Chemically and structurally diverse stimuli, ranging from the small molecules ATP or imidazoquinoline derivatives (R848, R837), to bacterial toxins and various crystals, can activate the NALP3 inflammasome4
. Undoubtedly, the list of NALP3 inflammasome activators will grow, yet the mechanisms by which the NALP3 inflammasome can sense different activators are not yet fully understood. One possibility is that NALP3 acts as a pattern recognition receptor that can directly bind and respond to various ligands. Indeed, recent reports suggest that NALP3 activators could gain access to the cytoplasmic space20, 21
. A precedent for such a mechanism is given by the large spectrum of ligands that can be sensed by certain Toll like receptors22
Another possibility is that the NALP3 inflammasome detects an intermediate molecule that is generated by a common mechanism evoked by many different ligands. It has been shown that potassium efflux is required for NALP3 activation upon various inflammasome stimuli23, 24
. However, it is currently unclear whether potassium efflux alone represents a NALP3 inflammasome stimulus per se
or whether low intracellular potassium acts as an additional requirement for inflammasome assembly after appearance of an as of yet undefined primary stimulus. An example of this potential mechanism is found in the formation of the closely related apoptosome25, 26
. The loss of intracellular potassium during apoptosis is an early, essential step for successful assembly of the apoptosome, which forms after cytochrome c is released from mitochondria in stress situations. It is therefore possible, that formation of the NALP3 inflammasome is highly favored at low intracellular potassium levels, which may be required yet not sufficient for activation.
We show here the novel finding that NALP3 inflammasome activation by crystals or alternatively induced lysosomal damage requires uptake and acidification of lysosomes and that the ingested crystals lead to lysosomal swelling and leakage. Notably, inhibition of a single lysosomal protease - cathepsin B – led to substantial, yet not complete, reduction of NALP3 inflammasome activation by lysosomal damage. In this context it is noteworthy that the NALP3 stimulus nigericin, which is thought to solely activate via potassium efflux, has induced lysosomal leakage and subsequent caspase-1 activation via the function of cathepsin B27
. Since it was also shown that pannexin 1, which helps establishing larger lysosomal channels, was required for nigericin-mediated NALP3 activation28, 29
, it is conceivable that pannexin 1 does not only allow access of bacterial ligands to the cytoplasm20, 21
but could also initiate lysosomal destabilization. Others have also suggested a role for cathepsin B in NALP3 inflammasome function, yet their data implied a role for cathepsin B downstream30, 31
rather than upstream of NALP3, as we have demonstrated here. It will be important to define molecular targets of cathepsin B and potentially other pH-activated proteases, as this may guide us to common upstream NALP3 activating factors. It was recently shown that a related cathepsin (cathepsin K) is also involved in innate immune recognition. Cathepsin K acts upstream of the innate signaling receptor TLR932
, which signals from within endo-lysosomal compartments33, 34
Collectively, our data suggest the hypothesis that lysosomal damage or leakage is perceived by the immune system as an endogenous danger signal. The NALP3 inflammasome can respond to internal membrane perturbations and thereby respond to different stimuli, which all have in common that they can induce lysosomal destabilization. Phagocytes that ingest endogenous or exogenous crystallized material or potentially also aggregated proteins or peptides are thus capable of sensing lysosomal damage induced by the phagocytosed cargo. Following NALP3 inflammasome activation highly inflammatory cytokines are generated, which leads to the recruitment of other immune cells to the affected tissue and to the induction of inflammatory mechanisms that assist in the clearance of the crystallized material. However, it is also possible that NALP3 activation by this mechanism can lead to sustained inflammation and resulting tissue damage. For example, chronic inhalative exposure to silica or asbestos can lead to chronic inflammation, tissue damage and carcinogenesis.
NALP3 activation by particulate matter can also intentionally be exploited therapeutically. Aluminium salt preparations, which are commonly used as adjuvants, contain small crystalline materials. We demonstrated here that alum activates immune cells via the NALP3 inflammasome, by a mechanism that involves lysosomal destabilization. It is likely that other vaccine candidates that are of particulate nature or that can perturb lysosomal membranes act via a similar NALP3 activation mechanism.
Our findings should thus aid in the development of novel vaccines and can lead to novel therapeutic concepts for the treatment of NALP3 inflammasome related pathologies.