age - related macular degeneration ( amd ) is a neurodegenerative disease characterized by the deterioration of photoreceptors in the macula , a specialized region of the retina responsible for fine visual acuity that is required for tasks such as reading , facial recognition , and driving . according to the world health organization , amd currently ranks as the third global leading cause of blindness , second only to cataract and glaucoma . however , among the elderly , amd is the most common cause of irreversible vision loss in developed countries . the economic costs for treatment and care of individuals who suffer vision loss from amd are projected to be more than us$ 300 billion annually , a heavy toll that will significantly impact global social and public health systems and one that prompts an urgent need to decipher its underlying mechanisms . being a complex disease , the pathogenesis and progression of amd are influenced by a variety of risk factors . among them , advanced chronologic aging is thought to be the strongest [ 46 ] . the prevalence of amd steadily increases with age , affecting 2% of the population at age 40 and 25% by age 80 . besides aging , other risk factors such as cigarette smoking and diet also contribute to the development of the disease [ 811 ] . clinically , early stages of amd are defined by the presence of drusen , the extracellular deposits located between the retinal pigment epithelium ( rpe ) and bruch 's membrane ( bm ) ( figure 1 ) . despite the fact that early amd is usually not associated with appreciable vision loss , the number and the size of drusen deposits serve as signs of disease progression . when the disease progresses into the late stage , it takes one of two forms : geographic atrophy ( ga ) , featured by confluent regions of rpe and photoreceptor degeneration , and choroidal neovascularization ( cnv ) , characterized by the abnormal growth of leaky choroidal vessels invading retina . initially considered a supporting cell in the outer retina , rpe are active in a wide range of biological processes that maintain local homeostasis . these processes include recycling components of the visual cycle , secreting trophic factors , controlling cross - epithelium transport , and maintaining the outer blood - retinal barrier [ 13 , 14 ] . central to amd pathogenesis , the rpe undergoes significant changes in structure and function that predispose individuals to disease processes associated with amd . suggestive of an associated , and perhaps causal , role in rpe dysfunction is the finding that rpe cells overlying drusen appear swollen and vacuolated . it is further proposed that the spontaneous release of drusen components during drusen regression in amd development may result in rpe loss in ga . recent advances have highlighted the essential role of immune processes in the development , progression , and treatment of amd . both the innate and adaptive immune systems have been shown to contribute to amd pathogenesis ( for reviews , see [ 28 , 29 ] ) . the innate immune system is an evolutionarily conserved system that constitutes the first line of defense against pathogens . inflammasome activation is a key component of innate immunity , which when overactive has been linked with many human immune diseases [ 3033 ] . the canonical inflammasome complexes are assembled around protein members of the nod - like receptor ( nlrs ) or hin-200 protein families , converting the procaspase-1 zymogen into a catalytically active enzyme . the inflammasome family is further categorized based on the presence of an apoptosis - associated speck - like protein containing a caspase recruitment domain ( asc ) . the canonical inflammasome can be further categorized into asc - dependent ( nlrp3 and aim2 ) and asc - independent ( nlrp1 and nlrc4 ) subtypes . the noncanonical inflammasome complex with an , as yet , unknown structural composition is proposed to promote the activation of caspase-11 . despite the gap in knowledge of the structure of the noncanonical inflammasome , there is a wealth of evidence to firmly establish the mode of action for several canonical inflammasomes in the immune signaling pathway , especially the most widely studied nlrp3 inflammasome . the nlrp3 inflammasome senses and responds to a diversity of pathogen- or danger - associated molecular patterns ( pamps or damps ) , including bacterial / viral / fungal pathogens , pore - forming toxins , uric acid crystals , particulate aggregates , and adenosine triphosphate ( atp ) . to be activated , the nlrp3 inflammasome requires the presence of two signals , a  priming signal  and an  activation signal ,  both of which are vital to control the degree of immune response driven by the products of inflammasome activation . in most cases , the  priming signal  channels through the nuclear factor kappa b ( nf-b ) pathway , upregulating the transcription of nlrp3 and prointerleukin-1 ( pro - il-1 ) . it is a fact that , in both immune and rpe cells , pro - il-1 is not constitutively expressed and the endogenous level of nlrp3 appears to be inadequate for inflammasome activation , thus making the priming process critical [ 17 , 38 ] . in contrast , other inflammasome - related proteins , asc , procaspase-1 , and pro - il-18 , are constitutively expressed in rpe cells and therefore priming may , or may not , further increase their protein levels [ 17 , 20 , 39 , 40 ] . in addition to the classic , transcription - dependent priming , it is now known that nlrp3 inflammasome can be  primed common priming signals for immune cells of the body and human rpe cells are lipopolysaccharide ( lps ) , tumor necrosis factor- ( tnf- ) , nitric oxide , and il-1 [ 17 , 42 ] . in the presence of foreign or endogenous nlrp3 senses one or more of the following intracellular changes : k efflux , release of lysosomal resident cathepsin b , overproduction of reactive oxygen species ( ros ) , nlrp3 translocation to mitochondria , cell volume change , and ca disequilibrium . once the nlrp3 is activated , it recruits asc and mediates the proximity - induced procaspase-1 autoactivation . the assembled nlrp3 inflammasome then turns itself into a cytokine processing platform by cleaving pro - il-1/pro - il-18 into mature peptides and releasing them into extracellular space for downstream effects ( figure 2 ) . of note , the generalized nlrp3 inflammasome activation process summarized above is much simpler than what actually happens in a given cell . more recently , the role of the nlrp3 inflammasome in amd pathogenesis has been extensively investigated using amd related stimuli . being a hallmark of amd progression , drusen has a rich proteinaceous composition , including complement regulators , amyloid - beta ( a ) , and oxidation by - products [ 15 , 4754 ] , which makes drusen components ideal triggers for potential interactions with the nlrp3 inflammasome . as part of the innate immunity , the complement system is one of the first that responds to tissue damage during aging and is activated by cell death . in this regard , stressed , damaged , or dying rpe could trigger local complement activation , which is supported by the fact that activated complement components as well as complement regulators are present in drusen . the degree of complement activation is precisely regulated in healthy retina and is thus beneficial for tissue homeostasis and longevity . however , when the complement system is in overdrive due to either genetic polymorphisms   or chronic , sustained pathological stimulation , it generates undesirable amounts of activated complement factors , facilitates the formation of terminal membrane attack complex ( mac ) , and thus advances amd pathology . to bolster this notion , doyle and colleagues have showed that drusen extracts isolated from amd donor eye tissues are able to activate the nlrp3 inflammasome in lps - primed macrophages . they further revealed the role of complement factor 1q ( c1q ) as an nlrp3 inflammasome  activation signal  by showing caspase-1 cleavage and elevated il-1 secretion after c1q stimulation on lps - primed mouse bone marrow derived macrophages and thp1 human monocytic cells . moreover , in addition to c1q stimulation , other studies have suggested complement factor 3a ( c3a ) and mac may also mediate the activation of the inflammasome , further linking many components of the complement pathway with il-1 and il-18 production [ 24 , 25 , 58 ] by unique underlying mechanisms . for instance , sublytic mac is known to activate nlrp3 inflammasome through ca influx and/or k efflux , whereas c3a activation of the nlrp3 inflammasome is initiated by the release of atp into the extracellular space . these studies were primarily conducted on monocytes , dendritic cells , or lung epithelial cells . although the aforementioned mechanisms are yet to be confirmed in amd models , they do lend to the biological plausibility that similar processes can happen in ocular tissue . we recently tested the systemic administration of a small molecular inhibitor for mac , aurintricarboxylic acid complex ( atac ) , on nlrp3 inflammasome activation in rat rpe / choroid tissues at different ages . we found that atac suppressed the age - dependent increase in mac formation and caspase-1 cleavage ( a prominent indicator for nlrp3 inflammasome activation ) in rpe / choroid tissue homogenates , which implies potential benefits by targeting mac formation in amd pathogenesis ( figure 2 ) . amyloid - beta ( a ) is a drusen component found in amd eyes [ 51 , 59 , 60 ] ; more recently there is growing interest in a for its capacity to stimulate inflammasome activation and potentially contribute to amd pathogenesis . as