FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Mice

Rip3-/- (20), Rip3-/-caspase-8-/- (14), Rip3-/-Fadd-/- (21), Nlrp3-/- (22), Asc-/- (23), Casp1-/-Casp11-/-, Casp1-/-Casp11Tg (2), IFNAR2-/- (24), Tlr4-/- (25), Trif-/- (26), MyD88-/-(27), Nod2-/- (28), TNF-α-/- (29), TNFR1-/- (30) were all described previously. Caspase-8flx mice were bred with LysMCre (B6. 129P2-Lyz2tm1(cre)Ifo/J - Jackson) mice to generate conditional caspase-8 KO mice. C57BL/6 WT (Jackson), Rip3-/-Casp8-/- and Rip3-/-Fadd-/- mice, and littermate controls were bred at St. Jude Children's Research Hospital. Animal studies were conducted under protocols approved by St. Jude Children's Research Hospital and Ghent University Committee on Use and Care of Animals VSports在线直播.

Macrophage differentiation and stimulation

Bone marrow-derived macrophages (BMDMs) were prepared as described previously (5). In brief, bone marrow cells were grown in L-cell-conditioned IMDM medium supplemented with 10% FBS, 1% non-essential amino acid and 1% penicillin-streptomycin for 5 days to differentiate into macrophages. On day5 BMDMs were seeded in 6-well cell culture plates, and the next day stimulated with LPS (20 ng/ml) for 4h, the last 30 minutes of which in the presence of 5 mm ATP or 20 μM nigericin. Where indicated, BMDM were pretreated with the caspase-8 inhibitor Ac-IETD-fmk (Calbiochem; 20 μM) before or after LPS-priming, respectively. In other experiments, LPS-pretreated or untreated BMDMs were infected with C. rodentium or E V体育2025版. coli at a multiplicity of infection (m. o. i. ) 25 for 24h. 2h post-infection, gentamycin (100 μg/ml) was added to the culture medium.

In vitro transcription/translation

³⁵S-labelled procaspases were produced in vitro using the SP6 High-Yield Wheat Germ Protein Expression System (Promega), and incubated with 100 U recombinant mouse caspase-8 (Enzo Life Sciences) or 35 ng mouse caspase-3 (VIB) before caspase processing was analyzed by autoradiography.

Western blotting

Samples for immunoblotting were prepared by combining cell lysates with culture supernatants. Samples were denatured in loading buffer containing SDS and 100 mM DTT and boiled for 5 min. SDS-PAGE-separated proteins were transferred to PVDF membranes and immunoblotted with primary antibodies against caspase-1 (Adipogen, AG-20B-0042), pro-caspase-8 (Enzo Life Sciences, 1G12), cleaved caspase-8 (Cell Signaling Technology, D5B2), caspase-11 (Novus Biologicals, 17D9), FADD (Millipore, 1F7), Nlrp3 (Adipogen, AG-20B-0014), IL-1β (R&D Systems), IL-18 (31) and GAPDH (Cell Signaling Technology, D16H11) followed by secondary anti-rabbit or anti-rat or anti- mouse or anti-goat HRP antibodies (Jackson Immuno Research Laboratories), as previously described(22) VSports app下载.

V体育官网入口 - Flow cytometry and phagocytosis assay

BMDMs were stained with CD11b, F4/80 and CD86 antibodies (eBioscience), or preincubated with GFP-expressing C V体育官网. rodentium, FITC-labeled zymosan A or OVA (Molecular Probes) for 3h prior to analysis on a LSR-II (BD Biosciences) and FlowJo software.

Confocal immunofluorescence microscopy

WT, caspase-1^-/- and Rip3^-/- caspase-8^-/- macrophages grown on coverslips were either left untreated (control) or stimulated with LPS+ATP or infected with C. rodentium. Cells were fixed with 4% paraformaldehyde and stained with caspase-1 (Adipogen) or caspase-8 (Cell Signaling Technology) antibodies. Nuclei were counterstained with DAPI. Cells were mounted on glass slides using ProLong gold antifade reagent (Life Technologies) and micrographs taken on Nikon C1 confocal microscope using a 40× objective lens. The images were processed and analyzed with Image J software. The images were taken at Cell and Tissue Imaging Center Light Microscopy Facility (CTIC-LM) at St. Jude.

