairment progression to the lower limbs, upper limbs, and trunk; FAP 3 = wheelchair-bound or bedridden; severe sensory, motor, and autonomic involvement of all limbs. This disease, as most amyloidosis, is

The diagnosis of FAP is challenging, often relying on genetic screening to identify TTR mutations as well as on the identification of Congo red-positive amyloid deposits in biopsies. These biopsies are generally invasive, and tissue is usually taken from the sural nerve, abdominal fat, or salivary glands [59]. The main *go-to* treatment for FAP is liver transplantation (LT), since the liver is the major organ of TTR production. Unfortunately, LT presents mortality risks, and it is not available to all patients [60]. More recently, two new drug-based treatments have been FDA approved. One of these treatments use a new drug (Tafamidis) that works by stabilizing the TTR protein that is available in several countries showing effective results in controlling disease progression [61]. The other, just recently approved by the FDA, uses antisense oligonucleotides (ASOs) to target TTR production in the liver directly, decreasing the

Since the first study in 2001, the new concept that inflammation may play a role in the pathogenesis of FAP has emerged. Sousa and colleagues showed the presence of proinflammatory markers such as TNF-α and IL-1β in biopsies of FAP patients [63, 64]. Interestingly, the levels of proinflammatory and oxidative markers in *ex vivo* tissue positively correlate with the scoring stage proposed by Coutinho and colleagues in FAP patients, which is an index used to discriminate disease progression [65]. In addition, their study also showed the participation of the receptor RAGE, which can also bind Aβ fibrils, in the recognition of TTR amyloid fibrils [63]. In this first study, the authors suggest that Schwann cells, which are cells

amount of TTR in the plasma, thus reducing protein aggregation [62].

*Amyloid Diseases*

tion and increase the risk of AD.

*2.1.2 Role of peripheral inflammation in AD*

other immune system effector mechanisms as well [44].

70 years, AD risk increased by 36% in female patients [49].

that encodes a clathrin assembly protein and thus is involved in endocytosis, have been shown to be a risk factor for developing late-onset AD [41]. More convincing evidence of the significant role of neuroinflammation in AD is found in recent genome-wide association studies (GWAS). These studies have identified more than 20 gene variants as risk factors for developing late-onset AD. These diseasemodifying genes include genes involved in both innate and adaptive immune system responses: *CR1*, *CLU*, *CD33*, *MS4A, ABCA7 EPHA1, TREM2,* and *HLA*-*DRB5/ HLA*-*DRB1* [41, 42]. It is interesting to note that all of these genes are present in microglia cells as well [41]. It is thought that these genes can change microglia func-

An emerging concept based on recent work is that peripheral inflammation, in addition to local, brain inflammation, also affects AD pathogenesis. Studies suggest that myeloid cells, such as neutrophils, can enter the brain and may also involve in Aβ clearance [10, 43]. Neutrophils can recognize fibrillar Aβ and produce *in vitro* and *in vivo* extracellular traps (NETs; a defense mechanism that results in neutrophil cell death) [10, 43]. Extracellular traps are protein and DNA-made meshes that can immobilize Aβ particles, degrade fibrillar amyloids, but are known to modulate

Acute systemic inflammation, caused by bacterial infection, exacerbates AD pathology [45], and chronic systemic inflammation, occurring in diseases such as rheumatoid arthritis (RA), depression, and obesity, has also been reported to modify the amyloid phenotype of AD mice and is considered common co-morbid states of AD patients. In autoimmune disease, chronic inflammation can increase the risk of developing AD. Patients with the autoimmune disease Sjögren's syndrome (SS) are twice as likely to develop AD [46]. In RA, it has been shown that anti-TNF-α therapy has a protective effect on dementia [47]. In the case of depression, Aβ accumulation in AD mouse models induces depressive-like behavior, which is dependent on inflammation [23]. Inflammatory cytokine production reduces serotonin levels and contributes to behavioral changes in mice with AD [23]. Again, this can be prevented by anti-TNF-α therapy [23]. Obesity is a known comorbidity of AD and a low-grade chronic inflammatory disease. In humans and AD mouse models, cafeteria diet consumption and a higher BMI are known to accelerate AD pathology [48, 49]. For example, in humans, for every 1.0 increase in BMI at age

In an attempt to cure AD, active immunization against Aβ was performed in mice and humans [50, 51]. Studies reported that immunization had a therapeutic effect on mouse models of AD [50–52]. Unfortunately, clinical Aβ vaccination trials have been interrupted due to the development of meningoencephalitis in 6% of the patients, likely involving the appearance of pro-inflammatory macrophages, CD4+ and CD8+ T cells [50]. As well as myeloid cells, T cells can enter the brain. There are myriads of T-cell subtypes surveilling the CSF and the meningeal membranes [53], and they can enter the brain parenchyma upon cell injury [53]. It is not only brain-local T cells that react to Aβ but also blood T cells have been shown to have hyperreactivity to the Aβ peptide [54], specifically to epitopes within the residues 15–42 [54]. This evidence together suggests that the immune system and inflammation play significant roles in AD: not only helping with homeostatic Aβ clearance and preventing AD plaque formation but also by contributing to cell injury. Unfortunately, to date, tackling the immune system to prevent AD has yet to

**44**

prove clinically effective.