a pathological peptide best known for its neurotoxicity in alzheimer 's disease ( ad ) , a is generated through the amyloidogenic pathway by cleaving the amyloid precursor protein ( app ) into the intramembrane a domain of 3643 amino acids in length . the accumulation of a in tissue results from its disturbed balance between production and clearance , the latter of which is largely controlled by the membrane - bound degradation enzyme , neprilysin [ 62 , 63 ] . a 's intrinsic cytotoxicity lies in its aggregated forms as soluble oligomers or insoluble fibrils . originally thought as a primary toxic structure , a fibrillar plaques are now considered less harmful to brain neurons than the small spherical oligomers that damage cell membranes and cause cell death [ 61 , 6468 ] . a 's ocular presence has been reported in studies of postmortem human donor eyes [ 54 , 60 , 69 ] showing specific deposition within drusen from amd eyes . the age - dependent deposition of a in the outer retina [ 59 , 70 ] can be , at least partially , attributed to local rpe synthesis . in this regard , transgenic animals lacking neprilysin exhibited a accumulation in both rpe and sub - rpe deposits , concomitant with significant rpe atrophy . when incubated with a oligomers , human primary rpe cells demonstrated a prominent decrease in cell viability . the exact mechanism by which a contributes to rpe atrophy is still poorly understood but may involve inflammasome - related caspase-1 dependent cell death . in addition to its cytotoxicity , a is also a major proinflammatory factor that has been extensively studied in the context of amd . the presence of a in drusen is found to overlap with complement activation sites [ 47 , 51 ] . in an rpe cell culture model , kurji et al . discovered that inflammation - associated genes and immune response pathways were the predominant responses of rpe to oligomeric a stimulation . proinflammatory responses to a stimulation were also verified further in an in vivo model using intravitreal injections of a in rodents , in which the rpe demonstrated upregulation of nlrp3 , il-1 , and il-18 . in this intravitreal a injection model , inflammasome activation was demonstrated by caspase-1 cleavage , il-1 , and il-18 immunoreactivity in the rpe and surrounding tissue including the vitreous , which was further suppressed by an nf-b inhibitor , vinpocetine . the secreted inflammasome effector cytokines , il-1 and il-18 , are known to exert potent cytotoxic effects on rpe cells , which might provide a possible explanation for a induced rpe cell death in vitro . these findings in rpe are consistent with a multitude of studies that implicate a and nlrp3 inflammasome activation in glial cells and central nervous system disease , specifically ad . using fibrillar a , halle et al . reported the activation of nlrp3 inflammasome by lysosomal destabilization which increased release of il-1 in murine microglia   ( figure 2 ) . to further substantiate the link between inflammasome activation and the deleterious effect of a in the brain , app / ps1 mice with nlrp3 deficiency demonstrated improved a clearance by microglia and preserved memory and behavior patterns . a-induced nlrp3 inflammasome activation in glial cells may rely on activation of cathepsin family of proteases and the degradation of nlrp10 . having the most abundant polyunsaturated fatty acids in the eye , photoreceptors are protected by rpe cells from excessive high - energy light exposure . as part of the visual cycle , photoreceptors shed their oxidized tips of outer segments , which are then phagocytosed by rpe cells for the recycling of 11-cis - retinal . with age , the ability of rpe cells to recycle the  waste  from photoreceptors decreases significantly , leading to the accumulation of lipid peroxidation by - products , lipofuscin , in the rpe . it has been previously suggested that lipofuscin accumulation in rpe causes lysosome damage and directly triggers the formation of active nlrp3 inflammasome [ 17 , 21 , 75 ] . other lipid peroxidation end products , such as 4-hydroxynonenal ( hne ) and carboxyethylpyrrole ( cep ) , have also been shown to contribute to nlrp3 inflammasome activation [ 18 , 22 ] . by incubating arpe-19 cells , a cell line that possesses key features of human rpe cells , with hne alone or in combination with lps , kauppinen et al . demonstrated a substantial increase of secreted il-18 and il-1 , products of nlrp3 inflammasome activation . doyle and colleagues discovered a priming effect for cep , which promoted il-1 secretion when combined with an inflammasome  activation signal ,  such as atp and c1q   ( figure 2 ) . furthermore , the recently established cep - immunized murine model of amd may serve as a useful platform to substantiate the role of cep in nlrp3 inflammasome activation . a disease with complex etiology , amd , is also heavily influenced by genetic modifications . in ga patients , kaneko et al . reported the repetitive element - derived alu rna transcripts as an inducer for rpe degeneration . these retrotransposon elements are short interspersed nuclear elements in eukaryotic genome , containing internal promoter sequences for rna polymerase iii . thus , the alu mobile repeats are noncanonical targets of dicer1 , an evolutionarily conserved member of the rnase iii nuclease family , essential for the control of microrna biogenesis . junk dna  entities in the host genome , the alu elements are now recognized for their complex regulatory functions , such as transcriptional repression   and modulation of alternative splicing , and involvement in human genetic diseases by inducing insertion mutations , dna breaks , genome instability , and exonization [ 82 , 83 ] . it has been shown that the loss of dicer1 expression , possibly due to oxidative stress in the rpe , is responsible for the abnormal alu repeats accumulation in ga patients . these alu transcripts function as both priming ( toll - like receptors independent , tlrs ) and activating ( p2x7 receptor dependent ) signals to stimulate nlrp3 inflammasome activation , leading to the release of il-18 and subsequent caspase-8/caspase-3 dependent apoptotic rpe death via myd88 and/or fas ligand mediated signaling [ 20 , 40 , 84 ] . these findings are novel as they provide us with a new perspective into the apoptotic rpe death mechanism underlying ga . these findings are also unique and counter intuitive at first appearance , since myd88 is a versatile adaptor protein for the tlr / il-1r superfamily - mediated proinflammatory signalling ( reviewed in ) , whereas apoptosis is essentially a noninflammatory cell death mechanism . although very rare , there are cell - type specific reports connecting apoptosis with myd88 mediated proinflammatory events , presumably as a final attempt for the tissue to remove the severely damaged cells . in pancreatic islet cells , dupraz et al . showed that overexpression of a dominant negative form of myd88 spared the cells from il-1 induced apoptosis . moreover , using human kidney epithelial 293 cells and human monocytic thp-1 cells , aliprantis and colleagues found that myd88 was crucial to the activation of tlr2 signaling and its downstream induction of caspase-8 dependent apoptosis in response to bacterial lipoproteins . however , similar mechanisms have not been previously reported in ocular tissues or cells , including rpe , until recently . taken together , these lines of evidence suggest that damaged rpe cells can respond to danger signals by activating the inflammasome pathway that may further lead to rpe atrophy in ga ( figure 2 ) . insufficient activation makes the immune system vulnerable to pamps and damps , whereas overwhelming inflammasome activation targets the host itself . clinically , inflammasome antagonists are being explored as novel therapeutics for treating human immune diseases [ 42 , 88 ] . the inhibition of inflammasome can potentially be achieved at four different levels along its activation pathway . these include blocking cell membrane receptors ( e.g. , p2x7 receptor for atp ) , controlling cytoplasmic second messengers ( e.g. , k , cathepsin b , ros ) , preventing inflammasome components from assembling , and antagonizing released cytokine products and/or their cognate receptors . to better understand how inflammasome formation is regulated mitochondria , the symbolic  power house  in the cell , are also known for other biological events , including intrinsic or in addition to lipid and protein peroxidation mentioned earlier in this review , mitochondrial dna ( mtdna ) is considered particularly prone to ros relative to its nuclear counterpart . research showed that when treated with hydrogen peroxide or rod outer segments human rpe cells generated more damaged and unrepaired mtdna , leading to mitochondrial redox dysfunction , inefficient energy production , rpe dysfunction , and ultimate initiation of rpe apoptosis . moreover , it has been recently suggested that inherited mitochondrial dna variation can also impact pathways other than apoptosis , for instance , in the complement activation . by introducing mitochondria from individuals with either high- or low - risk haplogroups ( accumulations of specific single nucleotide polymorphisms ) for amd into arpe-19 cells devoid of mtdna , kenney et al . were able to map the relationship between mtdna polymorphisms and nuclear gene expression for a number of molecular pathways related to amd . arpe-19 cybrids ( cytoplasmic hybrids with native arpe-19 cell nuclear dna and extrinsic mtdna ) harboring high - risk mtdna haplogroup for amd demonstrated decreased energy production and lower gene expression levels for cfh and c3 , key components