**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 incurable and results in death [59].

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

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

that myelinate peripheral nerves, were responsible for the cytokine production observed in the neural tissue. A few years later, the presence of neutrophil-derived proteins in TTR amyloid deposits was described [66]. Proteins such as lipocalin and metalloproteinases were found together with TTR deposits in FAP patients [66]. The authors suggest that the sural nerve itself is the possible tissue producing these proteins for extracellular matrix remodeling and might be an effort to degrade amyloid fibrils deposited around them. Interestingly, a forgotten report in 1986 already reported the presence of neutrophil-derived proteins in amyloid-containing tissue [67]. The report in 1986 describes the presence of elastase, a neutrophil's granule enzyme, in amyloid-containing tissue from patients diagnosed with AA amyloidosis, primary amyloidosis caused by immunoglobulin light-chain aggregation and hATTR amyloidosis [67]. Notably, intact neutrophils were not found, which confirms the most pathologist reports of FAP tissues not having leukocyte infiltration. But how intracellular components from neutrophils appeared in amyloidcontaining tissue? Azevedo and colleagues reported in 2012 that a common epitope found in amyloid fibrils arising from different proteins, one of them being TTR, are able to activate neutrophils and induce elastase secretion in the form of extracellular traps [10]. These structures, called neutrophil extracellular traps (NETs), represent an important strategy to immobilize and kill invading microorganisms or in this case, aggregated proteins. The NET scaffold consists of DNA fibers associated with various granule proteins, one of them being elastase [10]. These elastase and DNA-traps accumulate in amyloid tissue and thus could explain why elastase and neutrophil-derived proteins are found around amyloid tissues in FAP patients [10]. This immune response could also be an effort to eliminate amyloid fibrils or oligomers from the affected tissue. In 2012, another important report by Buxbaum and colleagues used an animal model of FAP to study the disease progression in mice [68]. The study showed the increased levels of inflammationrelated transcripts in both liver and heart of transgenic mice, strengthening the concept that inflammation might play an important role in FAP progression [68]. Additionally, Kurian and colleagues have observed sex-specific changes in blood cell gene expression in FAP patients, suggesting that inflammatory gene markers in circulating blood cells might be influenced by sexual dimorphisms [69]. More recently, new evidence shows the presence of elevated levels of IL-6 in FAP carriers that may be produced by myeloid cells and T cells [70].

These studies altogether suggest that inflammation in FAP consists of two different phases. One phase in which inflammation possibly begins at the moment of TTR production in the liver. The synthesis and abnormal folding process of the mutated and unstable TTR in the liver requires a high energetic state and thus, may cause endoplasmic reticulum (ER) stress and the activation of the liver unfolding protein response (UPR). ER stress and the activation of UPR in liver were shown to cause pro-inflammatory cytokines production, such as IL-6 [71, 72]. IL-6 is known to increase the production of other proinflammatory intermediates and could enhance inflammation levels locally in the liver by activating liver-associated macrophages as seen in other nonamyloid diseases [73, 74]. It is ultimately important to understand whether the liver plays an important role in the inflammation observed in FAP patients due to the fact that most of these patients undergo domino liver transplant. In this procedure, a liver failure patient receives a liver from a FAP patient. However, a five-year study described that 35% of patients that underwent domino liver transplantation presented FAP symptoms earlier than donor FAP patients [75]. These data indicate that FAP patients may have altered liver capacity and a low-grade chronic inflammation, decreasing the success of liver transplants. The second phase occurs after unstable TTR reaches the bloodstream and aggregation starts. TTR oligomers have been found in blood from FAP patients [76] and could

**47**

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

ase-9 [10, 66].

**2.3 Other amyloid diseases**

elicit the production of various inflammatory cascades in circulating leukocytes and T cells. Amyloid oligomers are formed before fibril deposition and have been shown to be toxic to cells [77] and elicit inflammation when presented to immune cells [23]. Small, toxic oligomers can also be produced in situ after the cleavage of mature fibrils through the action of local proteases, such as elastase and metalloprotein-

So far, in FAP patients and hATTR mouse models as well as in vitro, TTR fibrils are able to elicit inflammation and activate a myriad of cell types. In a broader clinical context, the underlying inflammation that begins in asymptomatic patients and continues chronically might be important for the development of FAP-associated symptoms. Patients with FAP present symptoms other than neuropathy, such as gastrointestinal symptoms, cachexia, malnutrition, diarrhea, and others [59]. Inflammatory molecules are known to change neuroendocrine pathways leading to anorexia and thus cachexia in FAP patients. These new data point to an explanation for a lot of unknowns concerning the pathogenesis of FAP. Additionally, understanding the role of inflammation in hATTR will help improve the quality of life and disease management in affected patients. There are currently no studies showing if inflammation can increase the risk of developing hATTR. However, it is possible that an inflammatory environment could decrease liver function and predispose an individual for the production of misfolded proteins, such as TTR.

