The PYRIN Domain-Only Protein POP1 Inhibits Inflammasome

Article

The PYRIN Domain-only Protein POP1 Inhibits Inﬂammasome Assembly and Ameliorates Inﬂammatory Disease

Graphical Abstract Authors Lucia de Almeida, Sonal Khare, Alexander V. Misharin, ..., Hal M. Hoffman, Andrea Dorﬂeutner, Christian Stehlik

Correspondence a-dorﬂ[email protected] (A.D.), [email protected] (C.S.)

In Brief Inflammatory responses need to be tightly controlled to maintain homeostasis. Stehlik and colleagues demonstrate that the PYRIN domain-only protein POP1 inhibits ASC-containing inflammasome assembly and consequently caspase-1 activation, IL-1b and IL-18 release, pyroptosis, and the release of ASC particles in macrophages. Importantly, transgenic POP1 expression protects mice from systemic inflammation. Highlights d POP1 inhibits inflammasome-mediated responses to PAMPs and DAMPs d POP1 prevents IL-1b and IL-18 release and pyroptosis d POP1 prevents ASC danger particle-mediated response propagation to bystander cells d Transgenic POP1 expression protects mice from systemic inflammation

de Almeida et al., 2015, Immunity 43, 264–276 August 18, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.immuni.2015.07.018 Immunity Article

The PYRIN Domain-only Protein POP1 Inhibits Inﬂammasome Assembly and Ameliorates Inﬂammatory Disease

Lucia de Almeida,1 Sonal Khare,1 Alexander V. Misharin,1 Rajul Patel,1 Rojo A. Ratsimandresy,1 Melissa C. Wallin,1 Harris Perlman,1 David R. Greaves,2 Hal M. Hoffman,3 Andrea Dorﬂeutner,1,* and Christian Stehlik1,4,* 1Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA 2Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK 3Division of Rheumatology, Allergy, and Immunology, Department of Pediatrics, School of Medicine, University of California at San Diego (UCSD) and San Diego Branch, Ludwig Institute of Cancer Research, La Jolla, CA 92093, USA 4Robert H. Lurie Comprehensive Cancer Center, Interdepartmental Immunobiology Center and Skin Disease Research Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA *Correspondence: a-dorﬂ[email protected] (A.D.), [email protected] (C.S.) http://dx.doi.org/10.1016/j.immuni.2015.07.018

SUMMARY (ALR) and Nod-like receptor (NLR) families facilitate the activation of the pro-inflammatory caspase-1 within large macro- In response to infections and tissue damage, ASC- molecular protein complexes in macrophages, referred to as containing inflammasome protein complexes are inflammasomes. Inflammasomes promote the proteolytic matu- assembled that promote caspase-1 activation, ration and release of the leaderless pro-inflammatory cytokines IL-1b and IL-18 processing and release, pyroptosis, interleukin (IL)-1b and IL-18 and the induction of pyroptotic cell a and the release of ASC particles. However, excessive death, which causes the release of IL-1 and HMGB1 (Martinon or persistent activation of the inflammasome causes et al., 2002; Khare et al., 2010; Wen et al., 2013). Inflammasomes are composed of a sensory PRR, which is linked to caspase-1 via inflammatory diseases. Therefore, a well-balanced the adaptor protein ASC (Srinivasula et al., 2002; Stehlik et al., inflammasome response is crucial for the mainte- 2003a; Martinon et al., 2002). This complex exhibits specific nance of homeostasis. We show that the PYD-only protein-protein interactions, which are mediated by homotypic protein POP1 inhibited ASC-dependent inflamma- PYRIN domain (PYD) interactions between PRRs and ASC and some assembly by preventing inflammasome nucle- by homotypic caspase recruitment domain (CARD) interactions ation, and consequently interfered with caspase-1 between ASC and pro-caspase-1. Within this protein complex, activation, IL-1b and IL-18 release, pyroptosis, and pro-caspase-1 is activated by induced proximity-mediated olig- the release of ASC particles. There is no mouse omerization (Martinon et al., 2002; Lu et al., 2014). The underlying ortholog for POP1, but transgenic expression of assembly mechanism of this ternary complex has recently been human POP1 in monocytes, macrophages, and delineated and indicates that PRR activation triggers prion-like, dendritic cells protected mice from systemic inflam- self-perpetuating ASC polymerization into filaments, which is initialized by PRR-induced ASCPYD nucleation (Cai et al., 2014; mation triggered by molecular PAMPs, inflamma- Lu et al., 2014; Franklin et al., 2014; Dorfleutner et al., 2015). some component NLRP3 mutation, and ASC danger PRRs localize to the end of the hollow ASC filaments and allow particles. POP1 expression was regulated by TLR efficient self-propagation of the ASCPYD, whereby the ASCCARD and IL-1R signaling, and we propose that POP1 pro- is flexibly linked to the outside of the filaments, allowing recruit- vides a regulatory feedback loop that shuts down ment and clustering of pro-caspase-1 (Lu et al., 2014). Eventu- excessive inflammatory responses and thereby pre- ally, these filaments can assemble into a spherical structure, vents systemic inflammation. where caspase-1 is placed in the hollow core (Man et al., 2014). Thus, the PYD is essential for inflammasome assembly after activation of NLRP3 and other PYD-containing PRRs, and INTRODUCTION disassembly of this ternary signaling complex is required for its termination. NLRP3 is one of the best-studied inflammasome- Inflammation is an essential and tightly controlled process activating PRRs and the NLRP3 inflammasome is regulated by initiated by the innate immune system in response to infection a two-step mechanism, referred to as priming and activation and tissue damage and is responsible for pathogen clearance, (Khare et al., 2010). After priming with LPS or other NF-kB- wound healing, and restoring homeostasis. It is triggered by inducing signals, NLRP3 is activated in a second step by a the sensing of pathogen-associated molecular patterns (PAMPs) wide variety of PAMPs that cause potassium (K+) efflux, or damage-associated molecular patterns (DAMPs or danger including exogenous ATP, nigericin, and uric acid crystals, as signals) by germline-encoded pattern recognition receptors well as environmental and endogenous mediators released (PRRs). Particularly, cytosolic PRRs of the AIM2-like receptor in response to stress and tissue damage (Khare et al., 2010;

