Our current understanding of the mechanisms sensing cytoplasmic DNA is limited9
. A candidate receptor called DAI (D
ctivator of I
FN-regulatory factors) has been implicated in the DNA-induced type I IFN pathway4
. The NLR family member, NLRP3 has also been shown to activate caspase-1 in response to internalized adenoviral DNA6
. Caspase-1 activation in response to transfected bacterial, viral, mammalian or synthetic DNA however, does not involve NLRP3, although the adapter molecule ASC is required6,7
We hypothesized that an upstream activator of this dsDNA activated ASC pathway would contain a pyrin domain (PYD) for homotypic interaction with ASC and at least one additional domain, for direct binding to DNA or for association with an upstream receptor. In addition to NLRP310
have previously been shown to associate with ASC. Although ASC-deficient macrophages failed to activate caspase-1 and trigger IL-1β release in response to poly(dA-dT) · poly(dA-dT) [hereafter referred to as poly(dA-dT)])2
, macrophages lacking NLRP3, −6 and −12 responded normally (). Surprisingly, we found that macrophages lacking ASC had higher levels of IFNβ and IL-6 in response to poly(dA-dT), which was not observed in cells lacking NLRP3, the IL1R or to a lesser extent caspase-1 (Supplementary Fig. 1a–d
). Poly(dA-dT) induced cell death also occurred in an ASC-dependent manner (Supplementary Fig. 1e–f
). We speculate, that the increased cytokine production in ASC-deficient cells relates to their resistance to poly(dA-dT) induced cell death. In addition to poly(dA-dT), dsDNA from natural sources activated caspase-1 cleavage (Supplementary Fig. 2a–b
). In contrast, a small immunostimulatory oligonucleotide (ISD)3
, long ssDNA (poly(dI)), transfected dsRNA or the ssRNA virus Sendai virus failed to trigger this response in NLRP3-deficient macrophages (Supplementary Fig. 2c
poly(dA-dT)-induced inflammasome activation
Searching the PFAM database13
we identified several PYD-domain containing proteins, which also contained a HIN200 domain, previously shown to bind DNA14
,. In humans, the HIN200 family consists of four members15
. A multiple sequence alignment of PYD domains from these proteins with PYD domains from some of the NLRs is shown (). Sequence analysis of IFIX, IFI16 and MNDA predicted their nuclear localization, in contrast to AIM2, which was predicted to be cytosolic (, lower panel). Consistent with these predictions, fluorescent protein chimeras of IFIX, IFI16 and MNDA localized to the nucleus, while AIM2 was almost exclusively cytoplasmic ().
To study the possibility that these PYHIN proteins associated with ASC, we generated C-terminally tagged CFP PYD-domain fusions (which lacked the putative nuclear localization sequences identified above). Indeed, all of the PYD-CFP fusions were localized in the cytoplasm (Supplementary Fig. 3
). To test whether induced clustering of the PYD-CFP fusions leads to association with ASC-YFP, we utilized a HEK293 cell line that stably expressed ASC-YFP at low enough levels to be polydispersed throughout the cytoplasm (, Mock). Indeed, overexpression of the NLRP3-CFP-tagged PYD domain led to the formation of large cytosolic aggregates, which co-aggregated ASC-YFP (). Notably, in most transfected cells, extensive intracellular co-localization with ASC-YFP was observed with a complete loss of cytoplasmic distribution of ASC-YFP10,11,20
. Of all the PYHIN-PYD proteins tested, only AIM2-PYD led to complex formation with ASC ( and Supplementary Fig. 4
). Similar results were obtained with full-length AIM2-CFP but not full-length IFIX, IFI16 or MNDA, which were all localized to the nucleus ( and Supplementary Fig. 5a, b
). Additionally, only AIM2-PYD and NLRP3-PYD were found to bind HA-tagged ASC in co-immunoprecipitation studies (). Finally, endogenous ASC associated with endogenous AIM2, but not IFI16 in primed THP-1 cells ().
