Modulation of Innate Immunity by Amyloidogenic Peptides

Trends in Immunology

Review

Modulation of Innate Immunity by Amyloidogenic Peptides Clara Westwell-Roper

1,2,

* and C. Bruce Verchere1,3,4

Amyloid formation contributes to the development of progressive metabolic and neurodegenerative diseases, while also serving functional roles in host defense. Emerging evidence suggests that as amyloidogenic peptides populate distinct aggregation states, they interact with different combinations of pattern recognition receptors (PRRs) to direct the phenotype and function of tissue-resident and infiltrating innate immune cells. We review recent evidence of innate immunomodulation by distinct forms of amyloidogenic peptides produced by mammals (humans, non-human primates), bacteria, and fungi, as well as the corresponding cell-surface and intracellular PRRs in these interactions, in human and mouse models. Our emerging understanding of peptide aggregate-innate immune cell interactions, and the factors regulating the balance between amyloid function and pathogenicity, might aid the development of anti-amyloid and immunomodulating therapies.

Highlights Amyloidogenic peptides of both bacterial and mammalian origin transition through multiple aggregation states with distinct effects on mononuclear phagocyte function, commonly mediated by TLR2 and NLRP3. Promising therapeutic agents target such macrophage/peptide interactions. Disease-associated amyloidogenic peptides may act as antimicrobial peptides. Soluble oligomers bind microbial cell walls, protofibrils limit adhesion to host cells, and fibrils wall off invading pathogens. Infectious or sterile inflammatory stimuli may partially drive amyloidosis, a hypothesis supported by cross-seeding of endogenous amyloid formation by products of innate immune cell activation: apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks and neutrophil extracellular traps (NETs).

Amyloidogenic Peptides: Ubiquitous and Promiscuous Triggers of the Innate Immune Response Amyloid (see Glossary) diseases are the most common protein misfolding diseases in humans, with at least 50 amyloidogenic peptides linked to hereditary and acquired disorders [1]. Systemic amyloid diseases include secondary amyloidosis, in which fragments of serum amyloid A (SAA) accumulate in the kidney, liver, and spleen. Peptides that contribute to local amyloid formation include amyloid-β (Aβ) in Alzheimer’s disease (AD), tau in AD and frontotemporal dementia, prion protein (PrP) in the transmissible spongiform encephalopathies, α-synuclein in Parkinson’s disease (PD) and dementia with Lewy bodies, S100A9 in AD and traumatic brain injury (TBI), and islet amyloid polypeptide (IAPP) in type 2 diabetes (T2D). In each disease, protein monomers convert from a soluble native state to insoluble, nonbranching fibrils through nucleationdependent polymerization. The cross-β-sheet structure, detectable in vitro by X-ray diffraction and defined by parallel β-sheets lying perpendicular to the fibril axis, confers several features to pathogenic proteins: self-propagation, protease resistance, and sometimes, transmissibility [1]. Many amyloid-forming peptides follow similar aggregation pathways; intermediate aggregates (prefibrillar oligomers) and fibrils derived from different peptides can share common exposed epitopes and dye-binding properties [2]. Fibrils disrupt tissue architecture and perturb cellular membranes, while oligomers are associated with pore formation, calcium dysregulation, reactive oxygen species (ROS), and apoptosis in a variety of cell types ranging from neurons to pancreatic β cells [1]. Within the same patient, a single peptide can demonstrate remarkable aggregate polymorphism; indeed, multiple fibrillar structures have been recently extracted from postmortem cortical tissues of patients with AD [3]. Most organisms have evolved to make use of amyloid conformations; for instance, functional amyloids (such as enterobacterial curli fibrils comprised of the monomer CsgA) transition through some of the same oligomeric intermediates as pathogenic amyloids but with multiple tightly controlled regulatory mechanisms, including dedicated chaperone proteins. Bacterial and fungal amyloids serve important functions in biofilm formation, the fungal life cycle, virulence, antimicrobial activity, and epigenetic inheritance [4]. 762

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https://doi.org/10.1016/j.it.2019.06.005

Cross-seeding can also occur between amyloids in different tissues, such as the gut and brain, and across species. Manipulation of microbial amyloids might be a potential therapeutic approach to treat amyloid and inflammatory diseases.

1

BC Children’s Hospital Research Institute, Vancouver, BC, Canada Department of Psychiatry, University of British Columbia, BC, Canada 3 Department of Pathology and Laboratory Medicine, University of British Columbia, BC, Canada 4 Department of Surgery, University of British Columbia, BC, Canada 2

*Correspondence: [email protected] (C. Westwell-Roper).

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Mounting evidence over the past 20 years from both cell and animal models suggests that amyloidogenic peptides of both microbial and mammalian origin act as potent proinflammatory stimuli for tissue-resident and infiltrating macrophages (Box 1). Comprehensive reviews have addressed the role of innate immunity and amyloid-induced inflammation in AD [5], PD [6], secondary amyloidosis [7], and T2D [8]. Studies have begun to elucidate the role of myeloid cells in the regulation of amyloid plaque morphology and modulation of the inflammatory microenvironment. Specifically, interaction of diverse amyloidogenic peptides with phagocytic cells leads to uptake of peptide aggregates in vitro, in isolated tissues such as pancreatic islets and brain sections ex vivo, and in mouse models of amyloid disease that can be monitored by increasingly sophisticated imaging techniques such as two-photon microscopy. Recent reports, synthesized in this review, have revealed remarkable similarities in the repertoire of pattern recognition receptors (PRRs) activated by distinct human amyloidogenic peptides and their various oligomeric and fibrillar forms, both in vitro and in mouse models of amyloid disease. Moreover, developments in fibril purification, imaging, and spectroscopy have now revealed fibril structures in near-atomic detail and suggest a wide diversity of amyloid structures for any given peptide [3,9]; this diversity highlights the need to better understand how distinct structures (despite potential shared features, such as exposed epitopes or PRR binding sites) can modulate immune cell phenotypes. Furthermore, evidence of cross-seeding among microbial and mammalian amyloids points to a previously unappreciated role for gut microbiota and microbial infections in amyloid formation in mouse models of human neurodegenerative diseases, including AD and PD [10,11]. Consequently, this review integrates recent knowledge linking microbial and mammalian amyloids to innate immune function in human and rodent models, with a focus on the nature of amyloid/immune cell interactions. We discuss key findings suggesting that common mechanisms of innate immune cell activation by functional and pathogenic peptides, involving the sequential activation of scavenger receptors, Toll-like receptors (TLRs), and inflammasomes, might underlie host defense, reciprocal host–microbiome interactions, tissue homeostasis, and chronic amyloid disease.

Amyloid Uptake by Macrophages and Monocytes An effect of amyloid on macrophage phenotype was first surmised based on altered microglial morphology associated with evolving amyloid plaques in postmortem para-hippocampal tissue

Box 1. Early Evidence for Amyloid-Induced Inflammation in Mammals Early in vivo evidence for amyloid-associated inflammation was based on autopsy specimens from patients with AD, which demonstrated activated microglia and increased expression of proinflammatory cytokines, including IL-1β, associated with amyloid plaques [106]. Like Aβ, IAPP was shown in cell culture experiments to induce IL-6 and IL-8 secretion by human astrocytoma cells [107] and release of IL-1β, TNF-α, IL-6, IL-8, CCL3, and CCL4 by monocytes [108]. IAPP deposition was associated with increased islet macrophage secretion of proinflammatory cytokines in mouse models of T2D with transgenic human IAPP expression [61,62], and islets from patients with T2D were found to express increased IL-1 compared with controls with no amyloid [109]. Similarly, in vitro studies of extracellular α-synuclein demonstrated activation of microglia from rat primary mesencephalic cultures [110] and human astrocytes from surgically resected temporal lobe tissue [110]. Following the initial observation of upregulated MHC class II expression in tissue from PD patients [111], additional studies reported elevated IL-1, TNF-α, and IL-6 within the basal ganglia and cerebrospinal fluid [112]. Some studies using rodent models also found elevated expression of IL-6 and macrophage colony-stimulating factor at sites of amyloid deposition in systemic AA amyloidosis and SAA-containing epithelioid granulomas [24]. Network analysis of gene expression in rodent models of prion disease (also characterized by activation of astrocytes and microglia that release IL-1β and other cytokines) identified a core of immune response-related genes that regulate prion protein replication, accumulation, and neuronal cell death [113]. Similarly, amyloidogenic S100 proteins, a family of low-molecular weight DAMPs with variable propensity to form amyloid, are found in deposits associated with activated macrophages in normal prostate [114] and in the human brain following traumatic injury [95]. Amyloids can also trigger systemic inflammation in animal models of severe infection; for example, the amyloid-forming peptide CsgA, which aggregates to form curli fibrils, is required for Escherichia coli-induced septic shock in mice [115]. These data set the stage for studies seeking a mechanistic understanding for modulation of microglial, astrocyte, monocyte, and macrophage phenotype by amyloidogenic peptides.