LPS-induced endotoxemia and in vivo C. rodentium infection

In some experiments, cohorts of WT, Rip3^-/-, Rip3^-/-Casp8^-/- and Rip3^-/-Fadd^-/- mice were injected intraperitoneally with 35 mg/kg LPS (Sigma-Aldrich) for 5h before serum was collected for cytokine analysis. In other experiments, groups of WT, Rip3^-/-, Rip3^-/-Casp8^-/- and Rip3^-/-Fadd^-/- mice were infected with C. rodentium (ATCC 51459; 1 × 10¹⁰ CFU) by oral gavage. Food and water intake were stopped 8 h prior to infection and allowed to resume 1 h after infection. To determine bacterial counts, serial dilutions of homogenized feces were plated on MacConkey agar plates and incubated at 37 °C for 24 h. Stool consistency were scored (stool score) according to standard protocol as described previously (3). Briefly, 1= well formed pellets, 2= semiformed pellets that do not adhere to the anus, 3= soft stool that do not form pellets and adhere to the anus, 4= liquid stool that adhere to the anus, 5= liquid stool with blood.

Cytokine analysis

Concentrations of cytokines and chemokines were determined by multiplex ELISA (Millipore), or classical ELISA for IL-1β (eBioscience), IL-18 (MBL international).

VSports手机版 - Real-time PCR

Total RNA was extracted from cells stimulated with LPS or infected with C. rodentium using Trizol (Life Technologies) according to the manufacturer's instructions. RNA was quantified and 1 μg total RNA was reverse-transcribed to complementary DNA with poly(dT) primers using the first-strand cDNA synthesis kit (Life Technologies). Transcript levels of proIL-1β and Nlrp3 were quantified by RT-qPCR on an ABI7500 real-time PCR instrument with SYBR-Green (Applied BioSystems). Gapdh expression was used for normalization, and results are presented as fold induction over levels untreated control cells.

Statistics

GraphPad Prism 5.0 software was used for data analysis. Data are represented as mean ± standard errors of mean (SEM). Statistical significance was determined by Student's t test; p<0.05 was considered statistically significant.

"VSports手机版" FADD is critical for potent canonical and non-canonical Nlrp3 inflammasome activation

Conditional deletion of FADD in hematopoietic progenitor cells affects myeloid cell differentiation into bone marrow-derived macrophages (BMDMs) (32). However, simultaneous deletion of RIP3 and FADD did not cause global defects in macrophage differentiation as Rip3^-/-Fadd^-/- macrophages looked morphologically normal, and expressed normal levels of the myeloid cell/macrophage surface markers CD11b, F4/80 and CD86 (Supplemental Fig. 1A-1D). In addition, phagocytosis and pinocytosis of respectively GFP-labeled C. rodentium, fluorescently-labeled zymosan and FITC-ovalbumin was not affected (Supplemental Fig. 1E-1G), indicating that Rip3^-/-Fadd^-/- macrophages were not functionally impaired. To understand the roles of RIP3 and FADD in canonical Nlrp3 inflammasome activation, LPS-primed wildtype, Rip3^-/- and Rip3^-/-Fadd^-/-macrophages were stimulated with ATP or nigericin, and caspase-1 processing was monitored. Under these conditions, ATP- and nigericin-induced caspase-1 activation was significantly blunted in LPS-primed macrophages that lacked FADD in a RIP3-deficient background, but not in cells lacking RIP3 only (Fig. 1A). ATP-induced caspase-1 processing was also significantly reduced in Pam3CSK4-primed Rip3^-/-Fadd^-/- macrophages (Fig. 1B), indicating that the requirement for FADD was not restricted to TLR4-stimulated cells. In addition to these canonical Nlrp3 stimuli, FADD-deficiency hampered non-canonical Nlrp3 inflammasome activation in macrophages infected with the enteropathogens C. rodentium and E. coli because cells lacking RIP3 and FADD were defective in procaspase-1 maturation, whereas wildtype and Rip3^-/- macrophages responded to these enteropathogens with robust activation of caspase-1 (Fig. 1C). Because caspase-11 is required for caspase-1 maturation in enteropathogen–infected macrophages (2), defective caspase-1 maturation in Rip3^-/-Fadd^-/- macrophages may be consequent to their inability to activate caspase-11. Indeed, C. rodentium- and E. coli-infected Rip3^-/-Fadd^-/- macrophages also were blunted in maturation of procaspase-11 into the large catalytic subunit (p30) (Fig. 1C). In addition, they failed to secrete significant amounts of IL-1β (Fig. 1D) and IL-18 (Fig. 1E) in the culture medium. The role of RIP3 and FADD in enteropathogen-induced Nlrp3 inflammasome activation is specific because RIP3 and FADD were dispensable for Salmonella typhimurium-induced caspase-1 activation (Fig. 1F), which proceeds through the Nlrc4 inflammasome (23). Together, these results suggest a specific role for FADD in potent activation of the canonical and non-canonical arms of the Nlrp3 inflammasome.

