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The protein ASC is a central component of most inflammasome complexes, forming functional oligomeric filaments that activate large amounts of pro-caspase-1 for further IL-1β processing and the induction of Gasdermin D-dependent cell death. The central role of inflammasomes in the innate immune response pose them as new molecular targets for therapy of diverse acute, chronic and inherited autoinflammatory pathologies. In recent years, an increasing number of molecules were proposed to modulate inflammasome signalling by interacting with different components of inflammasome complexes. However, the difficult in vitro reconstitution of the inflammasome has limited the development of specific on-target biochemical assays for compound activity confirmation and for drug discovery in high throughput screening setups. Here we describe a homogeneous, pH-based ASC oligomerization assay that employs fluorescence anisotropy (FA) to monitor the in vitro filament formation of the PYD domain of human ASC. The absence of additional solubility tags as well as of proteolytic enzymes to initiate the filament reaction makes this assay suitable for testing the direct effect of small molecules on filament formation in high throughput format. The ability of the assay to detect modulators of filament formation was confirmed by using a non-filament forming PYD mutant. The high and reproducible Z'-factor of 0.7 allowed to screen 10,100 compounds by high-throughput screening (HTS) aiming to identify inhibitors of ASC filament. While none of these molecules was able to inhibit ASC filament formation in vitro, the assay is directly amenable to screen other compound classes or validate candidate molecules from other screens.

The pro-inflammatory cytokine IL-1β is well known for its role in host defense and the initiation of potent inflammatory responses. It is processed from its inactive pro-form by the inflammatory caspase-1 into its mature bioactive form, which is then released from the cell via an unconventional secretion mechanism. Recently, gasdermin-D has been identified as a new target of caspase-1. After proteolytical cleavage of gasdermin-D, the N-terminal fragment induces pyroptosis, a lytic cell death, by forming large permeability pores in the plasma membrane. Here we show using the murine system that gasdermin-D is required for IL-1β secretion by macrophages, dendritic cells and partially in neutrophils, and that secretion is a cell-lysis-independent event. Liposome transport assays in vitro further demonstrate that gasdermin-D pores are large enough to allow the direct release of IL-1β. Moreover, IL-18 and other small soluble cytosolic proteins can also be released in a lysis-independent but gasdermin-D-dependent mode, suggesting that the gasdermin-D pores allow passive the release of cytosolic proteins in a size-dependent manner.

Summary Inflammasomes activate the protease caspase-1, which cleaves interleukin-1β and interleukin-18 to generate the mature cytokines and controls their secretion and a form of inflammatory cell death called pyroptosis. By generating mice expressing enzymatically inactive caspase-1C284A, we provide genetic evidence that caspase-1 protease activity is required for canonical IL-1 secretion, pyroptosis, and inflammasome-mediated immunity. In caspase-1-deficient cells, caspase-8 can be activated at the inflammasome. Using mice either lacking the pyroptosis effector gasdermin D (GSDMD) or expressing caspase-1C284A, we found that GSDMD-dependent pyroptosis prevented caspase-8 activation at the inflammasome. In the absence of GSDMD-dependent pyroptosis, the inflammasome engaged a delayed, alternative form of lytic cell death that was accompanied by the release of large amounts of mature IL-1 and contributed to host protection. Features of this cell death modality distinguished it from apoptosis, suggesting it may represent a distinct form of pro-inflammatory regulated necrosis., Graphical Abstract, Highlights • Interplay of caspase-1 and caspase-8 revealed by analysis of Caspase1C284A mice • GSDMD-dependent pyroptosis suppresses caspase-8 activation at the inflammasome • The inflammasome engages caspase-8-driven secondary pyroptosis and IL-1 release • GSDMD-independent mechanisms contribute to inflammasome-mediated host protection, Schneider et al. show that, in the absence of GSDMD or caspase-1 protease activity (e.g., in Casp1C284A mice), the inflammasome engages an alternative type of lytic cell death and IL-1 release that contributes to immunity against infection. This secondary form of pyroptosis is dependent on apoptotic caspase activity but distinct from apoptosis.

International audience; Pathogenic and commensal Gram-negative bacteria produce and release outer membrane vesicles (OMVs), which present several surface antigens and play an important role for bacterial pathogenesis. OMVs also modulate the host immune system, which makes them attractive as vaccine candidates. At the cellular level, OMVs are internalized by macrophages and deliver lipopolysaccharide (LPS) into the host cytosol, thus activating the caspase-11 non-canonical inflammasome. Here, we show that OMV-induced inflammasome activation requires TLR4-TRIF signaling, the production of type I interferons, and the action of guanylate-binding proteins (GBPs), both in macrophages and in~vivo Mechanistically, we find that isoprenylated GBPs associate with the surface of OMVs or with transfected LPS, indicating that the key factor that determines GBP recruitment to the Gram-negative bacterial outer membranes is LPS itself. Our findings provide new insights into the mechanism by which GBPs target foreign surfaces and reveal a novel function for GBPs in controlling the intracellular detection of LPS derived from extracellular bacteria in the form of OMVs, thus extending their function as a hub between cell-autonomous immunity and innate immunity.