Although recent papers have confirmed that amyloid fibrils present polymorphisms in topology, amyloids still possess an unchangeable structural fingerprint that is shared across species [78]. A lot of different proteins are able to form amyloid fibrils and not all amyloids are pathogenic. Various hormones are present in amyloid form in the pituitary gland [79], and melanocytes possess amyloids, which contribute to melanin formation [80], etc. What makes an amyloid pathogenic or not is still unclear. However, amyloids also possess another universal characteristic: they are able to activate the immune system and induce inflammation. This suggests that inflammation may be an important component of many other amyloid diseases. Indeed, inflammation has been described in many other amyloid diseases. In Parkinson's disease (PD), the involvement of inflammation in the disease process is supported by data showing the infiltration of activated microglia and T cells in post-mortem PD brains [81, 82] Additionally, there is accumulation of proinflammatory cytokines such as TNF-α, IFN-γ, and IL-6 IL-1β in the brain and cerebrospinal fluid of PD patients [83, 84]. The PD culprit protein, α-synuclein, is able to bind to several immune receptors and elicits *in vitro* and *in vivo* inflammatory response [85]. Local inflammation has been thoroughly reported for PD patients, mainly derived from activated microglia [82, 85]. Protein aggregation in PD extends well beyond the CNS and also affects peripheral autonomic neuronal circuits, such as the enteric nervous system [86]. Gut inflammation has been recently reported in PD

and is thought to be an important component of the disease [86].

Prion disease is another widely studied amyloid disease and is also known as Creutzfeldt-Jakob disease, fatal insomnia, spongiform encephalopathy, and Kuru. Prion diseases are rare, progressive neurodegenerative disorders that affect both humans and animals [87]. They are caused by the aggregation of PrPc (cellular prion protein) into transmissible, pathogenic prions [87]. These diseases are accompanied by long incubation periods and brain changes associated with neuronal loss [87]. Identifying a role of inflammation in these diseases is rather recent and begun with studies showing that the pathological hallmarks of the prion diseases are associated with the presence of activated astrocytes and microglia [88]. CD8+ T cells are

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

*Amyloid Diseases*

that myelinate peripheral nerves, were responsible for the cytokine production observed in the neural tissue. A few years later, the presence of neutrophil-derived proteins in TTR amyloid deposits was described [66]. Proteins such as lipocalin and metalloproteinases were found together with TTR deposits in FAP patients [66]. The authors suggest that the sural nerve itself is the possible tissue producing these proteins for extracellular matrix remodeling and might be an effort to degrade amyloid fibrils deposited around them. Interestingly, a forgotten report in 1986 already reported the presence of neutrophil-derived proteins in amyloid-containing tissue [67]. The report in 1986 describes the presence of elastase, a neutrophil's granule enzyme, in amyloid-containing tissue from patients diagnosed with AA amyloidosis, primary amyloidosis caused by immunoglobulin light-chain aggregation and hATTR amyloidosis [67]. Notably, intact neutrophils were not found, which confirms the most pathologist reports of FAP tissues not having leukocyte infiltration. But how intracellular components from neutrophils appeared in amyloidcontaining tissue? Azevedo and colleagues reported in 2012 that a common epitope found in amyloid fibrils arising from different proteins, one of them being TTR, are able to activate neutrophils and induce elastase secretion in the form of extracellular traps [10]. These structures, called neutrophil extracellular traps (NETs), represent an important strategy to immobilize and kill invading microorganisms or in this case, aggregated proteins. The NET scaffold consists of DNA fibers associated with

various granule proteins, one of them being elastase [10]. These elastase and DNA-traps accumulate in amyloid tissue and thus could explain why

that may be produced by myeloid cells and T cells [70].

elastase and neutrophil-derived proteins are found around amyloid tissues in FAP patients [10]. This immune response could also be an effort to eliminate amyloid fibrils or oligomers from the affected tissue. In 2012, another important report by Buxbaum and colleagues used an animal model of FAP to study the disease progression in mice [68]. The study showed the increased levels of inflammationrelated transcripts in both liver and heart of transgenic mice, strengthening the concept that inflammation might play an important role in FAP progression [68]. Additionally, Kurian and colleagues have observed sex-specific changes in blood cell gene expression in FAP patients, suggesting that inflammatory gene markers in circulating blood cells might be influenced by sexual dimorphisms [69]. More recently, new evidence shows the presence of elevated levels of IL-6 in FAP carriers