FADD mediates caspase-8 maturation with canonical and non-canonical Nlrp3 inflammasome stimuli

FADD modulates apoptosis and necroptosis signaling through its associated effector protease caspase-8 (21). We therefore analyzed the expression and activation status of caspase-8 under conditions well-known to elicit activation of the canonical and non-canonical Nlrp3 inflammasomes, respectively. Wildtype, RIP3-deficient and Rip3^-/-Fadd^-/- cells expressed comparable levels of procaspase-8, but procaspase-8 processing into the large catalytic (p17) subunit was markedly reduced in LPS-primed Rip3^-/-Fadd^-/- macrophages that have been treated with the canonical Nlrp3 inflammasome stimuli ATP and nigericin (Fig. 2A). Notably, combined stimulation with LPS and ATP was needed to induce caspase-8 maturation, because wildtype, RIP3-deficient and Rip3^-/-Fadd^-/- macrophages that were treated with only LPS or ATP failed to process caspase-8 (Supplemental Fig. 2A). Silica-treated macrophages also required FADD for potent induction of caspase-1 maturation and caspase-8 processing (Supplemental Fig. 2B). In addition, we found caspase-8 activation to be significantly reduced in C. rodentium- and E. coli-infected Rip3^-/-Fadd^-/- cells, but not in Rip3^-/- macrophages (Fig. 2B). Together, these results suggest that caspase-8 is processed under inflammasome-activating conditions. To further understand the relationship between FADD-induced caspase-8 maturation and Nlrp3 inflammasome activation, we monitored caspase-8 activation status in inflammasome-deficient macrophages. As reported for LPS+nigericin-treated macrophages (19), neither loss of caspase-11, nor combined loss of caspase-1 and -11 affected caspase-8 activation in LPS+ATP-stimulated cells, whereas loss of Nlrp3 or ASC inhibited LPS+ATP-induced caspase-8 processing (Fig. 2C). As reported (2), C. rodentium- and E. coli-induced caspase-1 processing was abolished in Nlrp3^-/-, Asc^-/-, and caspase-1/11-deficient macrophages in which caspase-11 expression was restored from a C57BL/6 bacterial artificial chromosome (Casp1^-/-Casp11^Tg) (Fig. 2D). Caspase-11 expression and maturation were not affected in these cells. Notably, unlike during canonical Nlrp3 signaling, loss of neither Nlrp3, ASC, caspase-1, caspase-11 nor the combined loss of caspase-1 and caspase-11 affected enteropathogen-induced caspase-8 activation (Fig. 2D and Supplemental Fig. 2C), positioning FADD-dependent caspase-8 activation in enteropathogen-infected macrophages upstream of caspase-11 and the Nlrp3 inflammasome.