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.

The AIM2 inflammasome detects double-stranded DNA in the cytosol and induces caspase-1-dependent pyroptosis as well as release of the inflammatory cytokines interleukin 1β (IL-1β) and IL-18. AIM2 is critical for host defense against DNA viruses and bacteria that replicate in the cytosol, such as Francisella tularensis subspecies novicida (F. novicida). The activation of AIM2 by F. novicida requires bacteriolysis, yet whether this process is accidental or is a host-driven immunological mechanism has remained unclear. By screening nearly 500 interferon-stimulated genes (ISGs) through the use of small interfering RNA (siRNA), we identified guanylate-binding proteins GBP2 and GBP5 as key activators of AIM2 during infection with F. novicida. We confirmed their prominent role in vitro and in a mouse model of tularemia. Mechanistically, these two GBPs targeted cytosolic F. novicida and promoted bacteriolysis. Thus, in addition to their role in host defense against vacuolar pathogens, GBPs also facilitate the presentation of ligands by directly attacking cytosolic bacteria.

A hallmark of inflammasome activation is the ASC speck, a micrometre-sized structure formed by the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which consists of a pyrin domain (PYD) and a caspase recruitment domain (CARD). Here we show that assembly of the ASC speck involves oligomerization of ASCPYD into filaments and cross-linking of these filaments by ASCCARD. ASC mutants with a non-functional CARD only assemble filaments but not specks, and moreover disrupt endogenous specks in primary macrophages. Systematic site-directed mutagenesis of ASCPYD is used to identify oligomerization-deficient ASC mutants and demonstrate that ASC speck formation is required for efficient processing of IL-1β, but dispensable for gasdermin-D cleavage and pyroptosis induction. Our results suggest that the oligomerization of ASC creates a multitude of potential caspase-1 activation sites, thus serving as a signal amplification mechanism for inflammasome-mediated cytokine production., Inflammasomes regulate IL-1β family maturation and pyroptosis. Here the authors show that ASC oligomerization and the formation of ASC specks are needed for IL-1β processing, but are not required for pyroptosis, indicating distinct inflammasome regulatory pathways.

The co-ordinated expression of virulence factors is a critical process for any bacterial pathogen to colonize its host. Here we investigated the mechanisms of niche adaptation of the zoonotic pathogen Bartonella henselae by combining genetic approaches and shotgun proteomics. We demonstrated that expression of the VirB/D4 type IV secretion system (T4SS) and its secreted effector proteins require the alternative sigma factor RpoH1, which levels are controlled by the stringent response (SR) components DksA and SpoT. The RpoH1-dependent activation requires an active BatR/BatS two-component system (TCS) while BatR expression is controlled by RpoH1 and the SR components. Deletion of spoT results in a strong attenuation of VirB/D4 T4SS expression whereas dksA, rpoH1 or batR deletion fully abolishes its activity. In contrast to their activating effect on the VirB/D4 T4SS, which is critical at the early stage of host infection, SpoT and DksA negatively regulate the Trw T4SS, which mediates host-specific erythrocyte infection at a later stage of the colonization process. Our findings support a model where the SR signalling and the physiological pH-induced BatR/BatS TCS conjointly control the spatiotemporal expression of B. henselae adaptation factors during host infection.

We have characterized the Drosophila homologue of the proto-oncogenic RAC protein kinase (DRAC-PK). The DRAC-PK gene gives rise to two transcripts with the same coding potential, generated by the use of two different polyadenylation signals. Each transcript encodes two polypeptides because of the presence of a weaker initiator ACG codon, upstream from the major AUG, such that the larger protein contains an N-terminal extension. Like the human isoforms, DRAC-PKs possess a novel signaling region, the pleckstrin homology domain. DRAC-PK proteins have a similar expression pattern, being regulated both maternally and zygotically, and are expressed throughout Drosophila development. Antisera specific for recombinant DRAC-PK and for its C terminus detected two polypeptides of 66 and 85 kDa in Drosophila extracts. The antirecombinant antisera also recognized a polypeptide of 120 kDa from Drosophila, which apparently shared an epitope related to DRAC-PK sequences. The role of p120 appears to be restricted compared with that of DRAC-PK, since it was not detected in larvae or adult flies. There was no spatial restriction of DRAC-PK expression during embryogenesis, suggesting that localized activation might be a regulatory mechanism for its function. DRAC-PK possesses an intrinsic kinase activity that is approximately 8-fold higher in adult flies than in 0-3-h embryos undergoing rapid mitotic cycles.

Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death. In mouse macrophages, activation of this pathway requires the production of type-I interferons, indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol.