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. 2008 Jun 3;105(22):7803-8.
doi: 10.1073/pnas.0802726105. Epub 2008 May 29.

A NOD2-NALP1 complex mediates caspase-1-dependent IL-1beta secretion in response to Bacillus anthracis infection and muramyl dipeptide

Li-Chung Hsu  1 , Syed R Ali, Shauna McGillivray, Ping-Hui Tseng, Sanjeev Mariathasan, Eric W Humke, Lars Eckmann, Jonathan J Powell, Victor Nizet, Vishva M Dixit, Michael Karin
Affiliations

A NOD2-NALP1 complex mediates caspase-1-dependent IL-1beta secretion in response to Bacillus anthracis infection and muramyl dipeptide

Li-Chung Hsu et al. Proc Natl Acad Sci U S A. .

Abstract

NOD2, a NOD-like receptor (NLR), is an intracellular sensor of bacterial muramyl dipeptide (MDP) that was suggested to promote secretion of the proinflammatory cytokine IL-1beta. Yet, the molecular mechanism by which NOD2 can stimulate IL-1beta secretion, and its biological significance were heretofore unknown. We found that NOD2 through its N-terminal caspase recruitment domain directly binds and activates caspase-1 to trigger IL-1beta processing and secretion in MDP-stimulated macrophages, whereas the C-terminal leucine-rich repeats of NOD2 prevent caspase-1 activation in nonstimulated cells. MDP challenge induces the association of NOD2 with another NLR protein, NALP1, and gel filtration analysis revealed the formation of a complex consisting of NOD2, NALP1, and caspase-1. Importantly, Bacillus anthracis infection induces IL-1beta secretion in a manner that depended on caspase-1 and NOD2. In vitro, Anthrax lethal toxin strongly potentiated IL-1beta secretion, and that response was NOD2 and caspase-1-dependent. Thus, NOD2 plays a key role in the B. anthracis-induced inflammatory response by being a critical mediator of IL-1beta secretion.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Caspase-1 and RIP2 are essential for MDP-induced IL-1β production in macrophages. (A) IL-1β and TNF-α secretion by WT and Nod2−/− macrophages. Macrophages were left untreated or primed with LPS (0.5 ng/ml) for 6 h before treatment with TiO2 microparticles without (−) or with MDP (10 μg/ml). For NOD2-independent inflammasome activation, macrophages were primed with high levels of LPS (500 ng/ml) followed by incubation with ATP (5 mM). Supernatants were collected, and secreted cytokines were measured by ELISA. This and all similar experiments were repeated at least three times, and one representative experiment done in triplicates is shown. Results are expressed as the mean ± SD, and the statistical analysis was performed by using a two-sided unpaired Student's t test. Significant differences, **, P < 0.01; *, P < 0.05. (B) Reconstitution of Nod2-deficient macrophages with NOD2 restores MDP-induced IL-1β release. Bone marrow of Nod2−/− mice was transduced with retroviruses encoding NOD2 or GFP, differentiated into macrophages and stimulated as in A. IL-1β levels in supernatants were determined by ELISA and NOD2 protein amount was monitored by immunobot. (C) Caspase-1 and RIP2 dependence of MDP-induced IL-1β secretion. Peritoneal macrophages from the indicated strains were treated as above and IL-1β release was measured.
Fig. 2.
Fig. 2.
NOD2 binds to and activates caspase-1. (A) Caspase-1 activation by MDP is NOD2-dependent. Peritoneal macrophages from the indicated strains were treated as described in Fig. 1A, and supernatants were analyzed by immunoblotting for the presence of activated caspase-1 (p20) and TNF-α. L/A = macrophages were primed with LPS + ATP as in Fig. 1A. (B) NOD2 interacts with caspase-1 upon MDP stimulation. TDM were treated with MDP (10 μg/ml). At the indicated time points, cells were lysed, caspase-1 was immunoprecipitated (IP), and the presence of NOD2 in immunoprecipitates and original lysates was examined by immunoblotting (IB). (C) The NOD2 CARD motif binds caspase-1. Myc-tagged caspase-1 was coexpressed in HEK293T cells with indicated FLAG-tagged NOD2 constructs. After 36 h, cells were lysed and caspase-1 was immunoprecipitated. The presence of FLAG-tagged NOD2 and caspase-1 in the immunoprecipitates was examined by immunoblotting. (D) Caspase-1 binds NOD2 via its CARD motif. FLAG-tagged NOD2 was coexpressed in HEK293T cells with the indicated Myc-tagged caspase-1 constructs. After 36 h, cell lysates were immunoprecipitated with Myc antibody and analyzed as above. (E) The NOD2-CARD motifs are required for MDP-induced IL-1β secretion. FLAG-tagged NOD2 expression vectors were cotransfected into HEK293T cells along with caspase-1 and pro-IL-1β expression vectors as above. After 36 h, culture supernatants and cell lysates were examined for mature IL-1β and the different NOD2 proteins, respectively. (F) The NOD2-LRR prevents caspase-1 activation in the absence of MDP. The different NOD2 constructs were coexpressed in HEK293T cells as shown. Secretion of mature IL-1β and activated caspase-1 was examined as above.