Previous studies implicated TLR signaling in the canonical and non-canonical Nlrp3 inflammasome pathways (4, 5, 7). This prompted us to analyze whether TLR signaling modulated expression of FADD and/or caspase-8 activation in LPS+ATP-treated or enteropathogen-infected macrophages. FADD and procaspase-8 were constitutively expressed, and their expression levels did not change significantly in LPS+ATP-treated or C. rodentium-infected Tlr4^-/-, Trif^-/- and MyD88^-/- macrophages, respectively (Fig. 2E and 2F). However, both LPS+ATP- and C. rodentium-induced caspase-8 maturation were markedly reduced in TLR4- and TRIF-deficient macrophages (Fig. 2E and 2F). Moreover, we found caspase-8 processing to be reduced in MyD88^-/- macrophages (Fig. 2E and 2F), suggesting that - in addition to FADD - caspase-8 activation further required TLR-dependent signaling. Notably, C. rodentium- and E. coli-induced caspase-8 activation also was partially affected in IFNAR1^-/- macrophages (Fig. 2G), suggesting type I interferon signaling to contribute to efficient caspase-8 activation in enteropathogen-infected macrophages. Together, these results implicate TLR- and type I interferon signaling in caspase-8 activation by stimuli of the canonical and non-canonical Nlrp3 inflammasome, respectively. In addition, our observations that caspase-8 activation is defective in Rip3^-/-Fadd^-/- cells, and that Nlrp3 and ASC mediate caspase-8 maturation in response to canonical Nlrp3 stimuli suggest that caspase-8 may operate downstream of FADD in regulating Nlrp3 inflammasome responses.

Caspase-8/RIP3 deletion inhibits canonical Nlrp3 inflammasome activation

Having established a role for FADD in activation of caspase-8 and the Nlrp3 inflammasome, we next sought genetic confirmation of the potential role of caspase-8 in canonical and non-canonical Nlrp3 inflammasome activation, respectively. To this end, we differentiated BMDMs from caspase-8-deficient mice in a RIP3-deficient background (14), as well as from animals with a conditionally-targeted deletion of caspase-8 in myeloid progenitors cells (casp8^LysM-Cre) (33). As reported for an independently-generated casp8^LysM-Cre mouse line (34), BMDM cultures of casp8^LysM-Cre+ mice mainly contained cells in which deletion of the floxed caspase-8 allele had failed to occur (Supplemental Fig. 2D and 2E). In contrast, caspase-8 was successfully deleted in Rip3^-/-caspase-8^-/- BMDMs (Supplemental Fig. 2D and 2E), suggesting that caspase-8-deficient myeloid progenitor cells might be sensitive to RIP3-mediated necroptosis. To phenotypically characterize Rip3^-/-caspase-8^-/- BMDMs, we confirmed that they appeared morphologically normal, that they expressed normal levels of the monocytes/macrophage markers CD11b, F4/80 and CD86 (Supplemental Fig. 1A-1D), and that phagocytosis of pathogenic bacteria, fungal cell wall components and antigenic peptides was not impaired (Supplemental Fig. 1E-1G). In line with the hypothesized role for caspase-8 in mediating efficient canonical Nlrp3 inflammasome activation, ATP- and nigericin-induced caspase-1 maturation were significantly affected in LPS-primed Rip3^-/-caspase-8^-/- macrophages, but not in macrophages lacking RIP3 alone (Fig. 3A). ATP-induced maturation of caspases-1 and -8 was already evident in wildtype and Rip3^-/- macrophages 5 minutes after ATP exposure and further increased with similar kinetics in these cells (Fig. 3B). In addition to inducing rapid caspase-1 maturation, potent canonical Nlrp3 inflammasome stimulation induces rapid pyroptotic cell death, while induction of caspase-8-dependent apoptosis proceeds with slower kinetics and prevails in caspase-1-deficient macrophages (19, 35). Accordingly, we noted that both ATP-induced pyroptosis (Fig. 3C) and IL-1β secretion (Fig. 3D) followed suit and were evident already 10 minutes after ATP exposure. Consistent with an upstream requirement for FADD and caspase-8 in potent induction of caspase-1 activation (Fig. 1A and 3B), the time-dependent induction of pyroptosis and IL-1β secretion were specifically reduced in LPS+ATP-treated Rip3^-/-Fadd^-/- and Rip3^-/-caspase-8^-/- macrophages (Fig. 3C and 3D). Rip3^-/-caspase-8^-/- macrophages were similarly defective in ATP-induced caspase-1 processing and IL-1β secretion after priming with the TLR2 ligand Pam3CSK4 (Fig. 3E and 3F), demonstrating that the role of caspase-8 in Nlrp3 inflammasome activation was not limited to LPS-primed macrophages. Both FADD and caspase-8 mediate cellular responses to TNF-α (36), but their role in ATP-induced Nlrp3 inflammasome activation was independent of TNF-α signaling because TNF-α^-/- and TNF-R1^-/- BMDMs responded to LPS+ATP stimulation with normal caspase-1 and caspase-8 activation, and secreted normal levels of IL-1β (Supplemental Fig. 3A-3C). Together, these results demonstrate that Rip3^-/-caspase-8^-/- macrophages are significantly impaired in canonical Nlrp3 inflammasome activation.

Enteropathogen-induced Nlrp3 activation is impaired in V体育官网入口 - Rip3/caspase-8 knockout macrophages

To explore the role of caspase-8 in non-canonical Nlrp3 inflammasome signaling, macrophages were infected with the enteropathogens C. rodentium- and E. coli. Notably, caspase-1 maturation was significantly impaired in C. rodentium- and E. coli-infected Rip3^-/-caspase-8^-/- macrophages (Fig. 4A and Supplemental Fig. 4A). In contrast, caspase-1 processing was normal in enteropathogen-infected macrophages lacking RIP3 alone (Fig. 4A). As was recently reported (37), caspase-1 maturation also was not affected in S. typhimurium-infected Rip3^-/-caspase-8^-/- cells (Fig. 4B), demonstrating specificity of these results. As with canonical Nlrp3 inflammasome stimuli, TNF-α signaling also was dispensable for enteropathogen-induced caspase-1 and caspase-8 activation in macrophages infected with C. rodentium (Supplemental Fig. 3D-3F). Analysis of caspase-11 processing in C. rodentium- and E. coli-infected Rip3^-/- and Rip3^-/-caspase-8^-/- macrophages further supported an upstream role for caspase-8 in mediating non-canonical Nlrp3 inflammasome activation because caspase-11 maturation was specifically affected in Rip3^-/-caspase-8^-/- BMDMs (Fig. 4C). Concurrently, the time-dependent secretion of IL-1β (Fig. 4D), and the induction of IL-1α release and pyroptosis (Fig. 4E and F) were specifically reduced in C. rodentium-infectedRip3^-/-caspase-8^-/- macrophages, but not in macrophages lacking only RIP3. Together, these results show that enteropathogen-induced activation of caspase-11 and the non-canonical Nlrp3 inflammasome is severely hampered in Rip3^-/-caspase-8^-/- macrophages.