These studies altogether suggest that inflammation in FAP consists of two different phases. One phase in which inflammation possibly begins at the moment of TTR production in the liver. The synthesis and abnormal folding process of the mutated and unstable TTR in the liver requires a high energetic state and thus, may cause endoplasmic reticulum (ER) stress and the activation of the liver unfolding protein response (UPR). ER stress and the activation of UPR in liver were shown to cause pro-inflammatory cytokines production, such as IL-6 [71, 72]. IL-6 is known to increase the production of other proinflammatory intermediates and could enhance inflammation levels locally in the liver by activating liver-associated macrophages as seen in other nonamyloid diseases [73, 74]. It is ultimately important to understand whether the liver plays an important role in the inflammation observed in FAP patients due to the fact that most of these patients undergo domino liver transplant. In this procedure, a liver failure patient receives a liver from a FAP patient. However, a five-year study described that 35% of patients that underwent domino liver transplantation presented FAP symptoms earlier than donor FAP patients [75]. These data indicate that FAP patients may have altered liver capacity and a low-grade chronic inflammation, decreasing the success of liver transplants. The second phase occurs after unstable TTR reaches the bloodstream and aggregation starts. TTR oligomers have been found in blood from FAP patients [76] and could

**46**

elicit the production of various inflammatory cascades in circulating leukocytes and T cells. Amyloid oligomers are formed before fibril deposition and have been shown to be toxic to cells [77] and elicit inflammation when presented to immune cells [23]. Small, toxic oligomers can also be produced in situ after the cleavage of mature fibrils through the action of local proteases, such as elastase and metalloproteinase-9 [10, 66].

So far, in FAP patients and hATTR mouse models as well as in vitro, TTR fibrils are able to elicit inflammation and activate a myriad of cell types. In a broader clinical context, the underlying inflammation that begins in asymptomatic patients and continues chronically might be important for the development of FAP-associated symptoms. Patients with FAP present symptoms other than neuropathy, such as gastrointestinal symptoms, cachexia, malnutrition, diarrhea, and others [59]. Inflammatory molecules are known to change neuroendocrine pathways leading to anorexia and thus cachexia in FAP patients. These new data point to an explanation for a lot of unknowns concerning the pathogenesis of FAP. Additionally, understanding the role of inflammation in hATTR will help improve the quality of life and disease management in affected patients. There are currently no studies showing if inflammation can increase the risk of developing hATTR. However, it is possible that an inflammatory environment could decrease liver function and predispose an individual for the production of misfolded proteins, such as TTR.

### **2.3 Other amyloid diseases**

Although recent papers have confirmed that amyloid fibrils present polymorphisms in topology, amyloids still possess an unchangeable structural fingerprint that is shared across species [78]. A lot of different proteins are able to form amyloid fibrils and not all amyloids are pathogenic. Various hormones are present in amyloid form in the pituitary gland [79], and melanocytes possess amyloids, which contribute to melanin formation [80], etc. What makes an amyloid pathogenic or not is still unclear. However, amyloids also possess another universal characteristic: they are able to activate the immune system and induce inflammation. This suggests that inflammation may be an important component of many other amyloid diseases. Indeed, inflammation has been described in many other amyloid diseases. In Parkinson's disease (PD), the involvement of inflammation in the disease process is supported by data showing the infiltration of activated microglia and T cells in post-mortem PD brains [81, 82] Additionally, there is accumulation of proinflammatory cytokines such as TNF-α, IFN-γ, and IL-6 IL-1β in the brain and cerebrospinal fluid of PD patients [83, 84]. The PD culprit protein, α-synuclein, is able to bind to several immune receptors and elicits *in vitro* and *in vivo* inflammatory response [85]. Local inflammation has been thoroughly reported for PD patients, mainly derived from activated microglia [82, 85]. Protein aggregation in PD extends well beyond the CNS and also affects peripheral autonomic neuronal circuits, such as the enteric nervous system [86]. Gut inflammation has been recently reported in PD and is thought to be an important component of the disease [86].

Prion disease is another widely studied amyloid disease and is also known as Creutzfeldt-Jakob disease, fatal insomnia, spongiform encephalopathy, and Kuru. Prion diseases are rare, progressive neurodegenerative disorders that affect both humans and animals [87]. They are caused by the aggregation of PrPc (cellular prion protein) into transmissible, pathogenic prions [87]. These diseases are accompanied by long incubation periods and brain changes associated with neuronal loss [87]. Identifying a role of inflammation in these diseases is rather recent and begun with studies showing that the pathological hallmarks of the prion diseases are associated with the presence of activated astrocytes and microglia [88]. CD8+ T cells are

#### *Amyloid Diseases*

also present in prion-affected brains and usually are found near activated microglia and prion amyloid plaques [89]. As inflammation progresses, inflammatory cytokines are also detected in prion-containing brains [88], and these are thought to play an important role in behavioral changes and neuronal loss observed in affected mice. And this is yet another example of inflammation being widely present and contributing to pathogenesis in an amyloid disease which was first thought to not have an inflammatory component.