Glossary α-synuclein: presynaptic protein found in Lewy bodies; forms amyloid in PD. Amyloid: ordered arrangement of peptides defined by cross-β-sheet conformation. Amyloid-β (Aβ): peptide derived from cleavage of amyloid precursor protein; forms amyloid in AD. Antimicrobial peptide (AMP): host defense peptide with immunomodulatory and membranedestabilizing effects. APP/PS1 mice: model of AD expressing human APP with a KM670/ 671NL mutation and PSEN1 with an L166P mutation, both under control of the Thy1 promoter. Autophagy: regulated process by which cytosolic components are recycled through lysosomes. Biofilm: microbial community of adherent cells that produce extracellular matrix polymers. Cross-β-sheet: structural motif consisting of β-strands perpendicular to the fibril axis; stabilized by interstrand hydrogen bonds and dry steric zipper interfaces. CRND8: mouse model of AD in which human APP695 (with KM670/671/NL and V717F mutations) is driven by the hamster prion promoter. Cross-seeding: facilitation of fibrillization by interaction between two different peptides. Curli: amyloid fibril produced by enteric bacteria. Danger-associated molecular pattern (DAMP): sterile proinflammatory signal released by dysfunctional or dying cells. Fluid-phase pinocytosis: mode of endocytosis in which small particles are internalized with extracellular fluid. Granuloma: focal collection of inflammatory cells formed in response to a persistent stimulus. Inflammasome: complex comprised of a sensor, adaptor, and enzymatic component; processes pro-IL-1β and pro-IL-18 into their mature forms. Islet amyloid polypeptide (IAPP): hormone secreted by β cells; forms amyloid in T2D. Long-term potentiation: strengthening of neuronal synapses following high-frequency stimulation. Lysozyme: antimicrobial enzyme; forms amyloid in hereditary systemic amyloidosis.

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from AD patients [12]. Similarly, IAPP-immunoreactive fibrils were observed within resident macrophages in human insulinomas and monkey pancreatic islets [13]. Moreover, uptake and degradation of fibrils was demonstrated in vitro by electron microscopy showing aggregates becoming condensed in lysosomes to form protofilaments that were resistant to proteolysis in mouse peritoneal macrophages [14]. Recently, imaging in two transgenic AD mouse models expressing human amyloid precursor protein (APP) (5xFAD and CRND8) revealed the formation of a barrier by microglia around small amyloid plaques [15]. In addition, lower rates of dystrophic neuronal process formation in areas with microglial coverage compared with those without suggested that microglia-mediated peptide clearance protected neurons from Aβ toxicity [15]. This study also attempted to characterize the peptide aggregates by using the purported protofibrilbinding properties of curcumin, as well as exogenous labeled soluble Aβ, and suggested that accumulation of protofibrillar ‘hotspots’ or areas rich in pre-amyloid aggregates occurred in areas lacking microglial processes compared with microglia-covered regions. These areas were distinct from soluble peptide, antibody-binding oligomers, or larger thioflavin-binding aggregates [15]. Moreover, expansion of microglial coverage by Cx3cr1 gene deletion and anti-Aβ immunotherapy in this model prevented accumulation of neurotoxic Aβ aggregates. Taken together, these data point to a critical role for microglia in protecting neurons from Aβ amyloid formation. The pathways for peptide uptake depend on its aggregation state, as well as binding by soluble factors, including scavenger receptors (Box 2). On the one hand, small soluble aggregates are taken up nonspecifically by fluid-phase pinocytosis and internalized soluble Aβ localizes to late endolysosomal compartments for degradation [16]. On the other hand, fibril uptake depends on scavenger receptor-mediated particle recognition and results in segregation into distinct subcellular compartments in cultured rodent microglia and human monocytes [17]. Consequently, innate immune cells can constitutively clear small aggregates under homeostatic conditions. However, the peptide may transition through multiple aggregation states with different biological activities following uptake; for example, intralysosomal oligomerization has been observed in mouse bone marrow-derived macrophages (BMDMs) for human α-synuclein, IAPP, and Aβ [18]. As a result, multiple mechanisms leading to downstream inflammatory signaling, including endolysosomal dysfunction, may depend not only on the process of fibril formation, but also on factors affecting the cell’s phagocytic efficiency. The latter include the cell’s metabolic state, which, as demonstrated in cultured mouse microglia subjected to extracellular metabolic flux analysis, is driven toward glycolysis by interaction with Aβ aggregates [19]. Presumptive Box 2. Soluble Scavenger Receptors and Peptide Aggregation A number of soluble scavenger receptors, including serum amyloid P (SAP), CRP, and complement, appear to colocalize with amyloid deposits and peptides in vivo in rodent and human tissues based on histological analyses and in vivo antibody studies. For example, a small Phase I trial (NCT01777243) of an anti-SAP antibody in 15 patients with systemic amyloidosis showed reduced amyloid load, which may be secondary to subsequent complement activation and recruitment of macrophages that destroy the deposits [116] or to blockade of SAP binding to amyloid fibrils, rendering them more susceptible to proteolytic cleavage [117]. Several in vitro studies have suggested specificity in the interactions between soluble amyloidogenic peptides and complement proteins (e.g., C1q interacts through its globular domains with small prion protein oligomers [118]), mannose-binding lectin (e.g., the cysteine-rich domain may be responsible for binding of Aβ as demonstrated by ELISA [119]), and the pentraxins serum amyloid P component and CRP (both of which have been demonstrated by ELISA and crosslinking experiments to interact with monomeric and oligomeric Aβ [120]). Other in vitro studies have confirmed that these interactions are dependent on the amyloid-forming capacity of the binding peptide; for example, SAP binds yeast expressing an amyloid-forming version of the adhesin Als5p, but not a nonamyloid version [120]. Both CRP and SAP can also inhibit Aβ and β2-microglobulin aggregation under some conditions, as determined by thioflavin T binding and electron microscopy, although in the presence of calcium, SAP can accelerate the later stages of fibrillization; this raises the possibility that SAP might serve a chaperone-like function [120]. While the structural basis for their binding remains to be confirmed, the dominant hypothesis suggests an interaction between hydrophobic surface domains of extracellular chaperones and exposed hydrophobic residues of oligomers, primarily because hydrophobicity correlates with aggregate toxicity and binding is disrupted at lower pH [120].

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Myddosome: protein complex characterized by the presence of the adaptor protein MyD88. Neutrophil extracellular trap (NET): network of extracellular fibers comprised of DNA together with cytoplasmic and granular proteins. Nucleation-dependent polymerization: process by which soluble monomers organize to facilitate amyloid fibril formation. Pattern recognition receptors (PRRs): germline-encoded innate immune sensing proteins, both membrane-bound and intracellular. Prion protein (PrP): conserved cell membrane protein that exists in normal cellular (PrPc) and disease-associated scrapie (PrPSc) isoforms. Protofibril: transient intermediate aggregate with unstructured regions lacking the cross-β structure of mature amyloid fibrils. PS19 mouse model of tauopathy: characterized by human tau with the disease-associated P301S mutation under the control of the mouse prion protein promoter. RIP1/RIP3 necrosome: complex containing kinases involved in TNF-induced programmed necrosis Scavenger receptor: pattern recognition receptor involved in ligand uptake; originally identified based on binding oxidized low-density lipoprotein. Self-propagation: production of more copies, often by templating. Stereotactic injection: injection technique using skull features to determine coordinates required to reach a target brain location. Surface plasmon resonance: technique for studying molecular interactions based on changes in refraction of polarized light. Synucleinopathy: neurodegenerative disease characterized by aggregated α-synuclein. Templating: induction of a conformational rearrangement resulting in a new isoform. Thioflavin: benzothiazole salt that undergoes a red shift in emission spectrum upon β-sheet binding. Toll-like receptor (TLR): conserved family of membrane-bound PRRs. Transmissible spongiform encephalopathies: prion-associated diseases affecting the nervous system. TREM2: immunoglobulin superfamily member involved in myeloid cell activation.