Caspase-8 and FADD mediate transcriptional priming of the Nlrp3 inflammasome

Nlrp3 inflammasome activation is a highly regulated process at both the transcriptional and post-translational levels. At the transcriptional level, Nlrp3 inflammasome-mediated caspase-1 activation and IL-1β secretion requires TLR4/MyD88-mediated upregulation of Nlrp3 and proIL-1β expression (4, 5). In addition, C. rodentium- and E. coli-induced Nlrp3 inflammasome activation requires TLR4/TRIF-mediated expression of caspase-11 (5-7). To further determine the level at which FADD and caspase-8 regulated Nlrp3 inflammasome activation, we analyzed Nlrp3 and proIL-1β expression levels in LPS-primed macrophages. Notably, LPS-induced upregulation of Nlrp3 mRNA (Fig. 5A) and protein expression (Fig. 5B) levels were significantly reduced in Rip3^-/-Fadd^-/- macrophages. In agreement with FADD being responsible for these effects, loss of RIP3 alone did not significantly affect Nlrp3 expression (Fig. 5A and 5B). Concurrently, Rip3^-/-Fadd^-/- macrophages were defective in LPS-induced proIL-1β mRNA (Fig. 5C), as well as in LPS-induced protein expression (Supplemental Fig. 2A), and in LPS+ATP- and LPS+silica-induced proIL-1β maturation (Fig. 5D and Supplemental Fig. 4B), suggesting that FADD regulated TLR4/NF- κ B-dependent transcriptional priming of the Nlrp3 inflammasome. Indeed, LPS-induced phosphorylation of Iκ Bα and ERK was specifically reduced in Rip3^-/-Fadd^-/- macrophages, but not in cells lacking RIP3 only (Fig. 5E). In agreement, LPS-induced secretion of the NF-κB-dependent cytokines IL-6 (Fig. 5F) and KC (Fig. 5G) was significantly reduced in the absence of FADD. In line with caspase-8 operating downstream of FADD, proIL-1β mRNA (Fig. 5H) and protein expression (Supplemental Fig. 2A) were significantly downregulated in LPS-primed Rip3^-/-caspase-8^-/- macrophages as well. As a result, levels of both proIL-1β and mature IL-1β were significantly reduced in LPS+ATP- and LPS+silica-treated Rip3^-/-caspase-8^-/- macrophages (Fig. 5I and Supplemental Fig. 4B). To address whether FADD and caspase-8 were specifically involved in TLR-induced NF- κ B activation, we stimulated cells with the NOD2 agonist muramyl dipeptide (MDP). As expected, NOD2-deficient macrophages failed to induce proIL-1β mRNA levels in response to MDP (Fig. 5J). Notably, Rip3^-/-Fadd^-/- and Rip3^-/-caspase-8^-/- macrophages also were significantly affected in their ability to upregulate proIL-1β mRNA levels in response to MDP, albeit not to the extent of Nod2^-/- macrophages (Fig. 5J). Together, these results demonstrate an accessory role for FADD and caspase-8 in NF- κ B-dependent transcriptional upregulation of Nlrp3 and proIL-1β in cells primed with TLR and non-TLR agonists.

V体育2025版 - Role of caspase-8 in post-translational activation of the Nlrp3 inflammasome