264 Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. Mun˜ oz-Planillo et al., 2013). Besides inflammasome-linked two different shRNAs causing elevated IL-1b release in response cytokines, oligomeric ASC particles are also released and to LPS and POP1 silencing) was confirmed by qPCR (Figure 1A). phagocytized by neighboring cells to perpetuate inflammasome Similarly, siRNA-mediated silencing of POP1 in primary human responses (Franklin et al., 2014; Baroja-Mazo et al., 2014). Over- macrophages resulted in elevated IL-1b and IL-18, but not all, inflammasomes play an essential role in host defense. IL-6, release in response to LPS. POP1 silencing was again However, impaired inflammasome activation results in failure confirmed by qPCR (Figure 1B). Conversely, THP-1 cells stably to restrict colitogenic microbiota species and subsequently expressing GFP-POP1 displayed diminished IL-1b secretion in promotes metabolic dysfunction. In contrast, constitutive inflam- response to LPS and also in response to non-canonical inflammasome activation through disease-associated mutations in masome activation with LPS transfection or cytosolic delivery NLRP3 promotes excessive IL-1b release and causes inflamma- of LPS with cholera toxin subunit B (CTB) (Figure 1C). Cytokine tory diseases, including Cryopyrinopathies (or Cryopyrin-associ- release after non-canonical inflammasome activation requires ated periodic syndromes; CAPS) (Henao-Mejia et al., 2012; NLRP3. Consistently, POP1 blocked IL-1b release after NLRP3 Hoffman and Brydges, 2011). Therefore, a controlled and well- inflammasome activation with nigericin, calcium pyrophosphate balanced inflammasome response is essential for maintaining dehydrate (CPPD) crystals, or K+ depletion. Furthermore, POP1 homeostasis. However, the molecular mechanisms regulating blocked IL-1b release after AIM2 and NLRC4 activation with assembly and disassembly of inflammasomes are largely un- poly(dA:dT) or flagellin transfection, respectively (Figure 1D), known. We discovered a family of PYD-only proteins (POPs) and POP1 expression was confirmed by qPCR (Figure 1E). that are encoded in humans but not in mice (Stehlik and Dorfleut- Hence, POP1 inhibits inflammasome-dependent cytokine ner, 2007; Khare et al., 2014; Stehlik et al., 2003b; Dorfleutner release. Comparable results were also obtained for myc-tagged et al., 2007a, 2007b; Bedoya et al., 2007; Johnston et al., POP1, thus establishing that the GFP tag does not affect the 2005). We recently demonstrated that POP3 functions as a spe- function of POP1 (Figure S1A). cific inhibitor for ALR inflammasomes (Khare et al., 2014). POP3 binds to the PYD of ALRs, but not to ASC or NLRs, and thereby POP1 Impairs ASCPYD Nucleation and Caspase-1 prevents ALR interactions with the inflammasome adaptor ASC. Activation in Human Macrophages However, the function of endogenous POP1 (PYD-containing 1, Caspase-1 activation in canonical inflammasomes of macro- PYDC1) has not yet been established. POP1 is highly similar to phages is required for IL-1b and IL-18 release and for pyroptosis. the PYD of ASC and in vitro overexpression experiments showed Therefore, we determined caspase-1 activity in the presence that POP1 interacts with the PYD of ASC; however, its role in vivo and absence of POP1. Treatment of Pam3CSK4-primed shPOP1 is unknown. knockdown cells with nigericin resulted in elevated active Here we report that POP1 functions in an IL-1b-induced regula- caspase-1 p10 and mature IL-1b in culture supernatants when tory loop to inhibit the assembly and consequently the activity compared to control THP-1 cells (Figure 2A). Further, intracel- of ASC-containing inflammasomes by preventing nucleation of lular caspase-1 activation was also augmented in shPOP1 ASC. POP1 also prevented the release of oligomeric ASC danger THP-1 compared to control THP-1, as shown by caspase-1 particles, and incorporation of POP1 into ASC particles rendered FLICA assay (Figure 2B). Conversely, expression of GFP-POP1 them inactive, thereby preventing self-perpetuation of inflam- in primed THP-1 cells showed drastically reduced caspase-1 masome responses in neighboring cells. To demonstrate the activity in response to nigericin compared to GFP THP-1 cells importance of inflammasomes particularly in monocytes and (Figure 2C). Consequently, POP1 expression also caused macrophages, we engineered mice that usually lack POP1 to spe- reduced pyroptosis, as determined by LDH release (Figure 2D). cifically express transgenic POP1 in monocytes, macrophages, TLR-mediated priming is necessary for NLRP3 inflammasome and DCs. We used the NLRP3 inflammasome as an example to activation (Bauernfeind et al., 2009; Juliana et al., 2012; Schroder demonstrate that transgenic POP1 expression in the monocyte- et al., 2012; Lin et al., 2014). However, stable POP1 expression in macrophage-DC lineage was sufficient to blunt excessive sys- THP-1 cells did not affect transcription of IL1B (Figure S1B) and temic inflammation in response to PAMPs, ASC danger particles, resulted in mild increased transcription of NLRP3 or PYCARD and CAPS-associated mutations. Interestingly, CAPS patients (ASC) in response to LPS (Figure S1C), and contrary to transient exhibited reduced POP1 expression, suggesting that impaired overexpression in epithelial cell lines (Stehlik et al., 2003b), stable POP1 expression might contribute to excessive inflammasome- POP1 expression also did not affect phosphorylation of IkBa driven inflammation. Our data reveal a mechanism by which hu- (Figure S1D). Furthermore, silencing of POP1 in THP-1 cells man inflammasome assembly is regulated and detail how healthy also did not affect transcription of PYCARD and NLRP3 (Fig- tissue puts the brakes on inflammasome-induced systemic ure S1E). Thus, POP1 seems to directly regulate inflammasome inflammation. In addition, our results emphasize the crucial role assembly or activation in macrophages. Recruitment of ASC to of the monocyte-macrophage-DC lineage in this response. upstream sensors is essential for inflammasome activation and we hypothesized that POP1 interferes with this interaction. RESULTS POP1 specifically bound to endogenous ASC, but not to the PYD-containing PRRs NLRP3 and AIM2 in LPS-primed THP-1 POP1 Inhibits Inflammasome-Mediated Cytokine cells (Figure 2E). Binding of ASC to NLRP3 induces ASCPYD Release in Human Macrophages nucleation, which provides the oligomeric platform essential To investigate the role of POP1 in inflammasome signaling, we for caspase-1 activation (Cai et al., 2014; Lu et al., 2014). There- established stable shRNA-mediated POP1 silencing in human fore, we hypothesized that POP1 prevents ASCPYD-NLRP3PYD monocytic THP-1 cells (shPOP1 THP-1). POP1 knockdown (via interactions and thus ASCPYD nucleation (Chu et al., 2015).

Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. 265 Figure 1. POP1 Inhibits the NLRP3 Inflammasome in Human Macrophages (A) THP-1 cells stably expressing shRNAs targeting POP1 (POP1#1, #2) or a scrambled Ctrl were analyzed by real-time PCR for POP1 transcripts and for IL-1b release in culture supernatants (SN) in untreated cells (Ctrl) or in response to LPS. (B) Primary human macrophages transfected with a scrambled Ctrl or a POP1-specific siRNA were analyzed for POP1 transcripts by real-time PCR and culture SN for IL-1b, IL-18, and IL-6 release in response to LPS. (C and D) Culture SN from THP-1 cells stably expressing GFP or GFP-POP1 were analyzed for IL-1b release by ELISA in untreated cells (Ctrl) or in response to LPS treatment, LPS transfection, or incubation with LPS complexed with CTB (C) and nigericin or CPPD treatment or K+ depletion in LPS-primed cells or transfection of poly(dA:dT) or flagellin (D). (E) Real-time PCR of POP1 transcripts in above cells. Data are representative of two (A) or three (B–E) replicates; error bars represent SEM. *p = 0.0009, **p = 0.0128, ***p < 0.0001, ****p < 0.0001 in (A); *p = 0.0284, **p = 0.0315, ***p = 0.0001 in (B); *p < 0.0001, **p < 0.0001, ***p < 0.0001 in left panel of (D); *p < 0.0001, **p < 0.0001 in right panel of (D); and *p = 0.0024, **p = 0.0022, ***p = 0.0139 in (E); all two-tailed unpaired t test. See also Figure S1A.

Indeed, the nigericin-induced interaction of NLRP3 and ASC we generated GFP-POP1 transgenic (TG) mice. Caspase-1 is was abolished in LPS-primed GFP-POP1 THP-1 cells, indicating essential for IL-1b and IL-18 release in monocytes and macro- that POP1 might prevent NLRP3-mediated ASC nucleation phages and we detected POP1 expression in CD68+ macro- (Figure 2F). Upon ASC nucleation, the ASCPYD polymerizes in phages in inflamed lung tissue (Figure S2A). Therefore, to limit a prion-like self-perpetuating manner that further promotes POP1 expression to macrophages, we used the hCD68/IVS-1 caspase-1 activation (Lu et al., 2014). Because ASC-ASC, promoter/enhancer (Iqbal et al., 2014; Khare et al., 2014; ASC-POP1, and ASC-NLRP3 interactions utilize the same key Gough et al., 2001) for GFP-POP1 expression in TG mice. We residues (Vajjhala et al., 2012), POP1 binding to the ASCPYD confirmed POP1 expression specifically in CD68-POP1 TG could also directly prevent ASCPYD self-polymerization. Surpris- but not in wild-type (WT) mice by qPCR analysis of whole blood ingly, POP1 expression in HEK293 cells did not impair the cell RNA (Figure S2B). Accordingly, we also detected POP1 ASCPYD self-interaction, as determined by in coIP experiments expression in transgenic POP1 bone-marrow-derived macro- between Myc-ASC and HA-ASCPYD (Figure S1F). However, phages (BMDMs) by immunoblot (Figure 3A). Human and overexpression in HEK293 cells might lead to inaccurate results. mouse ASCPYD have a high degree of homology (Figure S2C) Our data indicate that, in the context of PRR-mediated inflam- and therefore it was not surprising that, similar to human masome activation, POP1 prevents the essential ASC nucleation ASC in THP-1 cells, POP1 also interacted with mouse ASC, step in human macrophages, which is required for inflamma- but not NLRP3 or AIM2 in BMDMs (Figure 3B). ASC polymeri- some assembly. zation can be captured by non-reversible cross-linking and functions as a read-out for inflammasome activation (Fer- POP1 Prevents Caspase-1 Activation and Cytokine nandes-Alnemri et al., 2007), which was markedly reduced in Release in Mouse Macrophages LPS-primed and ATP-treated POP1 BMDMs compared to WT Because the genes for all POPs, including POP1, are absent BMDMs (Figure 3C). Consequently, POP1 BMDMs lacked in mice (Stehlik and Dorfleutner, 2007; Khare et al., 2014), active caspase-1 p10 and mature IL-1 b in culture supernatants