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blockade of Aβ-induced TLR2 signaling using a blocking antibody restored oxidative metabolism and increased phagocytic efficiency, pointing to a potential strategy to promote aggregate clearance by modulation of specific Aβ-microglia interactions [19]. Cell-surface scavenger receptors are structurally heterogeneous, comprising eight classes based on their domain architecture. Ligand binding induces endocytosis or phagocytosis followed by lysosomal degradation. Targeting to endosomal compartments may promote further amyloid aggregation due to acidification of lysosomes and high intracompartment peptide concentrations, both factors that promote amyloid aggregation in a variety of model systems, as recently described for SAA, based on multiple in vitro spectroscopic, biochemical, and structural methods [20]. Some scavenger receptors also activate signaling pathways upon ligand binding, including the class B scavenger receptor CD36, which cooperates with TLR4 and TLR6 [21] as well as TLR2/6 heterodimers to induce MyD88-dependent NF-κB activation and chemokine upregulation in human and mouse mononuclear cells [22]. Scavenger receptors that have long been implicated in amyloid uptake by human and rodent macrophages or microglia include macrophage receptor with collagenous structure (SR-MARCO) and CD36 for α-synuclein [23], SAA [24], Aβ [25], and IAPP [18]; formyl peptide receptor-like 1 (FPRL1) for Aβ and SAA; formyl peptide receptor 2 (FPR2) for Aβ, tau, and SAA; and a complex of CD47 and α6β1 integrin for Aβ [26] (Figure 1). All rodent models of AD in which amyloid pathology or cognitive function have been measured in the context of class A or B scavenger receptor deficiency have pointed to a protective role for these PRRs. For example, APP/PS1 mice that are also deficient in scavenger receptor class A (Scara1–/–) demonstrate accelerated hippocampal Aβ accumulation and mortality [27] as well as increased vascular amyloidosis [28] compared with Scara1+/+ mice; this pathology has been associated with impaired microglial phagocytosis and increased interleukin (IL)-1β and tumor necrosis factor (TNF)-α, together with decreased IL-10 and transforming growth factor (TGF)-β secretion ex vivo [29].

5xFAD: mouse model of AD; both human APP695 with KM670/671/NL, V717I, and I716V mutations and human PSEN1 with M146L and L286V mutations are overexpressed under the Thy1 promoter.

By contrast, Cd36–/– mice overexpressing human APP with the KM670/671NL mutation under control of the hamster prion promoter (Tg2567) are protected against neurovascular dysfunction and display improved cognition compared with Cd36+/+ controls [25]. This finding has prompted efforts to develop inhibitors of CD36-Aβ binding as potential anti-inflammatory therapies for the treatment of AD [30]. CD36 is also required in mouse BMDMs for the in vitro activation of the cytosolic PRR NACHT, LRR, and PYD domains-containing protein 3 (NLRP3 inflammasome; see below) by IAPP and Aβ; through the process of PRR templating, CD36 facilitates the conversion of Aβ from a soluble to a fibrillar state, as determined by fluorescence microscopy, in turn leading to downstream IL-1β secretion [18]. Thus, CD36 may not only sense but also modulate amyloid formation. While this study also demonstrated increased IAPP binding to Chinese hamster ovary cells with ectopic expression of CD36 compared with empty vector, the peptide species and mechanisms involved in this potential interaction require further study in order to clarify its relevance for amyloid clearance in pancreatic islets in T2D [18]. A recent study characterizing the phagocytic properties of brain mononuclear phagocytes suggested that CD11b+CD45high microglia isolated from 5xFAD mice (AD model) have a high capacity for phagocytosis of Aβ fibrils in a manner speculated to be dependent on CD36, CD148, and macrophage scavenger receptor 1 based on inhibition of Aβ uptake by neutralizing antibodies in cultured BV2 microglia [31]. This same cell subset exhibits high expression of triggering receptor expressed on myeloid cells 2 (TREM2), a phagocytic receptor also shown to bind Aβ oligomers in solid phase binding, surface plasmon resonance, and immunoprecipitation assays with human TREM2 in vitro [32]. This is relevant, as AD-associated TREM2 mutations reduce its binding affinity for oligomeric Aβ; indeed, both complete deletion and haploinsufficiency due to some of these mutations have been shown to accelerate peptide accumulation and neuronal loss relative to wild type (WT)

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(A) Ligand heterogeneity in time and space Monomers PRR-mediated Unfolded facilitation of amyloidogenesis Native Aggregationinduced PRR activation

(B) Mechanisms of PRR activation Structural interactions • Binding to exposed hydrophobic epitopes • Stabilization or facilitation of cross-β-sheet structure Nonspecific structural changes • Recognition of other ligands liberated by amyloidogenesis (e.g., lipoproteins, ATP) • Response to membrane perturbation • Receptor oligomerization facilitated by amyloid Altered homeostasis • Mitochondrial dysfunction; reactive oxygen species • Endolysosomal dysfunction • Altered ion homeostasis • Autophagy

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Figure 1. Diverse Human Amyloidogenic Peptides Activate Toll-like Receptor 2 and NLRP3 Inflammasome Signaling. Check marks indicate evidence supporting activation of the indicated receptor, based on cell culture studies using ELISA, immunoprecipitation, or functional assays. Data from mouse models of amyloid disease with deficiency or blockade of the indicated receptor have suggested improvement (orange), exacerbation (blue), or unclear (grey) effects on the combination of amyloid or inflammatory pathology and measures of disease progression. All receptor–peptide interactions are discussed in the text, except for receptor for advanced glycation end products (RAGE) and amyloid-β (Aβ) [105]. Panel A demonstrates the structural diversity of peptide species present throughout the process of amyloidogenesis, beginning with the formation of nuclei within a mixture of soluble peptides (nucleation) followed by fibril elongation and ultimately, equilibrium among fibrils, monomers, and potentially smaller aggregates. Each species populated by the aggregating peptide has the potential to trigger pattern recognition receptor (PRR) activation via mechanisms described in Panel B. In turn, receptor-mediated uptake and templating may facilitate the process of amyloid formation. Abbreviations: α-syn, α-Synuclein; FPR, formyl peptide receptor; FPRL-, formyl-peptide-receptor-like-; MARCO, macrophage receptor with collagenous structure; NLRP3, NACHT, LRR, and PYD domains-containing protein 3; SR, scavenger receptor; TLR, Toll-like receptor; TREM2, triggering receptor expressed on myeloid cells 2.

TREM2 in mice receiving stereotactic injection of Aβ into the hippocampus [32]. Similarly, transdifferentiated microglia-like cells derived from TREM2+/– or TREM2–/– human pluripotent stem cells demonstrate impaired amyloid plaque clearance in vitro compared with TREM2+/+ controls [33]. This is consistent with reduced clustering of microglia around amyloid plaques in the temporal neocortex of patients with AD-associated TREM2 coding variants compared with those without [34], further pointing to a key role for this receptor in mediating amyloid/microglial interactions. A recent study emphasized opposing roles of TREM2 deficiency at different stages of disease in the APP/PS1 mouse model, with amelioration of plaque pathology in younger mice and exacerbation at a more advanced stage [35]. In addition, Trem2+/– mice show exacerbation of tau pathology, inflammation, and cortical atrophy in the PS19 mouse model of tauopathy, relative 766

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to Trem2+/+ and Trem2–/– mice [36]. In this model, complete loss of TREM2 might limit tauinduced neuronal damage by suppressing the proinflammatory response, or might lead to compensatory changes in microglial phenotype [36]; these differential effects of partial and complete loss of TREM2, and perhaps other PRRs with signaling capacity, suggest that the outcome of therapeutic blocking agents may be difficult to predict. Further work is required to understand how different structural forms of oligomeric peptides are recognized and processed by phagocytic cells in different tissues. An increased understanding of this type of structural heterogeneity has implications for strategic approaches to therapeutic modulation of peptide clearance to combat aggregate formation (see Outstanding Questions ).