We next addressed whether caspase-8 also regulated Nlrp3 inflammasome activation at the post-translational level. To formally explore this, wildtype macrophages were treated with the pharmacological caspase-8 inhibitor Ac-IETD-fmk prior to or after LPS-priming. As expected, both proIL-1β expression and secretion of mature IL-1β (Fig. 6A and 6B) were significantly induced in macrophages treated with LPS and ATP in the absence of the caspase-8 inhibitor. In line with our genetic evidence that caspase-8 mediated LPS-induced production of proIL-1β (Fig. 5H and Supplemental Fig. 2A), macrophages pre-incubated with the caspase-8 inhibitor prior to being exposed to LPS had reduced levels of proIL-1β expression relative to cells that were primed with LPS before incubation with the inhibitor (Fig. 6A). In agreement with caspase-8 regulating canonical Nlrp3 inflammasome activation post-transcriptionally, the caspase-8 inhibitor Ac-IETD-fmk markedly reduced ATP-induced proIL-1β maturation (Fig. 6A), and secretion of mature IL-1β (Fig. 6B) in these cells. Unlike proIL-1β, proIL-18 is constitutively expressed in macrophages and its levels were not reduced in cells receiving Ac-IETD-fmk prior to LPS (Fig. 6A). Nevertheless, IL-18 secretion in the culture medium was severely hampered in macrophages treated with Ac-IETD-fmk prior to ATP stimulation as well (Fig. 6C). In agreement with caspase-8 regulating Nlrp3 inflammasome activation at the post-translational level, the caspase-8 inhibitor prevented caspase-1 and caspase-8 maturation, regardless of whether it was provided before or after LPS-priming (Fig. 6D). In contrast, Ac-IETD-fmk failed to inhibit S. typhimurium-induced caspase-1 maturation (Fig. 6E), demonstrating that it did not target caspase-1 enzymatic activity directly. Our observation that Nlrp3 and ASC were required for LPS+ATP-induced caspase-8 maturation (Fig. 2C) suggests caspase-8 to interact with inflammasome components. We thus hypothesized that caspase-8 may induce canonical Nlrp3 inflammasome activation by directly processing procaspase-1. Indeed, recombinant caspase-8 potently matured procaspase-1 into p30, p20 and p10 subunits associated with active caspase-1 (Fig. 6F). Such role for caspase-8 appeared specific as recombinant caspase-3 failed to cleave procaspase-1 under conditions that allowed it to efficiently process the caspase-3 and -7 zymogens (Supplemental Fig. 4C and 4D). To further address whether endogenous caspase-8 mediated caspase-1 activation by the canonical Nlrp3 inflammasome, lysates of untreated and LPS+ATP-stimulated wildtype, Rip3^-/- and Rip3^-/-caspase-8^-/- macrophages were incubated with the caspase activity probe biotin-VAD-fmk that covalently links to the catalytic cysteine of enzymatically active caspases. The amount of active caspase-1 recovered from streptavidin beads was subsequently analyzed by immunoblotting to determine the levels of active caspase-1 retrieved from LPS+ATP-stimulated macrophages of the different genotypes. As expected, active caspase-1 was not recovered from lysates of untreated BMDMs, nor from streptavidin beads loaded with lysates of LPS+ATP-treated wildtype macrophages in the absence of biotin-VAD-fmk (Fig. 6G), demonstrating specificity of the experimental setup. Notably, both the large catalytic subunit (p20, lower panel) and full-length caspase-1 (p45, upper panel) were efficiently pulled down from LPS+ATP-stimulated wildtype and Rip3^-/- macrophage lysates, but not from lysates of LPS+ATP-treated Rip3^-/-caspase-8^-/- BMDMs (Fig. 6G). The observation that caspase-8 expression was necessary to recover biotin-VAD-labeled unprocessed and processed caspase-1 from LPS+ATP-stimulated macrophages suggests that it may promote caspase-1 activation through both cleavage and proximity-induced autoactivation of procaspase-1 in the Nlrp3 inflammasome. In agreement, confocal immunofluorescence analysis showed significant co-localization of caspase-1 and caspase-8 in macrophages that have been stimulated with LPS+ATP or infected with C. rodentium, but not in untreated cells (Fig. 6H). This observation is in line with a recent report demonstrating significant co-localization of caspase-8 and ASC in confocal micrographs of LPS+nigericin-treated caspase-1^-/-caspase11^-/- macrophages (19). Together, our results suggest FADD and caspase-8 interact with core components of the Nlrp3 inflammasome and promote activation of procaspase-1 in the complex.

In vivo role of FADD and caspase-8 in Nlrp3 inflammasome activation

To demonstrate the in vivo relevance of FADD and caspase-8 in Nlrp3 inflammasome signaling, cohorts of wildtype, Rip3^-/-, Rip3^-/-caspase-8^-/- and Rip3^-/-Fadd^-/- mice were challenged with a lethal dose of LPS. The LPS-induced endotoxemia model was selected because both caspase-1 (2) and caspase-11 (2, 38) are required for IL-1β production in LPS-challenged mice, whereas circulating IL-1α levels are regulated by caspase-11 independently of caspase-1 (2). Notably, circulating levels of both IL-1β (Fig. 7A) and IL-1α (Fig. 7B) were significantly reduced in LPS-challenged Rip3^-/-caspase-8^-/- and Rip3^-/-Fadd^-/- mice relative to those of LPS-treated wildtype and Rip3^-/- mice, thus extending the roles of FADD and caspase-8 in Nlrp3 inflammasome signaling to a relevant in vivo setting. The Nlrp3 inflammasome also plays a critical role in controlling C. rodentium replication in vivo (3). As in Nlrp3^-/- and caspase-1/11^-/- mice (3), bacterial burdens (Fig. 7C) and stool scores (Fig. 7D) were significantly elevated in C. rodentium-infected Rip3^-/-caspase-8^-/- and Rip3^-/-Fadd^-/- mice relative to those of infected wildtype and Rip3^-/- mice. Together, these results demonstrate that FADD and caspase-8 control pathological disease parameters in at least two in vivo mouse models of human disease that were previously attributed to Nlrp3 inflammasome activation.