266 Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. Figure 2. POP1 Impairs Inﬂammasome Nucleation in Human Macrophages

(A and B) Pam3CSK4-primed THP-1 cells stably expressing shRNA#1 targeting POP1 or a scrambled Ctrl were treated with nigericin and total cell lysates (TCL) and culture supernatants (SN) were analyzed for caspase-1 and IL-1b by immunoblot (A) or active caspase-1 by flow cytometry (B). (C) LPS-primed THP-1 cells expressing GFP or GFP-POP1 were treated with nigericin and active caspase-1 was determined by flow cytometry. (D) LPS-primed THP-1 cells expressing GFP or GFP-POP1 were treated with nigericin or CPPD crystals, and LDH release was quantified in culture supernatants. *p = 0.001, **p = 0.0004 by two-tailed unpaired t test. (E) Interaction of GST-POP1 with endogenous ASC in THP-1 TCL using GST as negative control and showing 10% TCL as input. Asterisk (*) marks a degradation product of GST-POP1. (F) Immunoprecipitation (IP) of proteins with antibodies to ASC or control immunoglobulin G (IgG) from LPS-primed and nigericin-treated THP-1 cells stably expressing GFP or GFP-POP1, followed by immunoblot analysis alongside TCL. Data are representative of two replicates; error bars represent SEM. See also Figures S1B–S1F. of LPS/ATP-treated cells to a similar extent as the caspase-1 activation (Figure 3J), but did not reveal any altered LPS-induced inhibitor zYVAD-fmk (Figure 3D). Reduced caspase-1 activity activation of NF-kB, p38, JNK, or ERK (Figure S2D) or altered was also detected by flow cytometry in intact cells (Figure 3E), transcription of Il1b, Il18, Pycard, and Nlrp3 (Figure S2E), indicating that POP1 also inhibits caspase-1 activation in ruling out POP1 effects on inflammasome priming in mouse mouse macrophages. macrophages. Collectively, these data indicate that POP1 im- As expected from impaired caspase-1 activation, LPS/ATP- pairs assembly of the NLRP3 inflammasome in human and treated POP1 BMDMs also displayed significantly reduced mouse macrophages by impairing the PRR-mediated nucleation levels of IL-1b, IL-1a, and IL-18 in culture supernatants by ELISA. of ASC and consequently the release of inflammasome-depen- However, secretion of TNF-a, which occurs independently of dent cytokines. caspase-1, was not affected (Figure 3F). Significantly, reduced IL-1b release in POP1 BMDMs was comparable to PycardÀ/À Monocyte-Macrophage-DC-Specific Expression of BMDMs and Nlrp3À/À BMDMs (Figure 3G). K+ efflux is the unify- POP1 Ameliorates LPS-Induced Peritonitis ing mechanism of NLRP3 activation in BMDMs (Mun˜ oz-Planillo NLRP3 senses endogenous danger signals and PAMPs and et al., 2013), and culturing POP1 BMDMs in K+-free medium promotes inflammatory responses that can be detrimental showed impaired IL-1b release compared to WT BMDMs (Fig- to the host. We therefore used the NLRP3 inflammasome to ure 3H). Similarly, peritoneal POP1 macrophages (POP1 PMs) investigate the role of POP1 in vivo. To initially characterize showed impaired IL-1b release in response to activation of the CD68-POP1 TG mice, we analyzed peripheral blood, which ASC-dependent inflammasomes containing NLRP3 with ATP, revealed POP1 expression selectively in monocytes (Figures AIM2 with poly(dA:dT), and NLRC4 with flagellin (Figure 3I). 4AandS3), with equal expression in classical Ly6ChiCD43–, in- POP1 BMDMs also showed reduced LDH release, and thus py- termediate Ly6CintCD43+, and non-classical Ly6CloCD43+ roptosis, when compared to WT BMDMs in response to NLRP3 monocytes (Figure S4A). POP1 was also expressed in myeloid

Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. 267 Figure 3. POP1 Inhibits the NLRP3 Inflammasome in Mouse Macrophages (A) Immunoblot of POP1 expression in BMDMs using a GFP antibody. (B) Interaction of GST-POP1 with endogenous ASC from LPS-primed BMDM total cell lysates (TCL) using GST as negative control and showing 10% TCL as input. Asterisk (*) marks a degradation product of GST-POP1. (C) Immunoblot analysis of ASC polymerization (oligomerization) in untreated or LPS/ATP-treated wild-type (WT) and POP1 BMDMs after cross linking of pellets (P) and in TCL. (D) Immunoblot analysis of caspase-1 and IL-1b in culture SN of LPS/ATP-treated WT and POP1 BMDMs. Pro-caspase-1 expression in TCL confirms equal loading. (E) Flow cytometric quantification of active caspase-1 in WT and POP1 BMDMs in response to LPS/ATP. (F) Analysis of culture supernatants (SN) for IL-1b, IL-18, IL-1a, and TNF-a by ELISA in LPS/ATP-treated WT and POP1 BMDMs. (G) Analysis of culture SN for IL-1b by ELISA in LPS-primed and ATP-treated WT, POP1, PycardÀ/À, and Nlrp3À/À BMDMs. (H) LPS-primed WT and POP1 BMDMs cultured in K+ depleted medium (I) WT and POP1 PMs treated with LPS/ATP or transfected with flagellin or poly(dA:dT). (J) LPS-primed WT and POP1 BMDMs were treated with nigericin or CPPD crystals and released LDH in culture SN was quantified. Data are representative of four (A, H, I) or two (B–G, J) replicates; error bars represent SEM. *p = 0.0027, **p = 0.003, ***p = 0.0403 in (F); *p = 0.0214, **p = 0.0043, ***p = 0.0052 in (G); *p = 0.01 in (H); *p = 0.0009, **p < 0.0001 in (I); and *p < 0.0001 in (J); all two-tailed unpaired t test. See also Figure S2. precursors (MPs), macrophage and DC precursors (MDPs), phage-DC-specific POP1 expression was also observed in and common DC precursors (CDPs) in the bone marrow (Fig- other tissues, with no detectable expression in CD45– cells ures S4B and S4C), large peritoneal macrophages (LPMs), (Figure S5C and data not shown). Collectively, these results small peritoneal macrophages (SPMs), and peritoneal DCs demonstrate POP1 expression in the monocyte-macrophage- (Figures S4D and S4E), as well as in splenic red pulp macro- DC lineage. phages (RPMs), monocytes, and CD11b+ DCs, but not plasma- Caspase-11 is responsible for LPS- and Gram-negative- cytoid DCs (pDCs) (Figures S5A and S5B). Monocyte-macro- bacteria-induced lethal shock, but ASC and NLRP3 are both

