We first determined whether induction of ER stress triggers the maturation of IL-1β. Human macrophages were exposed to tunicamycin, an inhibitor of GlcNAc-1-phosphate transferase and thus of glycoprotein synthesis, in order to induce ER stress. The presence of processed IL-1β was detected in the supernatant of the human monocytic cell line THP-1 and primary human monocytes – macrophages following stimulation with tunicamycin (). Similarly, induction of ER stress using Brefeldin A (BFA), which reversibly blocks protein transport from the ER to the Golgi, also caused IL-1β maturation and secretion. (). Thapsigargin, which induces ER stress by blocking ER SERCA pumps and thereby depleting ER Ca2+ stores, also led to IL-1β secretion, although exclusively in primary cells ( and data not shown). Taken together, these data suggest that induction of ER stress by three different compounds results in the release of mature IL-1β by human macrophages.
Figure 1 ER stress triggers the release of mature interleukin-1β by macrophages. (a) PMA-differentiated THP-1 cells were stimulated with uric acid crystals (MSU, positive control), BFA, thapsigargin or tunicamycin for 6h. Precipitated supernatant (more ...)
The NLRP3 inflammasome is composed of NLRP3, Cardinal, the adaptor ASC and caspase-1, and mediates the production of active IL-1β
in response to an ever-expanding list of stimuli.4
A second inflammasome is constituted of the NLR member NLRC4 and caspase-1, and senses the PAMP flagellin, while a third inflammasome senses DNA via AIM2. While all inflammasomes described thus far (the NLRP3, AIM2, NLRC4 and NLRP1 inflammasomes) sense various PAMPs, only the NLRP3 inflammasome has been shown to respond to a plethora of DAMPs as well. We therefore hypothesized that NLRP3 is the inflammasome sensor activated by ER stress.
To confirm the specific involvement of the NLRP3 inflammasome, we induced ER stress in THP-1 cells in which NLRP3, ASC and caspase-1 were knocked-down using shRNA. We found that secretion of IL-1β
was markedly reduced in cells deficient in NLRP3, ASC and caspase-1, but not NLRC4 (Supplementary Figure 1
, and data not shown). The specific involvement of the NLRP3 inflammasome was further confirmed by performing similar experiments in murine macrophages isolated from Nlrp3−/−
mice (). Moreover, ER stress-induced inflammasome activation is independent of toll-like or RIG-I-like receptors, as IL-1β
secretion was normal in MyD88
- and Mavs
-deficient murine macrophages (Supplementary Figures 2A, B and C
Figure 2 ER stress specifically activates the NLRP3 inflammasome. (a–c) LPS-primed bone marrow-derived macrophages from Nlrp3+/+, Nlrp3−/−, Asc+/+, Asc−/−, Nlrc4+/+ and Nlrc4 (more ...)
The mechanism by which the NLRP3 inflammasome is activated remains unclear,7, 8
though recent evidence suggests mitochondria sense and integrate various danger signals and relay the signal to the NLRP3 inflammasome.9
Nonetheless, both potassium (K+
) efflux and an increase of reactive oxygen species (ROS) are required for NLRP3 inflammasome activation in response to all stimuli tested thus far. We therefore examined whether these factors are required for ER stress-induced IL-1β
maturation. This was indeed the case as the addition of ROS scavengers () or the inhibition of K+
efflux () completely inhibited IL-1β
secretion. Additionally, cytochalasin D treatment, which blocks actin polymerization, failed to block ER stress-induced IL-1β
secretion, suggesting that phagocytosis is not required akin to other non-particulate NLRP3 agonists ().
Figure 3 Mechanism of ER stress-induced inflammasome activation is similar to that of other known NLRP3 activators. (a, b and c) PMA-differentiated THP-1 cells were pre-incubated for 30min with 50-μM ammonium pyrrolidinedithiocarbamate, 130-mM (more ...)