Amyloid-Induced TLR Activation Mammalian TLRs are a family of membrane-bound PRRs that recognize common microbial structural motifs, in addition to endogenous ligands. TLR activation by ligand recognition induces receptor recruitment of adaptor proteins such as myeloid differentiation factor-88 (MyD88) and Toll/IL-1R domain-containing adapter inducing interferon-β (IFN-β). Downstream activation of the transcription factors NF-κB, activator protein 1, and interferon regulatory family (IRF) members induces the expression of proinflammatory cytokines such as IL-1β and TNF-α; chemokines such as CCL2 and IL-8; and co-stimulatory molecules such as CD80 and CD86. As with scavenger receptors, a similar repertoire of TLRs interacts with distinct amyloidogenic peptides; indeed, to date, TLR1, TLR2, TLR4, and TLR6 have all been identified in such interactions (Figure 1). Specifically, in vitro studies using anti-TLR blocking antibodies and human NF-κB reporter cell lines suggest that TLR1/2 heterodimer formation is triggered by aggregates of Aβ [37], lysozyme [38], α-synuclein [39], and CsgA [40]. Moreover, lysozyme and IAPP can also activate TLR2/6 in HEK293 cells transfected with human TLRs, human THP-1 monocytes, and mouse BMDMs [41]. TLR4 and TLR4/6 appear to be involved in the clearance of α-synuclein [42], Aβ [21], and PrPSc [43] based on studies showing increased formation of the respective amyloid in Tlr4-/- relative to WT mice, together with in vitro studies demonstrating a requirement for TLR4/6 for peptideinduced NF-κB activation (based on cytokine secretion or ROS production) in human THP-1 cells and primary mouse microglia. In addition to CD36, other membrane-bound receptors can partner with TLRs to enhance ligand recognition. These include CD14, which interacts with both Aβ and curli fibrils [44] (as evidenced from ELISA-based binding assays using synthetic peptides and human recombinant CD14, activation of NF-κB reporter activity in HEK cells expressing recombinant human TLR1/2 with soluble CD14, and increased production of proinflammatory mediators such as IL-6 and nitric oxide) in WT versus CD14-deficient BMDMs [44]. CD14 may also be involved in the progression of prion disease; Cd14–/– mice infected intracerebrally with two pathogenic prion strains survived longer than WT mice and also showed increased microglial TGF-β and IL-10 and reduced IL-1β expression by immunohistochemical analysis of thalamic sections [45]. However, CD14 was not required for NF-κB activation by lysozyme [38], SAA [46], or IAPP [41] in HEK293 or HeLa cells expressing TLR1 and TLR2 or in mouse BMDMs. The shared capacity of microbial amyloids such as curli and mammalian amyloids such as Aβ, lysozyme, SAA, and IAPP to activate TLR2 (and likely CD36), suggests that innate immune recognition of dysregulated amyloidogenic self-peptides might be an evolutionarily conserved mechanism of host defense. Signaling via a single TLR induces expression of hundreds of genes, including proinflammatory cytokines, anti-inflammatory mediators, antimicrobial peptides (AMPs), metabolic regulators, and proteins involved in adaptive immunity. Rodent models of amyloid disease (primarily AD) with TLR or MyD88 deficiency have found variably protective [37,47–49] and detrimental [50–53] effects of the same TLR pathway components (Figure 1). While a detailed discussion of each animal model tested is beyond the scope of this review, we posit that these variations might be explained Trends in Immunology, August 2019, Vol. 40, No. 8

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by four primary factors: (i) the presence of other stimuli modifying the activation state of monocytes or microglia in the vicinity of amyloid aggregates, (ii) the age and gender of the mouse model, (iii) the amyloid load or stage of aggregation at which receptor blockade or deficiency is assessed, and (iv) any cell type-specific effects of receptor deficiency. Recent in vivo work employing clinically relevant methods of inhibition points to the potential of targeting TLR pathways therapeutically in amyloid disease. Specifically, biweekly intravenous administration of anti-TLR2 blocking antibody decreased microglial activation (as determined by MHC class II and CD68 immunoreactivity) in the APP/PS1 transgenic mouse model of AD, which was associated with reduced hippocampal plaque burden and improved spatial learning relative to mice treated with isotype control [47]. A selective TLR4 receptor antagonist similarly limited memory impairment relative to isotype control in mice following Aβ oligomer infusion into the lateral cerebral ventricle [49]. These data highlight the potential feasibility of therapeutic TLR blockade in the central nervous system (CNS) and presumably, in other amyloid diseases.

Amyloid-Induced Inflammasome Activation Inflammasomes are a group of multiprotein complexes that process pro-IL-1β and pro-IL-18 into their mature forms. Most include three major components: a sensor, often of the nucleotidebinding oligomerization domain and leucine-rich repeat-containing receptors (NLR) family, such as NLRP3; pro-caspase-1, which cleaves pro-IL-1β; and ASC, which bridges pro-caspase-1 and the NLR. IL-1 signaling and inflammasome activation in neurodegenerative diseases have been reviewed elsewhere [54]. Some diseases associated with protein aggregation but not amyloid formation also appear to be IL-1-mediated, suggesting that the unique structure of amyloid or oligomers is not required for this response. For example, treatment with IL-1 receptor antagonist extends the lifespan of the G93A-SOD1 transgenic mouse model of amylotrophic lateral sclerosis [55]. However, not all amorphous aggregates are capable of activating NLRP3; for example, human lysozyme fibrils with a cross-β structural signature [assessed by Fourier transform infrared spectroscopy and transmission electron microscopy (TEM)], but not nonfibrillar aggregates, can induce NLRP-3 dependent IL-1β secretion from human THP-1 monocytes and mouse BMDMs [38]. Ligands that prime the inflammasome by induction of pro-IL-1β can also trigger upregulation or post-translational modification of their components, including deubiquitination of NLRP3, phosphorylation of apoptosis speck-like protein containing a caspase-recruitment domain (ASC), and post-translational activation of NLRP3 mediated by IRAK1 [56]. The bulk of the evidence for these post-translational modifications comes from in vitro experiments in mouse BMDMs, both WT, and lacking components of NLRP3 and TLR signaling pathways, with rapid production of active caspase-1 as a read-out. A heterogeneous mixture of amyloidogenic peptides may therefore provide multiple signals contributing to IL-1β release. Early studies demonstrating NLRP3-dependent IL-1β in mouse BMDMs and microglia exposed to amyloidogenic peptides (Aβ, IAPP, SAA, α-synuclein, HLA-B27, TNFR1, and Huntingtin) and nonamyloid aggregates (SOD1) have been previously reviewed [57]. One of the first in vivo studies suggesting a role for NLRP3 in mouse models of amyloid disease demonstrated attenuation of IL1β secretion in the lungs of C57BL/6 mice following oropharyngeal exposure to human apo-SAA, a model of allergic airway inflammation. Neutrophil influx was diminished in Tlr2–/– but not Nlrp3–/– mice relative to WT controls, whereas NLRP3 was required for detection of IL-1β (but not IL-6 or MCP-1) in bronchiolar lavage fluid [58]. Multiple reports have now demonstrated NLRP3dependent IL-1β secretion in mouse models expressing other amyloidogenic peptides, including Aβ [59] and α-synuclein [60] (Figure 1). For example, APP/PS1 Nlrp3–/– and Casp1–/– mice are protected from loss of spatial memory compared with Nlrp3+/+ and Casp1+/+ controls; they also demonstrate reduced brain caspase-1 and IL-1β expression, as well as enhanced Aβ clearance in the hippocampus, frontal cortex, and motor cortex [59,72]. In the case of α-synuclein, 768