268 Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. Figure 4. Monocyte-Macrophage-DC-Specific Expression of POP1 Ameliorates LPS-Induced Peritonitis (A) Analysis of POP1 expression by flow cytometry in peripheral blood cell populations from wild-type (WT) and CD68-POP1 (POP1) mice. Data are representative of three replicates. (B) Quantification of MPO activity by in vivo imaging in mice 3 hr after i.p. injection of PBS or E. coli LPS (2.5 mg/kg body weight) in WT (n = 6) and CD68-POP1 (n = 5) mice. (C and D) Endotoxic shock was induced by i.p. injection of E. coli LPS (20 mg/kg body weight) and body temperature (C) and survival (D) was determined in WT (n = 5) and CD68-POP1 (n = 5) mice. (E) ELISA of IL-1b and TNF-a in the serum of WT (n = 5) and CD68-POP1 (n = 5) mice 3 hr after i.p. E. coli LPS challenge (20 mg/kg body weight). *p = 0.0036 by two-tailed unpaired t test in (B); *p < 0.005, **p < 0.005 by two-way ANOVA + Bonferoni posttest in (C); *p < 0.005 by asymmetrical Log-rank Mantel- Cox survival test in (D); *p = 0.0392 by two-tailed unpaired t test in (E); error bars represent SEM. See also Figures S3–S5. necessary for amplifying this response to LPS in vivo (Kayagaki Monocyte-Macrophage-DC-Specific Expression of et al., 2011). Accordingly, PycardÀ/À and Nlrp3À/À mice are POP1 Ameliorates CAPS protected from LPS-induced lethality in response to moderate Because sepsis is a rather complex disease, we investigated a LPS doses (Mariathasan et al., 2004, 2006; Kayagaki et al., disease model, for which the pathology is absolutely dependent 2011). NLRP3 inflammasome-released IL-1b is essential for on the NLRP3 inflammasome. Cryopyrinopathies (or Cryopyrin- neutrophil recruitment during sterile inflammation (McDonald associated periodic syndromes; CAPS) are caused by mutations et al., 2010). Therefore, we injected WT and CD68-POP1 TG in NLRP3 (Hoffman et al., 2001), are therefore directly linked to mice i.p. with a low dose of LPS and determined neutrophil the NLRP3 inflammasome, and can be recapitulated in mice infiltration 3 hr after LPS challenge by quantifying myeloperox- by knocking-in CAPS-associated NLRP3 mutations (Brydges idase (MPO) activity in vivo. Contrary to PBS, injection of LPS et al., 2009, 2013; Meng et al., 2009). We employed a mouse recruited a substantial number of neutrophils into the perito- model for Muckle Wells syndrome (MWS), where floxed neal cavity, which was completely abolished in CD68-POP1 Nlrp3A350V, corresponding to human NLRP3A352V, is expressed TG mice (Figure 4B). Consequently, CD68-POP1 TG mice exclusively in myeloid cells in the presence of lysozyme M-Cre experienced significantly less hypothermia (Figure 4C) and (CreL) (Brydges et al., 2009, 2013). Nlrp3A350V/+ CreL mice were significantly more protected from a lethal LPS dose develop systemic inflammation that affects multiple organs, (Figure 4D). Compared to 100% lethality in WT mice, only display characteristic skin inflammation, and die within 2 weeks 30% of CD68-POP1 TG mice died within 96 hr, which is similar of birth. This phenotype is caused by excessive IL-1b and IL-18 to PycardÀ/À mice (Mariathasan et al., 2004). Consistent with release as well as pyroptosis (Brydges et al., 2009, 2013). reduced neutrophil infiltration and increased survival, serum Nlrp3A350V/+ CreL mice had inflammatory skin abscesses and le- IL-1b was reduced, but TNF-a levels remained unchanged sions shortly after birth, which developed into scaling erythema (Figure 4E), indicating that expression of POP1 in the mono- (Brydges et al., 2009), but Nlrp3A350V/+ CreL CD68-POP1 mice cyte-macrophage-DC lineage is sufficient to impair inflamma- did not display this phenotype (Figure S6A). Histological analysis some activation in response to PAMPs in vivo, thereby block- revealed that POP1 expression prevented leukocytic infiltrates in ing the secretion of IL-1b and ameliorating an excessive host multiple organs, including the liver and the skin, and also response. restored skin architecture (Figure 5A). Also, the systemic IL-1b

Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. 269 Figure 5. Monocyte-Macrophage-DC-Specific Expression of POP1 Ameliorates CAPS (A) H&E staining of skin sections at day 8 in Nlrp3A350V/+ CreL CD68-POP1 and Nlrp3A350V/+ CreL mice. Scale bars represent 100 mm (original) and 10 mm (magnification). (B) Serum IL-1b levels in 8-day-old Nlrp3A350V/+ CreL (n = 4) and Nlrp3A350V/+ CreL CD68-POP1 (n = 5) mice. (C) Body weight of WT (n = 15), Nlrp3A350V/+ CreL CD68-POP1 (n = 11), and Nlrp3A350V/+ CreL (n = 11) mice. (D) Survival of WT (n = 11), Nlrp3A350V/+ CreL CD68-POP1 (n = 10), and Nlrp3A350V/+ CreL (n = 8) mice. (E) POP1 mRNA expression (relative to control) in whole blood drawn from healthy controls (n = 23), CINCA (n = 2), and MWS (n = 5) patients (GEO: GSE40561). Data are representative of two replicates in (A); error bars represent SEM. *p = 0.0394 in (B); *p < 0.005 by non-linear regression fit and Kruskal-Wallis one-way ANOVA (p = 0.0021) with Dunn’s Multiple Comparison post test in (C); *p < 0.0001 by asymmetrical Log-rank Mantel-Cox survival test in (D); *p = 0.0359 by two-tailed unpaired t test (E). See also Figures S6A and S6B. levels were reduced (Figure 5B). Significantly, POP1 expression which are released from macrophages through inflamma- rescued the severe growth delay (Figure 5C) and prevented mor- some-dependent pyroptosis and act as danger signals on tality of Nlrp3A350V/+ CreL mice from multi-system organ failure neighboring cells (Baroja-Mazo et al., 2014; Franklin et al., (Figure 5D). Because expression of POP1 efficiently ameliorated 2014). POP1 prevented ASC nucleation and the subsequent CAPS, we analyzed POP1 expression in a previously published ASC polymerization, caspase-1 activation, and pyroptosis, patient cohort (Boisson et al., 2012) and found that CAPS patients which are all required for the ASC particle response. Hence, displayed significantly lower POP1 expression compared to culture supernatants from LPS-primed and nigericin- or ATP- healthy controls (Figure 5E). We also observed the same trend treated GFP THP-1 cells and WT BMDMs contained ASC, in leukocytes from two large, independent septic patient cohorts, but supernatants from GFP-POP1 THP-1 cells (Figure 6A) or when compared to healthy controls (Figure S6B; Tang et al., POP1 BMDMs (Figure 6B) did not contain any ASC. Particu- 2008; Wong et al., 2009), although the reduced POP1 expression larly, the release of polymeric ASC was inhibited by POP1 in patients was less pronounced. These findings demonstrate (Figure 6C). Extracellular ASC particles are phagocytized by that POP1 inhibits excessive NLRP3 inflammasome activity macrophage and activate caspase-1 in an NLRP3- and ASC- in vivo and thereby ameliorates auto-inflammatory disease. We dependent process (Baroja-Mazo et al., 2014; Franklin et al., also identified reduced POP1 expression in CAPS patients, which 2014). FACS-purified ASC-GFP particles (Figure S6C) induced might circumvent appropriate inflammasome control. IL-1b release in LPS-primed GFP THP-1 cells, but not in GFP- POP1 THP-1 cells (Figure 6D), suggesting that POP1 prevents POP1 Prevents ASC Particle Release and Ameliorates ASC nucleation downstream of NLRP3 activation. POP1 is an ASC Particle-Induced Inflammation ASC binding protein, which can be incorporated into ASCPYD Recently, polymerized ASC particles were detected in the filaments (Figure S1F). This can result in reduced ASCCARD serum of active CAPS patients (Baroja-Mazo et al., 2014), density and subsequently prevent caspase-1 nucleation and

270 Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. Figure 6. Expression of POP1 Prevents ASC Particle Release and Ameliorates ASC Particle-Induced Inflammation (A) LPS-primed THP-1 cells expressing GFP or GFP-POP1 were treated with nigericin and released ASC determined by immunoblot in total cell lysates (TCL) and culture supernatants (SN). (B) Wild-type (WT) and POP1 BMDMs were treated with ATP and released ASC determined by immunoblot in TCL and SN. (C) Immunoblot analysis of ASC polymerization in untreated or LPS/ATP-treated WT and POP1 BMDMs after cross linking of pellets (P) and in TCL. (D) ASC-GFP particles were FACS purified and imaged by immunofluorescence microscopy, showing the characteristic filamentous structure. Scale bar represents 2 mm. Culture SN from THP-1 cells stably expressing GFP or GFP-POP1 were analyzed for IL-1b release by ELISA in LPS-primed cells before (Ctrl) and after treatment with 1 3 103 FACS-purified ASC-GFP particles. (E) Mixed ASC-GFP/RFP-POP1 particles were FACS purified as above, showing comparable size and overall structure. Scale bar represents 2 mm. Culture SN from THP-1 cells were analyzed for IL-1b release by ELISA in LPS primed cells before (Ctrl) and after treatment with 1 3 103 FACS-purified ASC-GFP and ASC-GFP/RFP-POP1 particles. (F) Quantification of MPO activity by in vivo imaging in WT and CD68-POP1 mice 4 hr after i.p. injection of PBS or 1 3 105 FACS-purified ASC-GFP particles (n = 2/genotype). (G) ELISA of total IL-1b in the peritoneal cavity from above mice 4 hr after ASC-GFP particle challenge. (H) 30 min after i.p. injection of TAT-GFP or TAT-POP1 (40 mg/kg), mice were i.p. injected with LPS (2.5 mg/kg). After 1 hr, MPO activity was determined by in vivo imaging and the MPO luminescent signal quantified in LPS-injected WT mice pre-treated with TAT-GFP (n = 2) and TAT-POP1 (n = 3) mice. Data are representative of two (A–E) replicates; error bars represent SEM. *p = 0.0488 in (D); *p = 0.0022 in (E); *p = 0.0488 in (F); *p = 0.0038 in (G); all two-tailed unpaired t test. See also Figures S6C–S6E. activation (Lu et al., 2014). To directly prove that POP1 incorpo- signaling pathways (Schwarze et al., 1999), and so we pro- ration into ASC particles renders them inactive, we generated duced recombinant POP1 and GFP as a control fused to the mixed ASC-GFP/RFP-POP1 particles (Figure S6C), which, in cell-penetrating HIV TAT sequence (TAT-POP1 and TAT-GFP) contrast to ASC-GFP particles, failed to cause IL-1b release (Figure S6D). TAT-GFP was efficiently taken up by PMs after in THP-1 cells (Figure 6E). Importantly, i.p. injection of ASC- i.p. injection in vivo (Figure S6E), and injection of TAT-POP1, GFP particles into WT mice resulted in neutrophil recruitment but not TAT-GFP, ameliorated LPS-induced peritonitis (Fig- (Figure 6F) and IL-1b release (Figure 6G), which was substan- ure 6H), reminiscent to transgenic POP1 expression. Collec- tially reduced in CD68-POP1 TG mice. tively, these data show that POP1 also blocks the release of Based on the reduced POP1 expression in CAPS patients ASC danger particles and consequently propagation of sec- and its inflammasome inhibitory function in macrophages, we ondary inflammasome responses in neighboring cells and designed a proof-of-concept treatment approach targeting that delivery of POP1 might be used as the basis for future ASC inflammasomes. Cell-penetrating peptides are frequently therapeutic approaches in patients with CAPS and other employed for the delivery of molecules targeting intracellular inflammasomopathies.

Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. 271 Figure 7. POP1 Expression Is Regulated by TLRs and IL-1b (A) POP1 and HMGB1 transcripts were measured by real-time PCR in LPS-treated primary human macrophages. (B) POP1 and PYCARD mRNA expression in leukocytes from LPS-infused human subjects. (C) POP1 mRNA expression in primary human macrophages after 4 hr IL-1b treatment. Data are representative of two (A, C, D) and eight (B) replicates; error bars represent SEM. *p = 0.0097, **p = 0.0443 (A); *p = 0.0284 (B); and *p = 0.0266 (C); all two-tailed unpaired t test. See also Figure S7.

POP1 Functions in an Inducible Inflammasome determined by measuring IL-18 levels 4 hr after i.p. LPS injection Regulatory Loop (R2 = 0.9316) (Figure S7F). Overall, our results demonstrate that Overwhelming evidence supports the necessity for a balanced POP1 regulates the inflammasome-mediated IL-1b and IL-18 inflammasome response to maintain tissue homeostasis (He- release, which in turn regulates POP1 expression. nao-Mejia et al., 2012). Therefore, we hypothesized that POP1 expression needs to be tightly regulated. We observed LPS- DISCUSSION induced late response gene expression of POP1 in human macrophages (Figure 7A) and THP-1 cells (Figure S7A), but in ASC-containing inflammasomes are responsible for cytokine contrast to POP3, POP1 was not upregulated in response to release through canonical and non-canonical inflammasomes IFN-b (Khare et al., 2014), which emphasizes the distinct function (Mariathasan et al., 2004; Kayagaki et al., 2011, 2013) and of individual POPs. Notably, POP1 expression peaked right consequently play a central role in facilitating the beneficial in- before the inducible expression of HMGB1, which is released flammatory responses to clear pathogen infections and initiate through pyroptosis (Lamkanfi et al., 2010; Willingham et al., wound healing after tissue damage. However, uncontrolled in- 2009) and contributes to inflammatory disease (Harris et al., flammasome responses cause inflammatory disease through 2012). Thus, the late response expression of POP1 potentially excessive cytokine release (Strowig et al., 2012; Hoffman and enables inflammasome functions in early host defense and might Brydges, 2011). Thus, a well-balanced inflammasome response provide a mechanism to counter excessive release of late medi- is crucial for maintaining homeostasis. Therefore, inflammasome ators that perpetuate systemic inflammation, such as HMGB1. regulatory proteins probably exist to maintain an appropriate Importantly, this LPS-inducible expression of POP1 was also level of activity and in particular to limit inflammasome activity observed in leukocytes isolated from human subjects after in vivo during the resolution phase of these responses. We proposed LPS infusion (Figure 7B; Calvano et al., 2005). TLR4 is required that POP1 is one of these proteins. Assembly of the inflamma- for inducible POP1 expression by LPS, as indicated by the fact some platform is initiated by nucleation of ASC through PYD in- that a TLR4 inhibitor reduced POP1 expression (Figure S7B). teractions with PYD-containing PRRs followed by prion-like ASC TLR signaling leads to NF-kB activation and blocking NF-kB self-polymerization (Cai et al., 2014; Franklin et al., 2014; Lu also reduced POP1 transcription (Figure S7B). Inducible POP1 et al., 2014; Chu et al., 2015). Hence, the PYD plays a crucial transcription was caused not only by TLR4, but also by TLR2 role in inflammasome assembly. We recently demonstrated activation by Pam3CSK4 (Figure S7C). IL-1R and IL-18R share that POP3 selectively regulates ALR inflammasomes by binding signaling components with TLRs and accordingly POP1 expres- to the PYD of ALRs but not to ASC, which is sufficient to sion was also elevated in human macrophages after IL-1b prevent ALR-ASC interactions (Khare et al., 2014). In contrast (Figure 7C) and IL-18 (Figure S7D) treatment. Thus, TLR, IL-1R, to POP3, our data support a model where POP1 acted more and IL-18R engagement contributes to NF-kB-dependent broadly by binding to the ASCPYD, which prevented ASC interac- inducible transcription of POP1. At the transcriptional level, we tion with PYD-containing PRRs and consequently the crucial observed 4-fold increased POP1 expression in human macro- ASC nucleation event. Similarly, POP1 prevented exogenous phages after TLR or IL-1R stimulation and our stable GFP-POP1 ASC particle responses, which are only partially dependent on THP-1 and Myc-POP1 THP-1 cells showed 3-fold and 7-fold NLRP3, but depend on ASC-nucleated ASC polymerization more POP1 compared to baseline expression levels, respec- (Cai et al., 2014; Franklin et al., 2014; Lu et al., 2014). Moreover, tively (Figures 1E, S1A, and S1E). Hence, our stable cell lines mixed particles composed of ASC and POP1 were inactive, closely mimicked the induced POP1 expression levels, which probably by reducing the necessary ASC-CARD density were therefore also sufficient to impair inflammasome activity. required for caspase-1 activation. Thus, POP1 expression pre- At the protein level, POP1 PM expressed less POP1 than GFP- vented not only inflammasome assembly and cytokine release, POP1 THP-1, as determined by flow cytometry and immunoblot but also release of ASC danger particles and propagation of sec- (Figure S7E), indicating that POP1 expression in our TG mice ondary inflammasome responses to bystander cells. Previous was decreased relative to human macrophages. Still, we found studies suggested that POP1 enhances IL-1b release in vitro that POP1 expression levels in CD68-POP1 TG mice varied (Stehlik et al., 2003b). However, based on our results, this is slightly and correlated inversely with inflammasome activity, as probably a consequence of overexpression in HEK293 cells,