Fig. 3.
Fig. 3.
NALP1 binds NOD2 and enhances MDP-induced IL-1β release. (A) NOD2 and NALP1 coelute with caspase-1 after MDP stimulation. TDM were left untreated or treated with MDP (10 μg/ml) for 2 h. Cell lysates were collected and separated on a Superdex 200 size exclusion column. Fractions were immunoblotted with antibodies against NOD2 (Genentech), caspase-1, and NALP1 (Abcam). Fraction number and molecular mass markers (in kDa) are shown above each image. (B) MDP enhances association of NOD2 with NALP1. HEK293T cells were transfected with NALP1 or NOD2 vectors in the absence or presence of MDP (2 μg/ml). After 36 h, cells were lysed, and NOD2 was immunoprecipitated. Presence of NALP1 in the immunoprecipitates and original lysates was examined by immunoblotting. (C) MDP induces binding of NALP1 to endogenous NOD2, and caspase-1. TDM were stimulated with MDP (10 μg/ml). NOD2 and caspase-1 were separately immunoprecipitated, and the presence of NALP1 in the immunoprecipitates was examined as above. (D) NALP1 is required for MDP-induced IL-1β release in TDM. TDM were infected with lentiviruses expressing either scrambled or NALP1-specific shRNA and cultured for 72 h before stimulation with 10 μg/ml MDP for 12 h. Secretion of mature IL-1β and expression of NALP1 were examined by immunoblotting. (E) NALP1 potentiates NOD2-induced IL-1β secretion by MDP. NOD2 and NALP1 were coexpressed as indicated with caspase-1 and pro-IL-1β in HEK293T cells with or without MDP. After 36 h, culture supernatants were examined for mature IL-1β. Caspase-1 was immunoprecipitated from cell lysates, and the presence of coprecipitated NALP1 was examined by immunoblotting. NOD2, NALP1, and caspase-1 expression was analyzed as above.
Fig. 4.
Fig. 4.
B. anthracis-induced IL-1β secretion depends on NOD2, NALP1, and caspase-1. (A) NALP1 is required for IL-1β release in B. anthracis-infected TDM. TDM were infected with scrambled or NALP1 shRNA lentivirus as above and cultured for 72 h before B. anthracis infection. Supernatants were collected 6 h postinfection, and secretion of mature IL-1β and expression of NALP1 were examined by immunoblotting. (B and C) NOD2 is necessary for IL-1β, but not TNF-α, secretion in B. anthracis-infected mouse macrophages. Peritoneal macrophages from WT, Nod2−/− and caspase1−/− mice were infected or not with the indicated B. anthracis Sterne strains (BaWT or BaΔLT) at a multiplicity of infection of 2. Macrophages were also pretreated with LPS and then pulsed with ATP as a positive control. Supernatants were collected 6 h postinfection, and secreted IL-1β (B) and TNF-α (C) were measured. (D and E) NOD2 is required for IL-1β, but not TNF-α, release by LT-treated macrophages. Macrophages were left untreated or treated with LT (200 ng/ml LF + 2.5 μg/ml PA63) in the absence or presence LPS (100 ng/ml) for 18 h. Supernatants were analyzed for secreted IL-1β (D) and TNF-α (E). Significant differences, **, P < 0.01; *, P < 0.05. (F) IL-1β secretion in B. anthracis infected mice requires caspase-1 and NOD2. Mice (n = 5) were injected i.p. with 107 cfu of early log-phase B. anthracis BaWT. IL-1β in plasma was measured 17 h after infection.
Fig. 5.
Fig. 5.
A model summarizing the control of IL-1β secretion by NOD2. We propose that in the context of a bacterial infection, TLR engagement provides the major input leading to activation of NF-κB and induction of cytokine precursors. The unique function of NOD2 in this context is to activate caspase-1 in response to bacterial MDP or intact B. anthracis. This function depends on the association of NOD2 with NALP1. Activated NF-κB inhibits caspase-1 activation through synthesis of PAI-2.
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