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recent work showed that oral administration of the NLRP3 inhibitor MCC950 in multiple PD models, including WT mice treated with preformed α-synuclein fibrils and MitoPark mice (with dopaminergic neuron-specific inactivation of mitochondrial transcription factor A), mitigated motor deficits, nigrostriatal dopaminergic degeneration, and accumulation of α-synuclein aggregates in both the nigrostriatal system and cortex [60]. Outside the CNS, the role of NLRP3 in the pathogenesis of islet β cell dysfunction in rodent models of T2D with islet amyloid formation has not been directly examined. However, synthetic human IAPP induces activation of caspase-1 and secretion of IL-1β in mouse BMDMs and dendritic cells, as well as islet-resident macrophages [61,62]. Moreover, intraperitoneal administration of IL-1 receptor antagonist improves glycemic control in human IAPP transgenic mice relative to vehicle control [63]. On the one hand, these studies highlight IL-1 signaling, and in particular NLRP3 activation, as potential common therapeutic targets among aging-associated amyloid diseases. On the other hand, no effect of NLRP3 or ASC deficiency has been noted on mortality or brain pathology in mouse models of prion disease, in which WT, Nlrp3–/–, or Pycard–/– mice were injected in the right hemisphere with pathogenic PrPSc [64]. This contrasted with preliminary data from cell culture models demonstrating IL-1β release in response to noninfectious neurotoxic peptides or in vitro-generated recombinant PrP aggregates in primary mouse microglia [64]. Thus, while some in vitro systems using protein fragments or recombinant peptide may be appropriate for studying peptide toxicity, they do not necessarily model the proinflammatory activity of bone fide peptide aggregates in vivo. The need for caution in extrapolating findings based on synthetic peptide activity is underscored not only by evidence of peptide aggregate heterogeneity in vivo [3] but also by evidence that protein modifications such as sialylation of PrP can modify microglial responses [65]. Recent work in other mouse models suggests potential for therapeutically relevant approaches employing blood–brain barrier-permeable small molecule inhibitors targeting the IL-1 pathway. For example, the caspase-1 inhibitor VX-765 reverses memory impairment in the J20 transgenic mouse model of AD expressing double mutant APP driven by the human platelet-derived growth factor beta polypeptide promoter [66]. This was associated with decreased Aβ deposition in the hippocampus and cortex as well as fewer Iba1-positive activated microglia and lower IL-1β relative to mice treated with vehicle control [66]. Inhibition of disease-associated pathology has also been observed with small molecule inhibitors of NLRP3, including MCC950, which abrogates Aβassociated inhibition of long-term potentiation in the brains of transgenic rats expressing double mutant human APP under the control of the Thy1.2 promoter [67]; and JC-124, which increases expression of the presynaptic marker synaptophysin and limits cortical and hippocampal plaque formation in CNRD8 transgenic mice [68]. Although NLRP3 appears to drive pathology in multiple rodent models of amyloid disease, caution is needed when blocking downstream targets that may also have protective effects. For example, APP/PS1 Il18–/– transgenic mice exhibit increased mortality due to seizure activity, together with increased expression of excitatory synaptic proteins, spine density, and basal excitatory synaptic transmission relative to Il18+/+ controls [69]. It is therefore important to consider that blockade of specific proinflammatory cytokines could have unanticipated consequences given their role in normal tissue function, in this case, suppression of aberrant neuronal transmission in the CNS [70]. Potential mechanisms for NLRP3 activation in amyloid disease include lysosomal damage, potentially occurring via aggregate-induced disruption of membrane lipids, based on the detergent-like effect of oligomeric peptides on lipid bilayers in vitro (characterized by atomic force and electron microscopy [70]). Other NLRP3 activation mechanisms may be associated with the release of lysosomal cysteine protease cathepsin B, potassium efflux, perturbation in calcium homeostasis, ROS generation, mitochondrial dysfunction, and production of oxidized

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mitochondrial DNA [71]. These mechanisms have been suggested for amyloidogenic peptides in general, based on the use of inhibitors in cultured murine macrophages and microglia, as well as the in vitro and in vivo characterization of their activity in other cell types, such as lysosomedependent autophagy of IAPP in β cells [73]. With respect to lysosomal damage, some peptides appear to act via distinct mechanisms; for example, SAA- and PrPSc-induced NLRP3 activation is associated with cathepsin B release but not with lysosomal destabilization (based on the maintenance of lysosomal pH and morphology) in human and mouse macrophages treated with peptide in vitro [74,75]. Autophagy may also be important, not only for survival of the peptide-producing cell, but also for inflammasome activation in mononuclear phagocytes. For example, a recent study of APP/PS1 mice with heterozygous loss of beclin-1 (Becn1+/–), a protein required for nucleation of autophagic vesicles, showed increased IL-1β protein in brain lysates relative to Becn1+/+ mice; ex vivo, beclin-1 was required for lipopolysaccharide- (LPS-) and ATP-induced inflammasome formation in microglia, as evidenced from ASC localization and caspase-1 activation assays in brain slices [76]. The mechanisms of amyloid-induced NLRP3 inflammasome activation described above (i.e., priming via PRR and TLR signaling followed by NLRP3 activation) may be concurrent, or occur in sequence, as reflected by the wide variation of reported kinetics for the same peptide. For example, one study suggested that membrane damage due to oligomerization within the lysosome of LPS-primed bone marrow-derived dendritic cells led to rapid caspase-1 activation and cleavage of pro-IL-1β in response to IAPP [61], whereas other in vitro studies have shown a requirement for a longer incubation period prior to caspase-1 activation, or suggested that LPS was not required for the priming step (induction of pro-IL-1β), occurring only in response to freshly dissolved IAPP [41]. These differences may relate to different methods of peptide preparation and specific aggregate species present in cell cultures (discussed below). While many in vitro studies have omitted assessment of viability and membrane permeability, incubation with amyloidogenic peptides at high concentrations may liberate other endogenous danger-associated molecular patterns (DAMPs) associated with cell death and membrane damage, leading to secondary signaling events. These DAMPs include membrane lipids, which may activate TLRs, as well as ATP, implicated in NLRP3 activation by SAA and Aβ [77] (Figure 2). It is likely that the combination of the peptide aggregate and some of these secondary signals are responsible for sequential priming (via scavenger receptor-mediated uptake, PRR-facilitated oligomerization, and TLR signaling) and activation of inflammasomes. This might partially explain their potent effects on tissue-resident macrophages in vitro and the consistent evidence of a local inflammatory response in both human tissue and mouse models of amyloid disease.

Nature of the Proinflammatory Amyloidogenic Peptide Species Knowledge of specific aggregate species is important for the development of therapies aiming to target amyloid/immune cell interactions without stabilizing toxic or proinflammatory intermediates. Few studies have compared PRR activation in response to multiple aggregate species, employing only fibril-forming peptides (often a heterogeneous mixture changing over time) and implicating a requirement for amyloid formation, but not necessarily the fibril itself. Like CD36, some receptors may alter the conformation of the interacting peptide, providing a template for aggregation of misfolded monomers or small oligomers [18]. For example, fungal HetS, a cell deathinducing protein containing a pore-forming domain, is activated by NLR templating, leading to propagation of the prion fold [78]. The NWD2 NLR displays an N terminal region homologous to the HetS amyloid motif, as determined from conformational analysis of fibrils by solid-state NMR. On ligand binding to NWD2, both in vitro and in Saccharomyces cerevisiae and Podospora anserina, these N terminal extensions adopt an amyloid fold and convert HetS to a pore-forming 770

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Prefibrillar oligomer

Monomer

Aggregation

TLR2, TLR4, TLR6, CD14, RAGE, TREM2

Fluid phase endocytosis ROS MAPK

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? Scavenger receptor e.g., CD36, SRA, SRB, FPR2, FPRL1

Intracellular aggregation (low pH, high intracompartmental concentration, PRR templating)

ASC NLRP3

Phagocytosis

Lysosomal instability Cathepsin B release

Membrane damage

K+ efflux Oxidized mtDNA Ca+2 influx ROS MAPKs

DAMPs ATP P2X7R

IL-1β Regulation of inflammatory response

• Acute secretion of proinflammatory cytokines • Enhanced phagocytosis Acute cell stress • Killing of pathogens

Microbial infection

• • • • • • •

• Chronic secretion of proinflammatory cytokines • Impaired phagocytosis • Oxidative tissue damage

IL-1R

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Modifying factors Amount of aggregate Duration of exposure Rate of production versus clearance Other proinflammatory stimuli Type and metabolic state of the cell Genetic/epigenetic susceptibility Age of host Trends in Immunology