272 Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. because we also observed a weak nucleation of ASC polymeri- recruitment and oligomerization of ASC and extracellular zation by POP1 in these cells. Other PYDs, including PYD from release of ASC danger particles proceed uncontrolled, due to NLRP3, can also induce weak nucleation of ASC polymerization reduced POP1 expression. The presence of POP1 might also in this cell type (Lu et al., 2014), and the NLRP3PYD also binds to increase the required threshold for inflammasome assembly. the same ASC region as POP1 (Vajjhala et al., 2012). At low expression levels, as observed in resting macrophages, Overexpression studies of several PYD proteins in HEK293 POP1 would probably not interfere with acute host defense cells have implicated PYDs in NF-kB regulation, but these results and maintenance of metabolic health. However, upon inducible were not observed in macrophages and in vivo. Similarly, we expression as a late response gene, POP1 might become previously observed an inhibitory effect of POP1 on NF-kB activ- involved in the resolution phase of inflammasome responses ity in HEK293 cells (Stehlik et al., 2003b) but did not observe (Figure S7G), which is still poorly understood. In particular, such activity in THP-1 cells, human macrophages, or BMDMs POP1 expression before the onset of HMGB1 expression might in response to all tested stimuli. However, POP1-expressing be important, because inflammasome-dependent release of THP-1 cells showed a slightly reduced IkBa phosphorylation HMGB1 is directly linked to inflammatory disease (Yang et al., in response to LPS, but this cannot be responsible for the potent 2013). Accordingly, in mice that normally lack POP1 and other inhibition of caspase-1 and we did not observe significant POP members (Stehlik and Dorfleutner, 2007; Khare et al., altered transcription of inflammasome components or release 2014), POP1 expression potently ameliorated systemic inflam- of IL-6 and TNF-a. Filament formation of prion activity containing masome-driven inflammation. Interestingly, CAPS is caused signaling components is one of the mechanisms of innate not only by excessive IL-1b secretion, but also by IL-18 secretion immune pathway activation. Although ASCPYD, AIM2PYD, and and pyroptosis (Brydges et al., 2009, 2013). Furthermore, ASC NLRP3PYD contain such intrinsic prion activity (Lu et al., 2014; inflammasomes are also responsible for cytokine release by Cai et al., 2014), POP1 does not appear to have this activity, the non-canonical inflammasome (Kayagaki et al., 2011). By because it does not form these characteristic filaments (Stehlik blocking all inflammasome-dependent mediators, POP1 ex- et al., 2003b), but POP1 expression levels probably are crucial hibited a potent anti-inflammatory function in peritonitis, for its inflammasome inhibitory function. Inflammasome activity sepsis, and CAPS, and this function can probably be extended increased upon POP1 silencing in human macrophages and to other ASC-dependent inflammatory diseases. Employing a THP-1 cells but decreased upon stable expression of POP1 at cell-permeable recombinant POP1 provided proof of concept levels that are comparable to its induced endogenous expres- that a POP1-based therapy could be effective in CAPS and other sion levels. POP1 transgene expression in mice was lower than inflammasomopathy patients with impaired POP1 expression, in our stable cells and by extension also probably lower than in where it might function as a novel broad-spectrum anti-inflam- human macrophages, and therefore closely mimicked physio- matory treatment strategy targeting all inflammasome effectors. logically relevant POP1 levels. Despite this lower expression, Assembly of inflammasomes is not limited to macrophages, POP1 was still able to inhibit the inflammasome, and variable but the cell types that are responsible for systemic inflammation POP1 expression levels in individual TG mice even correlated have not been elucidated yet. We demonstrated that mono- with its inflammasome inhibitory activity. An earlier study map- cyte-macrophage-DC-lineage-specific expression of POP1 ping the interaction of ASC with NLRP3 did not observe reduced was sufficient to prevent systemic inflammation in three different NLRP3-ASC interaction in the presence of POP1 when using inflammatory disease models, which strongly implicates that in- in vitro binding assays with recombinant proteins (Vajjhala flammasome activation and defects in inflammasome control in et al., 2012). However, protein folding and posttranslational this lineage are crucial for promoting systemic inflammation. modifications might not be recapitulated in vitro and might Many studies highlight the importance of inflammasomes for yield different results compared to our studies of endogenous homeostasis and disease pathology and suggest that under- inflammasome assembly in human and mouse macrophages. standing the mechanism by which healthy tissues put the brakes Posttranslational modifications, such as ubiquitination (Py on inflammasome-induced systemic inflammation will be the et al., 2013) and phosphorylation (Lin et al., 2015; Hara et al., next important step for designing future therapies. With the re- 2013), have been implicated in NLRP3 inflammasome activity, sults from our study, we started to provide some answers to and POP1 phosphorylation (Stehlik et al., 2003b) might also be this important question. POP1 provides a unique mechanism involved in the binding mechanism. that evolved in humans to possibly allow tighter control of an Excessive and uncontrolled release of inflammasome me- essential host defense system by guarding against excessive diators contributes to auto-inflammatory and auto-immune dis- and out-of-control responses that cause inflammatory disease. eases (Strowig et al., 2012). Hence, blocking IL-1b has proved beneficial in various inflammatory diseases in human and mice EXPERIMENTAL PROCEDURES (Dinarello, 2011). Furthermore, oligomeric ASC particles have been identified in CAPS and pulmonary disease (Franklin et al., Animals 2014; Baroja-Mazo et al., 2014). Despite the tight regulation of in- B6.TgN(CD68-POP1) TG mice were generated as described with GFP-POP1 flammasome responses, even a single point mutation in NLRP3 (Khare et al., 2014; Iqbal et al., 2014). C57BL/6 wild-type (WT) and Lysozyme Cre Cre can drive excessive systemic inflammation (Hoffman and M- knock-in mice ( L) were obtained from the Jackson Laboratories and Nlrp3À/À, PycardÀ/À, and floxed Nlrp3A350V knock-in mice were described pre- Brydges, 2011). Thus, inflammasome regulatory mechanisms viously (Mariathasan et al., 2004, 2006; Brydges et al., 2009). Mice were might also be impaired. In line with this possibility, we observed housed in a specific-pathogen-free animal facility and all experiments were reduced POP1 expression in CAPS patients. This observation performed on age- and gender-matched, randomly assigned 8- to 14-week- suggested that in addition to uncontrolled activation of NLRP3, old mice conducted according to procedures approved by the Northwestern

Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. 273 University Committee on Use and Care of Animals. Floxed Nlrp3A350V mice AUTHOR CONTRIBUTIONS (Brydges et al., 2009, 2013) were crossed with CreL and CD68-POP1 TG mice and male and female offspring were analyzed for body weight and sur- L.d.A., C.S., and A.D. designed the research; L.d.A., S.K., A.V.M., R.P., R.A.R., vival. Histological analysis was performed at day 8 after birth. A.D., C.S., and M.C.W. performed experiments; D.R.G., H.P., and H.M.H. provided essential reagents, expertise, and advice; L.d.A., S.K., A.V.M., Macrophage Isolation, Culture, and Transfection R.A.R., H.P., A.D., and C.S. analyzed results; L.d.A., A.D., and C.S. wrote Peripheral blood-derived human macrophages, BMDMs, and peritoneal mac- the paper; and A.D. and C.S. conceived the study and provided overall rophages (PMs) were isolated as described (Khare et al., 2012, 2014). direction.

Quantitative Real-Time PCR ACKNOWLEDGMENTS Total RNA was isolated and analyzed as described (Khare et al., 2012, 2014). This work was supported by the NIH (GM071723, HL097183, AI092490, LPS-Induced Peritonitis AI082406, AI099009, AI120625, and AR064349 to C.S.; AR057532 and -POP1 8- to 12-week-old female WT and CD68 TG mice had their abdomen AR066739 to A.D.; AR050250, AR054796, AI092490, and HL108795 to H.P.; shaved under anesthesia and were randomly selected for i.p. injection with AR061593 to A.V.M.; and AI52430 to H.M.H.), a Cancer Center Support Grant E. coli PBS or LPS (2.5 mg/kg, 0111:B4, Sigma). After 3 hr, mice were i.p. in- (CA060553), the Skin Disease Research Center (AR057216), and the American jected with XenoLight Rediject Inflammation probe (200 mg/kg, PerkinElmer) Heart Association (13GRNT17110117) to C.S., an ATS/Scleroderma Founda- (Gross et al., 2009) and in vivo bioluminescence was captured by imaging tion Grant to A.V.M., and The British Heart Foundation (RG/10/15/28578) to (IVIS Spectrum, PerkinElmer) 10 min after injection with a 5 min exposure on D.R.G. S.K. was an Arthritis Foundation fellow (AF161715), L.d.A. was sup- anesthetized mice (Khare et al., 2014). Images were quantified with Living Im- ported by the American Heart Association (11POST585000) and the NIH age software (PerkinElmer). Endotoxic shock was induced by i.p. injection of a (T32AR007611), and H.P. was supported by funds provided by the Solovy/ E. coli lethal dose of 20 mg/kg LPS ( 0111:B4) and mice were monitored four Arthritis Research Society Professor. Plasmids pMD2.G and psPAX2 were times daily for survival. Body temperature was measured with an animal rectal kindly provided by Didier Trono (E´ cole Polytechnique Fe´ de´ rale de Lausanne), probe. Blood was collected 3 hr after LPS injection by mandibular bleed, and Nlrp3A350V KI mice by H.M.H. (University of California at San Diego), and serum cytokine levels were quantified by ELISA. PycardÀ/À and Nlrp3À/À mice by Vishva M. Dixit (Genentech). This work was supported by the Northwestern University Transgenic and Targeted Mutagen- ASC-Particle-Induced Peritonitis esis Laboratory, Mouse Histology and Phenotyping Laboratory, and flow cy- ASC-GFP-containing particles were FACS purified and verified by micro- tometry facility. We thank Dr. C.M. Cuda for support with flow cytometry. scopy. 14-week-old male WT and CD68-POP1 TG mice had their abdomen shaved under anesthesia and were randomly selected for i.p. injection with Received: January 27, 2015 3 5 PBS or FACS-purified ASC-GFP particles (1 10 particles/mouse). After Revised: May 8, 2015 4 hr, MPO activity was determined as above. Peritoneal lavage fluids were Accepted: May 27, 2015 b collected and assayed for IL-1 by ELISA. Published: August 11, 2015