of the complement pathway . when confronted with sublethal ultraviolet radiation , these high - risk cybrids had a further decrease in cfh gene expression , which could potentially lead to a greater degree of complement activation . as discussed in previous sections , complement activation products and mac may act as potential triggers for inflammasome activation . however , the exact mechanisms bridging mitochondrial damage and rpe apoptosis remain elusive . bruey et al . reported a novel role of bcl-2 and bcl - xl , two mitochondria - associated antiapoptotic proteins , in the suppression of the nlrp1 inflammasome activation in atp stimulated macrophages . by comparing the macrophages isolated from bcl-2 overexpressing transgenic mice to their wild - type counterparts , zhou et al . showed a significant decrease of secreted il-1 levels in association with the increasing bcl-2 expression , under classic nlrp3 inflammasome activation conditions . separately , shimada and colleagues also reported bcl-2 attenuates nlrp3 inflammasome activity through inhibition of mtdna release from the dysregulated and apoptotic mitochondria in macrophages . however , another study using bcl-2 overexpressing mice does not support bcl-2 's role in regulating nlrp3 inflammasome function . clearly , further investigation on the relationship between mitochondria - associated antiapoptotic proteins and nlrp3 inflammasome activation is warranted . another example showcasing mitochondria 's involvement in nlrp3 inflammasome activation comes from the studies on mitochondrial ros , which has been proposed to be either necessary or facilitate the activation of the nlrp3 inflammasome , by two research groups , independently [ 45 , 97 ] . further proposed it was the dissociation between thioredoxin and thioredoxin - interacting protein ( txnip ) under ros stimulation that allowed txnip to bind to nlrp3 , resulting in nlrp3 conformational change and ultimate activation . moreover , the use of antioxidants to inhibit nlrp3 inflammasome activity has been extensively studied and proved effective , despite the fact that the detailed underlying signaling pathways remain unclear . nonetheless , there is ongoing debate as to whether mitochondrial ros only works as a facilitator of nlrp3 inflammasome activation , given the fact that increasing intracellular ros levels can as well inhibit caspase-1 activation and il-1 maturation [ 99101 ] . perhaps , a more comprehensive approach , such as a model reflecting intracellular antioxidant response to mitochondrial ros overproduction , might provide insights regarding the determinants of the level of nlrp3 inflammasome activity , taking into account that both sides of the redox axis could play a role . ubiquitylation is a common posttranslational modification of proteins through a cascade of enzymatic activity of ubiquitin ligases . it has multiple effects on proteasome- or lysosome - mediated protein degradation , cell signal transduction , and protein activity regulation . according to the type and length of ubiquitin linkage , there are three major forms of protein ubiquitylation currently studied , including lysine 48 ( k48 ) linked ubiquitylation , lysine 63 ( k63 ) linked ubiquitylation , and methionine 1 ( met 1 ) linked linear ubiquitylation . mounting evidence has implicated the involvement of ubiquitylation in amd pathophysiology . by looking at the retinal distribution of several class iii ubiquitin - conjugating enzymes in mice , mirza et al . reported robust protein expression of one such enzyme , ubcm2 , in murine photoreceptors and rpe cells . the authors further experimented ubcm2 's protective effects on photoreceptors using an acute bright - light - damage model . it was shown that mice with only one copy of functional ubcm2 allele were protected from acute excessive light damage to photoreceptors , suggesting a strong relationship between ubcm2-mediated ubiquitylation and photoreceptor survival . in human rpe cells , it is evident that there is an active ubiquitin - proteasome mediated protein degradation pathway , with low endogenous levels of ubiquitin to fight against cellular stressors . in the context of amd , tested the proteasome activity of primary human rpe cells in response to complement factor c3a , a known drusen component . c3a stimulation significantly reduced proteasome activity without changing its component at either protein or mrna levels , indicating a potential functional suppression of the proteasome in primary human rpe . on the other hand , glenn and colleagues reported altered proteomic profiling of arpe-19 cells cultured on advanced glycation end products ( ages , known drusen component ) modified matrigel bm extract compared to non - age modified control bm . of note , by immunocytochemistry , the authors were able to localize upregulated protein