A variety of structurally diverse molecules, including ATP12
, imidazoquinoline derivatives28
, bacterial toxins12
and various crystals11,13
, are known to activate the NALP3 inflammasome. Here we found that an endogenous peptide, Aβ, which forms insoluble fibrils in the brains of patients with Alzheimer’s disease, activated the NALP3 inflammasome. Aβ therefore seems to constitute another very clinically relevant endogenous ‘danger’ signal that is sensed by the NALP3 inflammasome. Although additional examples of fibrillar endogenous peptides that activate this pathway remain to be identified, we suspect that Aβ represents the first of a class of aggregated proteins that may share similar actions. It is conceivable that the NALP3 inflammasome may function as a general sensor for the recognition of peptide or protein aggregates that are also involved in the pathogenesis of diseases such as Parkinson’s and systemic amyloidosis and thus may be involved in inducing inflammatory responses in other disorders.
It has remained an open issue whether the NALP3 inflammasome is activated through direct binding to its ligands, similar to what has been described for Toll-like receptors29
, or through intermediary pathways that may be shared by different activators. A common structural finding among many different NALP3 activators is that they are of a particulate nature, and it thus seems possible that the mechanism of recognition is linked to the physical structure of the activator. In line with that hypothesis, our results have shown that the fibrillar state of Aβ was a requirement for IL-1β release.
Furthermore, we found that phagocytosis of Aβ was necessary for that pathway. We found that once Aβ was phagocytosed by microglial cells, Aβ-containing lysosomes adopted a swollen morphology and underwent structural damage, as indicated by loss of their pH-lowering capacity. Other studies have reported that microglial cells are incapable of efficiently degrading fibrillar Aβ for a period of weeks after its internalization30
. This ‘frustrated’ degradation of Aβ may contribute to the loss of lysosomal integrity, which in turn triggers the release of lysosomal components into the cytoplasm, as noted here. The release of such factors seems to be causally involved in the initiation of the Aβ-induced IL-1β pathway, as we found that IL-1β release depended on the lysosomal protease cathepsin B.
Cathepsin B has already been linked to the pathogenesis of Alzheimer’s disease. For example, larger amounts of cathepsin B have been reported in microglia surrounding senile plaques18
, and pharmacological inhibition of cathepsin B delays memory loss and decreases the Aβ plaque burden in a mouse model of Alzheimer’s disease19
. So far, most studies have focused on the function of cathepsin B in the cleavage of amyloid precursor protein in neurons19,31
and the extracellular removal of Aβ from senile plaques18
. Our study has suggested an additional effect of cathepsin B; that is, that cathepsin B serves as an intermediary among Aβ phagocytosis, lysosomal damage and microglial IL-1β release. Notably, lysosomal leakage after Aβ internalization has also been noted in neuronal cultures32
. However, in the cells surrounding senile plaques, NALP3 is expressed only by mononuclear phagocytes, not by neurons33
. It remains to be determined whether cathepsin B interacts directly with the inflammasome or whether this process involves intermediary molecules activated by cathepsin B. Our experiments have indicated that cathepsin B acts ‘upstream’ of NALP3 rather than influencing the production of pro-IL-1 or release of mature IL-1β. Furthermore, as lysosomes contain many other components, other phagosomal factors, including other cathepsins, could additionally contribute to inflammasome activation.
Unexpectedly, we also found that the NALP3–caspase-1 pathway was pivotal in the Aβ-induced secretion of critical proinflammatory, chemotactic and potentially neurotoxic cytokines in vitro
, including TNF and nitric oxide. The central importance of the inflammatory axis initiated by the NALP3–caspase-1 pathway and IL-1 was emphasized by our finding that activation and recruitment of microglia by exogenous fibrillar Aβ was much lower in vivo
in mice deficient in ASC, caspase-1, IL-1R or MyD88. Similar to our results, other studies have reported that in human monocytes, the release of TNF and IL-6 triggered by urate crystals, another endogenous ‘danger’ signal, is regulated by the IL-1β signaling pathway11
. This indicates that IL-1 signaling is essential or at least contributes to the production of various proinflammatory cytokines and chemokines. The receptors for IL-1and IL-18 contain an intracellular signaling domain (Toll–IL-1R) that is shared by all Toll-like receptors34
. Thus, it is not unexpected that the ‘downstream’ signaling events from IL-1, such as the induction of proinflammatory cytokines, overlap those noted after activation of Toll-like receptors.
The recruitment and activation of invading macrophages and microglia to senile plaques is a critical and early step in the initiation and progression of neuronal dysfunction and eventual neuronal death2
. Furthermore, several of the microglial cytokines we have identified here as being regulated by the NALP3–caspase-1 pathway have been shown before to induce neuronal cell death in vitro21,22
. Other studies, however, have reported that microglial recruitment and activation may also have beneficial effects on plaque removal and disease progression, which indicates that the involvement of inflammatory responses in the pathogenesis of Alzheimer’s disease in vivo
is more complex3,27,35
. The issues of whether the pathways we have described here are actually beneficial or deleterious to the brain and to what extent they interact with other pathways, such as those activated by Toll-like receptors36,37
or scavenger receptors25
, must be addressed by future studies with transgenic mouse models of Alzheimer’s disease. Our findings have suggested that pharmacological intervention aimed at this critical control point of microglial recruitment and activation may hold therapeutic promise. However, the complexity and interdependence on the cytokine network suggest that narrowly targeted anti-inflammatory therapy for patients with Alzheimer’s disease, such as the use of caspase-1 inhibitors, may have unanticipated effects.