Plasmids REFERENCES pcDNA3 and pGEX-based expression constructs for ASC, POP1, NLRP3, ASCPYD, and NLRP3PYD were described earlier (Khare et al., 2014; Stehlik Baroja-Mazo, A., Martıń-Sa´ nchez, F., Gomez, A.I., Martıńez, C.M., Amores- et al., 2003a, 2003b). Iniesta, J., Compan, V., Barbera` -Cremades, M., Yagu¨ e, J., Ruiz-Ortiz, E., Anto´ n, J., et al. (2014). The NLRP3 inflammasome is released as a particulate Antibody-Based Detection danger signal that amplifies the inflammatory response. Nat. Immunol. 15, Co-immunoprecipitations (IP), GST pull down, ASC cross-linking, immunohis- 738–748. tochemistry, ELISA, flow cytometry, and caspase-1 activity were performed as previously described (Khare et al., 2014; Fernandes-Alnemri and Alnemri, Bauernfeind, F.G., Horvath, G., Stutz, A., Alnemri, E.S., MacDonald, K., 2008). Speert, D., Fernandes-Alnemri, T., Wu, J., Monks, B.G., Fitzgerald, K.A., et al. (2009). Cutting edge: NF-kappaB activating pattern recognition and cyto- Cell-Penetrating Recombinant Proteins kine receptors license NLRP3 inflammasome activation by regulating NLRP3 183 6xHIS-POP1 and a 6xHIS-GFP cDNAs were fused with the HIV TAT sequence expression. J. Immunol. , 787–791. and purified from E. coli. 12-week-old male WT mice had their abdomen Bedoya, F., Sandler, L.L., and Harton, J.A. (2007). Pyrin-only protein 2 shaved under anesthesia were randomly selected for i.p. injection with TAT- modulates NF-kappaB and disrupts ASC:CLR interactions. J. Immunol. 178, GFP or TAT-POP1 (40 mg/kg) for 30 min prior LPS i.p. injection (2.5 mg/kg, 3837–3845. E. coli 0111:B4, Sigma) and were quantified for MPO activity in vivo 1 hr later, Boisson, B., Laplantine, E., Prando, C., Giliani, S., Israelsson, E., Xu, Z., as described above. Abhyankar, A., Israe¨ l, L., Trevejo-Nunez, G., Bogunovic, D., et al. (2012). Immunodeficiency, autoinflammation and amylopectinosis in humans with Statistics inherited HOIL-1 and LUBAC deficiency. Nat. Immunol. 13, 1178–1186. Graphs represent the mean ± SEM. A standard two-tailed unpaired t test was Brydges, S.D., Mueller, J.L., McGeough, M.D., Pena, C.A., Misaghi, A., used for statistical analysis of two groups with all data points showing a normal Gandhi, C., Putnam, C.D., Boyle, D.L., Firestein, G.S., Horner, A.A., et al. distribution and Kaplan-Meier survival curves were used to investigate differ- (2009). Inflammasome-mediated disease animal models reveal roles for innate ences in survival. Values of p < 0.05 were considered significant and listed in but not adaptive immunity. Immunity 30, 875–887. the figure legends (Prism 5, GraphPad). The investigators were not blinded to the genotype of the mice/cells. Sample sizes were selected on the basis of pre- Brydges, S.D., Broderick, L., McGeough, M.D., Pena, C.A., Mueller, J.L., and liminary results to ensure a power of 80% with 95% confidence between Hoffman, H.M. (2013). Divergence of IL-1, IL-18, and cell death in NLRP3 123 populations. inflammasomopathies. J. Clin. Invest. , 4695–4705. Cai, X., Chen, J., Xu, H., Liu, S., Jiang, Q.-X., Halfmann, R., and Chen, Z.J. SUPPLEMENTAL INFORMATION (2014). Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell 156, 1207–1222. Supplemental Information includes seven figures, one table, and Supple- Calvano, S.E., Xiao, W., Richards, D.R., Felciano, R.M., Baker, H.V., Cho, mental Experimental Procedures and can be found with this article online at R.J., Chen, R.O., Brownstein, B.H., Cobb, J.P., Tschoeke, S.K., et al.; http://dx.doi.org/10.1016/j.immuni.2015.07.018. Inflamm and Host Response to Injury Large Scale Collab. Res. Program