Figure 2. Possible Outcomes of Pattern Recognition Receptor Activation by Human Amyloidogenic Peptides. Peptide aggregation can occur intra- or extracellularly, leading to activation of cell-surface scavenger receptors, Toll-like receptors (TLRs), and the cytosolic NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. While both fibrillar and oligomeric peptide aggregates contribute to pattern recognition receptor (PRR) activation, discrepancies in the species reported to be responsible for TLR and NLRP3 activation need to be addressed by more careful studies. Scavenger receptors may facilitate aggregate uptake, promote TLR dimerization, and seed fibril formation within phagosomes. Interleukin-1 receptor (IL-1R) activates the same signaling pathways downstream of TLRs. Direct damage to parenchymal cells (e.g., via membrane disruption, oxidative damage, or altered calcium signaling) may lead to release of endogenous danger-associated molecular patterns (DAMPs) such as membrane lipids or ATP, or alteration in cellular homeostasis leading to activation of PRRs. The proinflammatory response to amyloidogenic peptides is determined by epigenetic modifications, altered gene transcription, changes in protein activity, and changes in the metabolic state of the cell. The net outcome depends on the nature and duration of the stimulus, the tissue environment, the cell type, and other modifying factors such as the age of the host and the presence of other proinflammatory stimuli. In the case of infection with an amyloid-producing microbe, effective phagocytosis and induction of antimicrobial mediators may lead to resolution of infection. However, chronic exposure to endogenous aggregates that are continually produced may impair phagocytosis and promote chronic proinflammatory cytokine secretion leading to parenchymal dysfunction. Abbreviations: ASC, Apoptosis-associated specklike protein containing a caspase recruitment domain; FPR, formyl peptide receptor; FPRL-, formyl-peptide-receptor-like-; MAPK, mitogen-activated protein kinase; mtDNA, mitochondrial DNA; RAGE, receptor for advanced glycation end-products; ROS, reactive oxygen species; SR, scavenger receptor; TREM2, triggering receptor expressed on myeloid cells 2.

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state [78]. Whether mammalian PRRs other than CD36 can promote oligomerization of microbial or mammalian amyloidogenic peptides remains to be determined. Recent studies comparing multiple aggregate species suggest that early soluble aggregates and late fibrillar species can modulate distinct signaling pathways (Figure 2). Specifically, IAPP [41] and α-synuclein [79] aggregates formed in vitro early in the process of amyloid formation (i.e., prior to fibril formation based on thioflavin T staining and TEM) activate TLR2 in HEK293 cells and require TLR2 for release of cytokines such as IL-1β in mouse BMDMs. In contrast, fibrils but not amorphous aggregates or monomers of lysozyme (as characterized by Fourier transform infrared spectroscopy, TEM, and thioflavin T binding) were reported to induce TLR2-dependent NF-κB activation in HEK 293 cells [38]. The authors of this study reported that oligomeric contamination was ruled out given a lack of binding of an anti-oligomer A11 antibody to peptide preparations, suggesting that intermediate aggregates (and not fibrils) of some peptides can trigger TLR signaling, while fibrils are required for others. However, techniques for characterization have not been consistent among studies; for example, the A11 antibody was not used to characterize the peptide preparations in experiments with IAPP and α-synuclein [41,79]. Consistent methods of preparation and characterization are thus required to determine whether different amyloidogenic peptides give rise to unique species with distinct PRR-activating activities. The ability of CD14, TLR2, and TLR4 to bind multiple ligands has been attributed to broad recognition of exposed hydrophobic domains [80]. Interactions with hydrophobic side chains that stabilize the cross-β-sheet structure may contribute to TLR binding by prefibrillar oligomeric species, which have similar exposed epitopes [2]. Both soluble and fibrillar preparations of Aβ [18] and IAPP [41] (based on timing of treatment following dissolution relative to a parallel thioflavin T binding assay and TEM) appear to trigger NLRP3 in vitro (peptide-induced caspase-1 activation and IL-1β secretion) by WT but not Nlrp3–/– mouse BMDMs. These findings suggest that fibrils are sufficient for inflammasome activation, although smaller contaminating aggregates or the process of fibrillization itself may be important contributors. Similarly, fibrils but not monomers or amorphous aggregates (again characterized by TEM, Fourier transform infrared spectroscopy, and thioflavin T binding) have been implicated in NLRP3 activation leading to induction of IL-1β by α-synuclein [81] and lysozyme [38] in human THP-1 cells [81] and PrPSc in primary mouse microglia [38]. Dramatic differences in geometry, residue exposure, and inherent stability between oligomers and fibrils make it unlikely that common receptors bind directly to entirely pure populations of both species. Taken together, these studies suggest that for all amyloidogenic peptides studied, NLRP3 activation in myeloid cells in vitro requires aggregates formed at the later stages of fibrillization. Recent progress in structural characterization of amyloid fibril preparations may shed light on the considerable variation in outcomes from in vitro assays and in vivo models, beyond technical aspects of peptide preparation. Over the past year, electron cryotomography (cryo-ET) structures have allowed for resolution at atomic detail of PrP [82], Aβ40 [83], and tau [84] and shown βstrand stabilization by multiple mechanisms, confirming that the same primary sequence can assemble into different structures even under identical conditions. These studies suggest that, in addition to variation in structure over time as they aggregate, amyloid peptides may generally exist as heterogeneous mixtures. Like a microbe equipped with multiple virulence factors, they may therefore trigger multiple distinct host signaling pathways with different kinetics over the course of a disease. Further improvements in cryo-ET may allow for the study of fibrils, plaques, and inclusions in situ with increased resolution, helping to determine how fibril polymorphism can influence disease phenotype and inflammation. Thus, studies are needed to determine the nature of the proinflammatory species and modulation of its activity by amyloid-inhibiting or stabilizing 772

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compounds. Generation of engineered variants that do not proceed past a particular aggregation stage or are otherwise stabilized in solution may prove useful in this regard [85]. An example of the utility of experimental and theoretical modeling of oligomer-receptor interactions is provided by a recent study of Aβ binding to human LilrB2, a neuronal receptor implicated in Aβ-induced synaptic loss based on observations of enhanced downstream signaling in human AD brains as well as rescue of memory defects in APP/PS1 mice upon genetic depletion of the murine homolog, PirB [86]. Binding experiments led to the generation of a model showing docking of a small molecule mimicking phenylalanine side chains of the Aβ binding core to the LilrB2 binding pocket, and the subsequent computational identification of small molecule inhibitors from a compound library [86]. A similar approach may be feasible in the development of inhibitors of amyloidogenic peptide interactions with innate immune cells.

Amyloid as a Product of the Innate Immune Response Some amyloid diseases such as AD and T2D share common inflammation-related risk factors. Recent subgroup analysis from the Atherosclerosis Risk in Communities (NCT00005131) Positron Emission Tomography Study suggests that midlife concentrations of over 3 mg/l of Creactive protein (CRP) (a pentameric acute phase reactant synthesized in the liver) are associated with elevated brain amyloid among white males, even in the absence of dementia [87]. Furthermore, elevated peripheral cytokines, including macrophage inflammatory protein-3, IL-17A, and IL-2, have recently been described in dementia with Lewy bodies [88]. This information is relevant as proinflammatory stimuli can regulate the formation of amyloids in at least three important ways: first, by changes in peptide processing [89]; second, by increased expression of the amyloidogenic peptide leading to increased amyloid formation [90]; and third, by modulation of microglial peptide clearance [15]. Thus, amyloidogenic peptides may accumulate gradually over the course of disease as a result of altered peptide homeostasis associated with a local or systemic proinflammatory state. Innate immune cells not only respond to but also produce amyloid aggregates capable of further modifying the inflammatory response. For example, neutrophils, eosinophils, mast cells, and monocytes/macrophages produce a fibrillar DNA network termed neutrophil extracellular traps (NETs); these networks have been suggested to have an amyloidogenic backbone that can entrap bacteria, fungi, protozoa, and viruses [91]. NETs have been shown to accumulate near amyloid plaques in 5xFAD transgenic mice (AD) and in postmortem human brain parenchyma [92]. Taken together with the observation that curli fibrils bind bacterial and eukaryotic DNA during biofilm formation in vitro, this suggests a potential DNA–amyloid interaction that may further modulate innate immune pathways driven by amyloid aggregation [93]. Amyloid formation is likely an evolutionarily conserved mechanism of immune response modulation; circulating immunocytes of the colonial ascidian Botryllus schlosseri, a type of invertebrate chordate, can also produce amyloid proteins involved in host defense against genetically incompatible colonies, as well as against microbial pathogens upon release of extracellular traps [94]. Analogous to neutrophil NETs, these extracellular traps contain an amyloid fibril backbone formed by aggregation of the protein BsAPP, an ortholog of APP [94]. Other multifunctional proteins involved in control and propagation of the acute phase and inflammatory responses can also undergo both intraand extracellular aggregation that further modulate monocyte/macrophage phenotypes. For example, S100A9 is a proinflammatory DAMP produced by neurons and microglia that forms both intra- and extracellular aggregates associated with neuronal loss following TBI [95]. Amyloid plaques comprised of S100A9, as well as Aβ (assessed by atomic force microscopy and costaining with amyloid-binding heptamer-formyl thiophene acetic acid) were detected by