The finding that ER stress, like other NLRP3 activators, activates the NLRP3 inflammasome in a K+
efflux- and ROS-dependent manner suggests that it may also be sensed by mitochondria. Thus, it is conceivable that ER stress initiates a signal that is transmitted to mitochondria and then relayed to the NLRP3 inflammasome. This notion is consistent with the subcellular localization of NLRP3. In resting cells, NLRP3 is associated with ER membranes, and then upon activation translocates to membranes that are positive for markers of both ER and mitochondria, which likely correspond to mitochondria-associated membranes (MAMs).9
VDACs are the major channels for the exchange of metabolites and ions between the mitochondria and other cellular compartments, including the ER. They are therefore important for mitochondrial oxidative metabolism and the generation of ROS. We therefore tested whether dampening mitochondrial activity by knocking down the expression of VDACs has an impact on inflammasome activation induced by ER stress. Akin to MSU, inflammasome activation by tunicamycin was impaired in cells with downregulated VDAC1 (). Interestingly, VDAC2 knock-down did not affect IL-1β
secretion by tunicamycin, while it substantially decreased inflammasome activity triggered by MSU or other NLRP3 activators, as previously reported ( and data not shown).9
This intriguing observation suggests the existence of different inflammasome activation mechanisms downstream of ER stress compared with other known NLRP3 activators, possibly at the mitochondrial level.
Figure 4 ER stress activates the NLRP3 inflammasome independently of the UPR. (a) PMA-differentiated THP-1 cells overexpressing an shRNA against IRE1α or an empty vector were stimulated with MSU, BFA or R837 for 6h, and analyzed by western blot. (more ...)
The cellular response to ER stress results in the activation of the UPR, which consists of the three main effector pathways that are initiated by ER-localized transmembrane proteins, namely IRE1α
, PERK and ATF6α
. We thus investigated the possible role of UPR effectors in inflammasome activation. THP-1 cells stably expressing shRNA against IRE1α
or PERK displayed no alteration in the secretion of mature IL-1β
in response to BFA or tunicamycin treatment ( and Supplementary Figure 3
). Additionally, macrophages derived from Atf6α−/−
mice did not differ from WT littermates in their response to thapsigargin or tunicamycin (). Of note, strong NLRP3 activators like MSU or R837 did not induce CHOP upregulation (), suggesting that they do not induce ER stress and their ability to activate the inflammasome is thus also independent of an ER stress response.
The UPR results in widespread blockage of the translation of most proteins through phosphorylation of eIF2α. Simultaneously, it selectively induces the transcription and translation of proteins initially involved in pro-survival pathways to help the cell cope with and resolve the cause of ER stress, and subsequently to initiate programed cell death if the ER stress is excessive or prolonged. Overall, most downstream effectors of the UPR are thus transcription-dependent. While synthesis of the ER stress response effector CHOP was blocked by the transcriptional inhibitor actinomycin D, inflammasome activation was not affected (). This observation further suggests that activation of the NLRP3 inflammasome by ER stress is not dependent on the UPR.
As signaling pathways triggered by the UPR appeared to be dispensable, we next investigated the role of alternative signaling pathways that were shown to emanate from a stressed ER.2
ER stress is proposed to trigger NF-κ
B activation through the interaction between IRE1α
and the IKK complex via the adaptor TRAF2.10, 11
In line with the observation that IRE1α
is not required (see above), THP-1 cells expressing an shRNA against TRAF2 responded normally to NLRP3 agonists (Supplementary Figure 4a
). We next investigated a possible role of the JNK signaling pathway. ER stress-induced JNK activation is thought to be triggered by a TRAF2- and ASK1- (a stress-activated MAP3K) dependent pathway. However, neither TRAF2 nor ASK1 were required for inflammasome activation in response to ER stress, as both THP-1 cells with knock-downed TRAF2 and macrophages from Ask1−/−
mice responded normally (Supplementary Figures 4A and B