To examine the functional relevance of AIM2-ASC complex formation, we examined NF-κB reporter gene activity in cells overexpressing the PYD-PYHIN proteins in the presence of ASC. Only NLRP3-PYD and AIM2-PYD led to potent NF-κB activation (). The effect of full length AIM2 was even more dramatic (, bottom panel). The full-length versions of IFIX, IFI16 and MNDA failed to activate NF-κB (Supplementary Fig. 5c
). ASC was absolutely required, since no substantial NF-κB reporter activity was observed in cells not transfected with ASC. No substantial activation of the IFNβ promoter reporter gene was observed with any of the PYHIN family members (Supplementary Fig. 6
We next examined whether the AIM2-ASC complex could lead to the formation of a functional inflammasome complex and caspase-1-dependent maturation of pro-IL-1β. We employed a transient transfection assay overexpressing the respective proteins of interest in the presence of ASC, caspase-1 and flag-tagged pro-IL-1β in 293T cells and monitored the cleavage of pro-IL-1β by immunoblotting. Among the PYD proteins tested, only that of NLRP3-PYD and AIM2-PYD induced maturation of pro-IL-1β, when ASC and caspase-1 were co-expressed (). Full-length AIM2 was even more potent than AIM2-PYD (, lower panel). Neither the PYD domain nor the full-length versions of IFIX, IFI16 or MNDA induced IL-1β cleavage (Supplementary Fig. 7
AIM2 is required for poly(dA-dT)- and vaccinia virus-triggered inflammasome activation
To study the role of AIM2 in cells with a functional poly(dA-dT)-triggered or dsDNA virus induced inflammasome complex, we used lentiviruses encoding shRNAs to knock down AIM2 in immortalized murine macrophage cell lines (B6-MCLs or N3-KO-MCLs)7
. AIM2 was expressed constitutively in both primary macrophages and in B6-MCLs and was further induced by poly(dA-dT) or Sendai virus (Supplementary Fig. 8
). Three different shRNAs were tested, of which two (shRNA AIM2 #2 and AIM2 #3) resulted in a strong reduction of AIM2 expression (). Knocking down AIM2, but not an unrelated gene, resulted in a strong attenuation of poly(dA-dT)-mediated IL-1β release () and caspase-1 cleavage (). Targeting AIM2 in THP1 cells using siRNA corroborated these findings (Supplementary Fig. 8d and e
). Moreover and consistent with what we had seen in ASC-deficient macrophages (Supplementary Fig. 1
), knocking down AIM2 resulted in a marked enhancement of poly(dA-dT)-mediated type I IFN induction (Supplementary Fig. 8b
). This effect was specific since the IFNβ response to Sendai virus was unaffected (Supplementary Fig. 8c
). In addition, and in agreement with the results obtained in ASC-deficient macrophages, macrophages that were targeted with AIM2 shRNAs were resistant to poly(dA-dT) triggered cell death (). We also examined the role of AIM2 in the recognition of the dsDNA virus vaccinia. Similar to what we had observed with transfected poly(dA-dT), vaccinia virus-induced caspase-1 cleavage occurred in an ASC-dependent but NLRP3-independent manner (). This effect was also dependent on AIM2, since shRNA-mediated knock down of AIM2 impaired vaccinia virus induced caspase-1 cleavage but not that induced by anthrax lethal toxin (). Moreover, knock down of a control protein did not affect caspase-1 cleavage after vaccinia virus infection. Vaccinia virus-triggered cell death was also strongly reduced in AIM2 shRNA targeted macrophages, but not in control macrophages (). Altogether, these results indicated that AIM2 controlled inflammasome activation and cell death in response to dsDNA and the dsDNA virus vaccinia.
To examine if AIM2 could be involved in the recognition of dsDNA directly, we generated fluorescein-labeled poly(dA-dT), (FITC-dsDNA) and co-transfected FITC-dsDNA together with CFP-tagged versions of full-length AIM2, AIM2-HIN domain, AIM2-PYD domain or full-length NLRP3. While cells expressing NLRP3 or AIM2-PYD showed no co-localization of the respective proteins with FITC-dsDNA, full-length AIM2 and AIM2-HIN domain showed extensive co-localization with FITC-dsDNA and led to the formation of DNA/protein aggregates in the cytosol (). We used single cell flow cytometry fluorescence resonance energy transfer (FRET) measurements to quantify these interactions (). A dose-dependent increase in FRET between full-length AIM2 and FITC-dsDNA was seen, while AIM2-PYD did not lead to measurable FRET. Other proteins such as NLRP3 or IFI16 did not show any FRET (data not shown). Additionally, binding studies using purified AIM2, AIM2-HIN domain and AIM2-PYD domain with biotinylated poly(dA-dT) (biotin-dsDNA) revealed that AIM2 directly interacted with poly(dA-dT) with high affinity; only full-length AIM2 or the AIM2-HIN domain were able to bind biotin-dsDNA (). Binding of poly(dA-dT) to AIM2 was specific, since AIM2 did not bind biotin-LPS, which bound to soluble CD14 under similar assay conditions ().
AIM2 binds dsDNA via its HIN domain
Collectively, these data identify AIM2 as a receptor for cytosolic dsDNA, which forms a novel inflammasome complex with ASC to activate caspase-1-mediated processing of IL-1β. Our data also indicate that the activation of the AIM2 inflammasome is important in innate immunity to vaccinia virus. Since bacterial pathogens such as Francisella tularensis
and aberrant host DNA in pathological autoimmunity22
also trigger the IL1β pathway, it will also be important to define the role of AIM2 in these responses. Further characterization of the AIM2 inflammasome as a sensor of microbial, as well as host DNA therefore, may enable the rational design of new therapies and treatments for infectious as well as autoimmune diseases.