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immunohistochemistry in the hippocampus of patients with AD or mild cognitive impairment and a history of TBI [95]. Some pathogenic amyloids have also been described as AMPs; these include lysozyme, a prototypical AMP capable of forming pathogenic amyloid, as well as Aβ [96,97] and α-synuclein [98]. In the case of Aβ, infection of the brains of 5xFAD mice with Salmonella sp. accelerated formation of cortical amyloid deposits, which colocalized with bacteria by immunostaining [96]. The authors of this study proposed a model in which protofibrils inhibited pathogen adhesion to the host while propagating fibrils mediated agglutination and entrapment of unattached microbes. While the fibrillar structure may be required for antimicrobial activity in vitro, nonamyloidogenic Aβ peptides may also modulate phagocyte activity. This was suggested by a study of transient Candida albicans cerebritis in C57BL/6 mice demonstrating focal gliosis with fungal cells in contact with Aβ peptides in the granuloma center and APP in the periphery [97]. Consistent with previous in vitro studies suggesting a fungistatic role for soluble Aβ peptides, the authors demonstrated impaired C. albicans clearance in App–/– mice relative to WT and increased phagocytic activity of BV2 microglia in response to soluble Aβ peptides relative to vehicle control [97]. Of note, both Aβ [99] and α-synuclein [9] aggregation can be potentiated by bacterial LPS in vitro. In the case of α-synuclein fibrils, the presence of LPS during aggregation alters fibril morphology (as evidenced from circular dichroism and electron microscopy); following injection of fibrils into mouse striatum, each fibril polymorph caused a distinct pattern of phosphorylated α-synuclein pathology. This difference in pathology due to peptide aggregates of the same primary sequence formed in the presence or absence of LPS, suggests that microbial components might modulate the diverse clinical phenotypes observed among humans with synucleinopathies [9]. Perhaps one of the most potent pathogen-derived modulators of amyloid formation is bacterial amyloid itself. Indeed, peptides such as CsgA derived from both commensal and pathogenic microbes may impact human amyloid-associated diseases by cross-seeding CNS amyloid [100] (Box 3), underscoring the potential for variable environmental factors such as diet or infection in influencing disease courses with considerable heterogeneity in clinical presentations. Finally, multiple intracellular signaling pathways in mammals can involve protein aggregation to form filamentous assemblies, including the Myddosome; mitochondrial antiviral signaling filaments; the RIP1/RIP3 necrosome, recently shown to be evolutionarily related to fungal Het/ HetS proteins [101]; and the ASC-dependent inflammasome, which functions homologously to the HetS pathway in fungi [101]. In fact, ASC specks released by activated microglia can bind Aβ in vitro and promote the spreading of aggregated peptide in vivo, as demonstrated by increased amyloid formation following intrahippocampal injection of ASC specks in APP/PS1 mice compared with vehicle control [102]. Moreover, brain homogenates from the transgenic mice increased intracerebral amyloid load when injected into other mice of the same genotype; however, lysates derived from APP/PS1, Asc–/– mice showed reduced capacity to cause Aβ seeding and spreading. Thus, inflammasome activation itself may cause cross-seeding of oligomers and aggregates. This activity was blocked by an anti-ASC antibody in the same study [102] suggesting a potential therapeutic target downstream of NLRP3 activation that might limit amyloid pathology and which should be investigated further.

Therapeutic Implications of Amyloid-Immune Cell Interactions An understanding of amyloid structure and proinflammatory properties is critical to the design of therapeutics aimed at inhibiting amyloid formation, deposition, and toxicity, and to the development of antimicrobial strategies for targeting biofilms. It is important to consider that a toxic peptide species may be distinct from other immune-activating or modulating species, particularly in 774

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Box 3. Amyloidogenic Peptides in the Mammalian Gut An estimated 40% of bacterial species produce amyloid, placing microbial amyloidogenic peptides at the interface between gut microbiota and systemic immunity. Commensal amyloids may have the capacity to cross-seed pathogenic amyloids, as suggested in rats with α-synuclein pathology treated orally with curli fibrils [121]. Seeding may involve propagation by retrograde transport via the vagus nerve or depend on amyloidogenic peptides entering the circulation [121]. Having crossed the epithelial barrier, microbiota-derived metabolites can affect myeloid cell phenotypes at distant sites, including the bone marrow. For example, myelopoiesis is decreased in germ-free compared with colonized mice and maintained by the transfer of sterile serum from colonized mice into germ-free WT but not Myd88–/–Ticam1–/– mice [122]. Moreover, stool samples from individuals with cognitive impairment and brain amyloid [assessed via positron emission tomography (PET)] contain increased abundance of Escherichia/Shigella species and reduced abundance of Eubacterium rectale compared to healthy controls, and to participants with cognitive impairment but lacking brain amyloidosis [123]. In rodent models, transgenic APP/PS1 mice have reduced cerebral Aβ amyloid under germ-free compared with conventional conditions; colonization of these mice with microbiota from conventionally raised transgenic mice increased cerebral Aβ compared with microbiota from WT mice [11]. The 16S RNA sequencing of fecal samples in this study revealed significantly different microbiome compositions between WT and transgenic mice [11], suggesting reciprocal regulation of host/ microbial amyloids. Microbial amyloids may also regulate gut epithelial immunity. Salmonella enterica serovar Typhimurium expressing mutant csgBA that does not form curli fibrils causes increased disruption of the gut epithelium in WT mice following oral administration, relative to the curli-producing serovar, an effect not observed in Tlr2–/– mice [124]. Infection with curli-producing Salmonella sp. enhances expression of IL-8, IL-17, and IL-22 in the cecal mucosa and reduces epithelial permeability compared with the mutant form [124,125]. Oral administration of purified curli can also confer protection against chemically induced colitis in WT but not Tlr2–/– mice [125]. It is unclear whether curli plays a protective or pathological role in chronic colitis or inflammatory bowel disease. Microbiota exhibit both top-down (host-driven) and bottom-up (microbial community-driven) evolution and their population is sensitive to environmental factors such as diet and antimicrobials. It is tempting to speculate that species-specific differences among pathogens and microbiota underlie differences in peptide amyloidogenicity. Organisms with a higher gut amyloid load, or increased abundance of amyloid-producing pathogens, might be in danger of increased pathogenic amyloid formation due to cross-seeding. A metaproteomic analysis of amyloidogenic proteins in mouth and gut will be an important first step in better understanding the normal commensal amyloid load and its potential role in health and disease.

the development of read-outs for the screening of potential small molecule inhibitors of amyloidogenic peptide activity. The central roles of both NF-κB and NLRP3 activation downstream of amyloid recognition point to these, and related pathways, as potential therapeutic targets to treating aging-associated amyloid disease. Indeed, the demonstration of therapeutic effects of small molecule inhibitors or antibodies targeting NLRP3, ASC, and IL-1β across multiple mouse models of amyloid disease suggest that that immunomodulation may be a viable strategy, even in the presence of extensive amyloid formation. Previous reviews have described inflammasome- and TLR-targeting therapies [103]. A few relatively nonspecific treatments, such as oral minocycline administration, can alter Aβmicroglia interactions in mouse models of neurodegenerative disease relative to controls, perhaps via mechanisms that modulate TLR2 and NLRP3 expression, together with Aβ phagocytosis [104]; however, whether similar mechanisms are at play in human disease is unclear. Given the diverse functional roles of each receptor, and the potential benefits of amyloidinduced proinflammatory responses, specific blockade is unlikely to lead to predictable in vivo results, as has been the case in animal models. Thus, promoting peptide clearance while limiting chronic inflammation is likely to require the modulation, rather than the elimination, of peripheral and tissue-resident innate immune cell functions. A major challenge in the clinical application of anti-amyloid therapies to date has been the advanced stage of pathology once clinical symptoms present. For example, a Phase III trial of the anti-Aβ antibody Aducanumab in mild cognitive impairment and AD (NCT02477800 and NCT02484547) was terminated in March 2019 following futility analysis suggesting the studies