expression of a dub protein , ubiquitin carboxyterminal hydrolase-1 ( uch - l1 ) , in age - stimulated arpe-19 cells , suggesting a potential role for dubs in amd pathogenesis . however , few reports exist on the roles of both ubiquitinating and deubiquitinating enzymes in nlrp3 inflammasome regulation . recently , py and colleagues demonstrated that brcc3 , a jamm domain - containing zinc metalloprotease dub , promotes nlrp3 inflammasome activation by deubiquitinating the mixed k64 and k48 ubiquitin chains on both the nacht and lrr domains of nlrp3 . the authors further suggested that the deubiquitylation of nlrp3 was critical for the inflammasome activation based on the facts that inhibiting brcc3 could abolish nlrp3 inflammasome activation under a diverse range of classic perhaps more intriguing is the report by rodgers et al . of the discovery that the linear ubiquitylation of the asc adaptor protein by the linear ubiquitin assembly complex ( lubac ) is also essential for nlrp3 inflammasome activation , independent of nf-b activity   ( figure 2 ) clinically , these studies provide potential alternative approaches for the treatment of inflammasome - related diseases by better controlling the ubiquitylation levels of separate nlrp3 inflammasome components , instead of targeting secreted levels of the mature proinflammatory cytokines . in this review , we summarize the key features of nlrp3 inflammasome activation and introduce newly discovered regulatory mechanisms . we also discuss the involvement of nlrp3 inflammasome in the pathogenesis of amd , with the focus on individual drusen components as potential facilitators for nlrp3 inflammasome activation in rpe cells . despite the rapid development of inflammasome research towards chronic inflammatory diseases such as amd , there are still many unsolved questions . on one hand , the mature product of inflammasome activation , il-18 , is hypothesized to carry out dual functions in different target cell types : as a destructive factor in ga   and as a protective , antiangiogenic factor in cnv . on the other hand , does the nlrp3 inflammasome activation in rpe lead to canonical caspase-1 dependent pyroptosis   or necrosis or apoptosis or a combination of these mechanisms ? it is well accepted that as amd progresses , rpe cells follow a common fate : ( 1 ) accumulation of lipofuscin ; ( 2 ) enlarged cell body ; ( 3 ) decrease in phagocytosis capacity ; ( 4 ) formation of drusen ; ( 5 ) morphological rounding ; ( 6 ) hyperpigmentation ; ( 7 ) hypopigmentation ; ( 8) rpe loss . in sections of human postmortem identified double - layered hyperpigmented rpe in the ga lesion , characteristic of necrosis , which was further supported by the discovery that rpe cells adjacent to excessive drusen accumulation die of necrosis . consistent with this description is the finding that necrosis , particularly the released atp , is a trigger for nlrp3 inflammasome activation . further studies are needed to understand , more fully , the combination of cell death mechanisms associated with ga . the potential involvement of an apoptotic mechanism is also supported by several studies discussed earlier in this review and by a recent transcriptome analysis on amd eyes . furthermore , much of our knowledge of the mechanisms associated with activation and regulation of the inflammasome comes from discoveries in nonocular immune cells . validation of these mechanisms in ocular cell types , such as rpe and photoreceptors , will be useful towards designing inflammasome - related treatment strategies for chronic inflammatory diseases of the retina , such as amd .
age - related macular degeneration ( amd ) is the leading cause of legal blindness in the elderly in industrialized countries . 
 amd is a multifactorial disease influenced by both genetic and environmental risk factors . 
 progression of amd is characterized by an increase in the number and size of drusen , extracellular deposits , which accumulate between the retinal pigment epithelium ( rpe ) and bruch 's membrane ( bm ) in outer retina . 
 the major pathways associated with its pathogenesis include oxidative stress and inflammation in the early stages of amd . 
 little is known about the interactions among these mechanisms that drive the transition from early to late stages of amd , such as geographic atrophy ( ga ) or choroidal neovascularization ( cnv ) . as part of the innate immune system 
 , inflammasome activation has been identified in rpe cells and proposed to be a causal factor for rpe dysfunction and degeneration . here , we will first review the classic model of inflammasome activation , then discuss the potentials of amd - related factors to activate the inflammasome in both nonocular immune cells and rpe cells , and finally introduce several novel mechanisms for regulating the inflammasome activity .