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would not reach their primary endpoints of slowing cognitive decline. While the identification of disease biomarkers allowing early detection of peptide aggregates might aid in the delivery of such agents to the appropriate patients, screening a large numbers of at-risk individuals is likely to be impractical. Targeting of secondary pathological processes, including the immune

Key Figure

Modulation of Human Amyloid Homeostasis by Innate Immune Recognition Templating of amyloidogenesis Uptake

Modulation of innate immune response e.g., cytokine secretion, antigen presentation Cross-seeding by aggregates released by myeloid cells (e.g., ASC)

Endolysosomal degradation

PRR-mediated aggregate recognition

Physical barrier e.g., NETs

Altered peptide synthesis, processing, or degradation

Microbial amyloids e.g., curli fibrils

Host amyloids e.g., Aβ, IAPP Parenchymal (dys)function

Antimicrobial defense (?)

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Amyloid disease

Pathogen

Infection Cross-seeding (extra/intracellular) Trends in Immunology

Figure 3. Both microbial and host amyloids are recognized by pattern recognition receptors (PRRs) expressed by macrophages, neutrophils, and other innate immune cells. These mammalian cells respond to peptide aggregates to modify the course of amyloid formation and regulate the local and systemic inflammatory milieu. Myeloid cells also use their own amyloid-forming peptides to create structural barriers that wall off amyloid plaques and as antimicrobial peptides that induce cell death. Outcomes of peptide uptake include degradation or, in some cases, facilitation of further amyloid formation (e.g., as described for CD36). The development and function of parenchymal cells is regulated by the complement of host and circulating immune cells. Proinflammatory stimuli, including peptide aggregates themselves, lead to increased production of aggregation-prone peptides, typically under tight regulation by normal protein homeostasis. A change in the cellular environment or cell damage in the setting of an inflammatory response may lead to release of amyloids that act as danger-associated molecular patterns, promote cell death, and upregulate mechanisms for clearance. When chronic inflammatory stimuli are present, as in aging-associated amyloidosis, excessive accumulation of amyloidogenic peptides and polarization of tissue macrophages towards a proinflammatory phenotype may tip the balance in favor of amyloid deposition and proinflammatory macrophage phenotypes. The possibility of cross-seeding between host and microbial amyloids opens new avenues for investigation of the role of microbiota in amyloid disease. Abbreviations: Aβ, Amyloid β; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; IAPP, islet amyloid polypeptide; NET, neutrophil extracellular trap; PRR, pattern recognition receptor.

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Clinician’s Corner Many common, age-related diseases, including T2D and neurocognitive disorders, are characterized by extra- and intracellular accumulation of peptides that aggregate to form amyloid fibrils, populating various species of oligomeric aggregates along this pathway. Rodent models and human tissue sections, as well as recent PET ligand studies in humans, suggest that monocytederived cells can be found in association with peptide aggregates. The outcomes of direct interactions between amyloidogenic peptides and innate immune cells may include homeostatic clearance and amplification of the proinflammatory response leading to tissue damage, dependent on the peptide’s aggregation state, the specific cell type, and the microenvironment. The lack of predictable outcomes in animal models of amyloid disease with PRR deficiency underscores the dual protective and detrimental roles of PRR/peptide interactions and is consistent with the variable results obtained with anti-amyloid vaccines to date. Recognition of endogenous amyloidogenic peptides by receptors of the innate immune system may be a byproduct of an evolutionarily conserved mechanism for detection of bacterial and fungal pathogens, which produce functional amyloids involved in their own host defense. Interactions of the host with microbial amyloid derived from both gut microbiota and invasive pathogens, may in the future represent an opportunity for therapeutic modulation of systemic infectious and inflammatory diseases, although caution is needed given the dual physiologic and pathologic nature of these interactions. Therapeutic agents targeting the phenotype of resident and circulating monocyte-derived cells may in turn modulate the clearance of and cytokine response to extracellular protein aggregates. Similarly, amyloid inhibitors may allow for modulation of the local and systemic inflammatory response, although further work is needed to allow for informed development of agents that avoid stabilizing proinflammatory species.

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response, may represent a more effective strategy for modulating the effects of amyloidogenic peptides later in the disease process. Other therapeutic approaches might also include the modulation of amyloid production by gut microbiota (Box 3) or the targeting/blockade of independent proinflammatory stimuli promoting amyloid accumulation. These and other therapeutic implications are summarized in Clinician’s Corner.

Concluding Remarks Given increasing evidence of crosstalk between TLR and NLRP3 signaling pathways, amyloidogenic peptides appear to act as particularly potent proinflammatory stimuli because of their capacity to induce both priming and activation of inflammasomes. Multiple amyloidogenic peptides of both bacterial and mammalian origin activate the same set of PRRs, with the strongest evidence stemming from rodent models of TLR2 and NLRP3. Furthermore, the recent characterization of amyloid aggregates from the CNS of patients with AD underscores the challenge of characterizing multiple proinflammatory peptide species and their effects, that may change over the course of a neurodegenerative disease. Functional amyloids also play a role in antimicrobial defense, amplification of proinflammatory signals, innate immune signaling, and host/microbiota interactions in humans. Recent evidence opens intriguing new avenues for research into the role of pathogenic and commensal microbes in the regulation of endogenous amyloid formation. Thus, improved techniques for the detailed characterization of aggregate structures, as well as the monitoring of innate immune responses in vivo, will likely give rise to strategies to modulate amyloid formation and activity (see Outstanding Questions). We suggest that the boundary between functional and pathogenic amyloids may be less clear than previously thought, and that, akin to innate defense regulator peptides, amyloidogenic peptides typically associated with human disease can also play critical roles in innate and adaptive immune modulation (Figure 3, Key Figure). Understanding this balance is critical to the development of putative anti-amyloid and immune-modulating therapies for the treatment of amyloid-associated diseases. Acknowledgments Related work in the laboratory of C.B.V. is supported by Canadian Institutes of Health Research grants MOP-123338, MOP130518, and PJT-156449. C.B.V. is supported by an investigatorship from the BC Children’s Hospital Research Institute and the Irving K. Barber Chair in Diabetes Research.

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Outstanding Questions Are the mechanisms by which microbial amyloid fibrils and host amyloidogenic peptides induce TLR activation fundamentally different? How can activation by the mature fibrillar form of lysozyme and curli be reconciled with the apparent requirement for the prefibrillar form of mammalian peptides? Are there common structural motifs shared by distinct aggregate species, or is TLR activation dependent on secondary signals? What is the mechanism by which amyloid fibrils and/or prefibrillar aggregates induce activation of NLRP3 and secretion of IL-1β? Are there distinct mechanisms of NLRP3 activation by different peptide forms? What is the composition of the gut ‘amyloidome’, and are there homeostatic interactions between microbial amyloids and host PRRs? Does variation in the composition of gut microbiota lead to amyloid-dependent changes in mucosal and systemic markers of immune activation? Is there a role for amyloidogenic peptides derived from pathogenic and commensal microbes in the modulation of amyloid formation in neurodegenerative and metabolic diseases, including cross-seeding? Could microbial components present in amyloid deposits contribute to local monocyte/ macrophage activation? Is there a role for clinical modulation of amyloid/macrophage interactions or IL-1 signaling (e.g., via NLRP3 inhibition) in the putative treatment of amyloid diseases in humans? Preclinical models suggest variable results with blockade of TLR signaling, but so far, have been relatively consistent with respect to improved pathology and functional measures with anti-IL-1 therapies.

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