274 Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. (2005). A network-based analysis of systemic inflammation in humans. Kayagaki, N., Warming, S., Lamkanfi, M., Vande Walle, L., Louie, S., Dong, J., Nature 437, 1032–1037. Newton, K., Qu, Y., Liu, J., Heldens, S., et al. (2011). Non-canonical inflamma- 479 Chu, L.H., Gangopadhyay, A., Dorfleutner, A., and Stehlik, C. (2015). An some activation targets caspase-11. Nature , 117–121. updated view on the structure and function of PYRIN domains. Apoptosis Kayagaki, N., Wong, M.T., Stowe, I.B., Ramani, S.R., Gonzalez, L.C., Akashi- 20, 157–173. Takamura, S., Miyake, K., Zhang, J., Lee, W.P., Muszynski, A., et al. (2013). Dinarello, C.A. (2011). Interleukin-1 in the pathogenesis and treatment of Noncanonical inflammasome activation by intracellular LPS independent of 341 inflammatory diseases. Blood 117, 3720–3732. TLR4. Science , 1246–1249. Dorfleutner, A., Bryan, N.B., Talbott, S.J., Funya, K.N., Rellick, S.L., Reed, J.C., Khare, S., Luc, N., Dorfleutner, A., and Stehlik, C. (2010). Inflammasomes and 30 Shi, X., Rojanasakul, Y., Flynn, D.C., and Stehlik, C. (2007a). Cellular pyrin their activation. Crit. Rev. Immunol. , 463–487. domain-only protein 2 is a candidate regulator of inflammasome activation. Khare, S., Dorfleutner, A., Bryan, N.B., Yun, C., Radian, A.D., de Almeida, L., Infect. Immun. 75, 1484–1492. Rojanasakul, Y., and Stehlik, C. (2012). An NLRP7-containing inflammasome Dorfleutner, A., Talbott, S.J., Bryan, N.B., Funya, K.N., Rellick, S.L., Reed, J.C., mediates recognition of microbial lipopeptides in human macrophages. Shi, X., Rojanasakul, Y., Flynn, D.C., and Stehlik, C. (2007b). A Shope Fibroma Immunity 36, 464–476. virus PYRIN-only protein modulates the host immune response. Virus Genes Khare, S., Ratsimandresy, R.A., de Almeida, L., Cuda, C.M., Rellick, S.L., 35, 685–694. Misharin, A.V., Wallin, M.C., Gangopadhyay, A., Forte, E., Gottwein, E., et al. Dorfleutner, A., Chu, L., and Stehlik, C. (2015). Inhibiting the inflammasome: (2014). The PYRIN domain-only protein POP3 inhibits ALR inflammasomes one domain at a time. Immunol. Rev. 265, 205–216. and regulates responses to infection with DNA viruses. Nat. Immunol. 15, 343–353. Fernandes-Alnemri, T., and Alnemri, E.S. (2008). Assembly, purification, and assay of the activity of the ASC pyroptosome. Methods Enzymol. 442, Lamkanfi, M., Sarkar, A., Vande Walle, L., Vitari, A.C., Amer, A.O., Wewers, 251–270. M.D., Tracey, K.J., Kanneganti, T.D., and Dixit, V.M. (2010). Inflammasome- dependent release of the alarmin HMGB1 in endotoxemia. J. Immunol. 185, Fernandes-Alnemri, T., Wu, J., Yu, J.W., Datta, P., Miller, B., Jankowski, W., 4385–4392. Rosenberg, S., Zhang, J., and Alnemri, E.S. (2007). The pyroptosome: a supra- molecular assembly of ASC dimers mediating inflammatory cell death via cas- Lin, K.-M., Hu, W., Troutman, T.D., Jennings, M., Brewer, T., Li, X., Nanda, S., pase-1 activation. Cell Death Differ. 14, 1590–1604. Cohen, P., Thomas, J.A., and Pasare, C. (2014). IRAK-1 bypasses priming and Franklin, B.S., Bossaller, L., De Nardo, D., Ratter, J.M., Stutz, A., Engels, G., directly links TLRs to rapid NLRP3 inflammasome activation. Proc. Natl. Acad. 111 Brenker, C., Nordhoff, M., Mirandola, S.R., Al-Amoudi, A., et al. (2014). The Sci. USA , 775–780. adaptor ASC has extracellular and ‘prionoid’ activities that propagate inflam- Lin, Y.-C., Huang, D.-Y., Wang, J.-S., Lin, Y.-L., Hsieh, S.-L., Huang, K.-C., and mation. Nat. Immunol. 15, 727–737. Lin, W.-W. (2015). Syk is involved in NLRP3 inflammasome-mediated cas- Gough, P.J., Gordon, S., and Greaves, D.R. (2001). The use of human CD68 pase-1 activation through adaptor ASC phosphorylation and enhanced oligo- transcriptional regulatory sequences to direct high-level expression of class merization. J. Leukoc. Biol. jlb.3HI0814-371RR, in press. A scavenger receptor in macrophages in vitro and in vivo. Immunology 103, Lu, A., Magupalli, V.G., Ruan, J., Yin, Q., Atianand, M.K., Vos, M.R., Schro¨ der, 351–361. G.F., Fitzgerald, K.A., Wu, H., and Egelman, E.H. (2014). Unified polymeriza- Gross, S., Gammon, S.T., Moss, B.L., Rauch, D., Harding, J., Heinecke, J.W., tion mechanism for the assembly of ASC-dependent inflammasomes. Cell 156 Ratner, L., and Piwnica-Worms, D. (2009). Bioluminescence imaging of mye- , 1193–1206. loperoxidase activity in vivo. Nat. Med. 15, 455–461. Man, S.M., Hopkins, L.J., Nugent, E., Cox, S., Glu¨ ck, I.M., Tourlomousis, P., Hara, H., Tsuchiya, K., Kawamura, I., Fang, R., Hernandez-Cuellar, E., Shen, Wright, J.A., Cicuta, P., Monie, T.P., and Bryant, C.E. (2014). Inflammasome Y., Mizuguchi, J., Schweighoffer, E., Tybulewicz, V., and Mitsuyama, M. activation causes dual recruitment of NLRC4 and NLRP3 to the same macro- 111 (2013). Phosphorylation of the adaptor ASC acts as a molecular switch that molecular complex. Proc. Natl. Acad. Sci. USA , 7403–7408. controls the formation of speck-like aggregates and inflammasome activity. Mariathasan, S., Newton, K., Monack, D.M., Vucic, D., French, D.M., Lee, Nat. Immunol. 14, 1247–1255. W.P., Roose-Girma, M., Erickson, S., and Dixit, V.M. (2004). Differential activa- 430 Harris, H.E., Andersson, U., and Pisetsky, D.S. (2012). HMGB1: a multifunc- tion of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature , tional alarmin driving autoimmune and inflammatory disease. Nat. Rev. 213–218. Rheumatol. 8, 195–202. Mariathasan, S., Weiss, D.S., Newton, K., McBride, J., O’Rourke, K., Roose- Henao-Mejia, J., Elinav, E., Strowig, T., and Flavell, R.A. (2012). Girma, M., Lee, W.P., Weinrauch, Y., Monack, D.M., and Dixit, V.M. (2006). Inflammasomes: far beyond inflammation. Nat. Immunol. 13, 321–324. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440, 228–232. Hoffman, H.M., and Brydges, S.D. (2011). Genetic and molecular basis of inflammasome-mediated disease. J. Biol. Chem. 286, 10889–10896. Martinon, F., Burns, K., and Tschopp, J. (2002). The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of Hoffman, H.M., Mueller, J.L., Broide, D.H., Wanderer, A.A., and Kolodner, R.D. proIL-beta. Mol. Cell 10, 417–426. (2001). Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nat. McDonald, B., Pittman, K., Menezes, G.B., Hirota, S.A., Slaba, I., Waterhouse, Genet. 29, 301–305. C.C.M., Beck, P.L., Muruve, D.A., and Kubes, P. (2010). Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 330, Iqbal, A.J., McNeill, E., Kapellos, T.S., Regan-Komito, D., Norman, S., Burd, S., 362–366. Smart, N., Machemer, D.E.W., Stylianou, E., et al. (2014). Human CD68 promoter directs GFP transgene expression in mouse myeloid cells, allowing Meng, G., Zhang, F., Fuss, I., Kitani, A., and Strober, W. (2009). A mutation in analysis of monocyte to macrophage differentiation in vivo. Blood 124, the Nlrp3 gene causing inflammasome hyperactivation potentiates Th17 cell- e33–e44. dominant immune responses. Immunity 30, 860–874. Johnston, J.B., Barrett, J.W., Nazarian, S.H., Goodwin, M., Ricciuto, D., Wang, Mun˜ oz-Planillo, R., Kuffa, P., Martıńez-Colo´ n, G., Smith, B.L., Rajendiran, G., and McFadden, G. (2005). A poxvirus-encoded pyrin domain protein inter- T.M., and Nu´ n˜ ez, G. (2013). K+ efflux is the common trigger of NLRP3 inflam- acts with ASC-1 to inhibit host inflammatory and apoptotic responses to infec- masome activation by bacterial toxins and particulate matter. Immunity 38, tion. Immunity 23, 587–598. 1142–1153. Juliana, C., Fernandes-Alnemri, T., Kang, S., Farias, A., Qin, F., and Alnemri, Py, B.F., Kim, M.-S., Vakifahmetoglu-Norberg, H., and Yuan, J. (2013). E.S. (2012). Non-transcriptional priming and deubiquitination regulate Deubiquitination of NLRP3 by BRCC3 critically regulates inflammasome activ- NLRP3 inflammasome activation. J. Biol. Chem. 287, 36617–36622. ity. Mol. Cell 49, 331–338.

Immunity 43, 264–276, August 18, 2015 ª2015 Elsevier Inc. 275 Schroder, K., Sagulenko, V., Zamoshnikova, A., Richards, A.A., Cridland, J.A., Tang, B.M., McLean, A.S., Dawes, I.W., Huang, S.J., Cowley, M.J., and Lin, Irvine, K.M., Stacey, K.J., and Sweet, M.J. (2012). Acute lipopolysaccharide R.C. (2008). Gene-expression profiling of gram-positive and gram-negative priming boosts inflammasome activation independently of inflammasome sepsis in critically ill patients. Crit. Care Med. 36, 1125–1128. sensor induction. Immunobiology 217, 1325–1329. Vajjhala, P.R., Mirams, R.E., and Hill, J.M. (2012). Multiple binding sites on the Schwarze, S.R., Ho, A., Vocero-Akbani, A., and Dowdy, S.F. (1999). In vivo pyrin domain of ASC protein allow self-association and interaction with NLRP3 protein transduction: delivery of a biologically active protein into the mouse. protein. J. Biol. Chem. 287, 41732–41743. Science 285, 1569–1572. Wen, H., Miao, E.A., and Ting, J.P.-Y. (2013). Mechanisms of NOD-like recep- Srinivasula, S.M., Poyet, J.-L., Razmara, M., Datta, P., Zhang, Z., and Alnemri, tor-associated inflammasome activation. Immunity 39, 432–441. E.S. (2002). The PYRIN-CARD protein ASC is an activating adaptor for caspase-1. J. Biol. Chem. 277, 21119–21122. Willingham, S.B., Allen, I.C., Bergstralh, D.T., Brickey, W.J., Huang, M.T.H., Stehlik, C., and Dorfleutner, A. (2007). COPs and POPs: modulators of inflam- Taxman, D.J., Duncan, J.A., and Ting, J.P.Y. (2009). NLRP3 (NALP3, masome activity. J. Immunol. 179, 7993–7998. Cryopyrin) facilitates in vivo caspase-1 activation, necrosis, and HMGB1 release via inflammasome-dependent and -independent pathways. Stehlik, C., Lee, S.H., Dorfleutner, A., Stassinopoulos, A., Sagara, J., and J. Immunol. 183, 2008–2015. Reed, J.C. (2003a). Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation. Wong, H.R., Cvijanovich, N., Allen, G.L., Lin, R., Anas, N., Meyer, K., Freishtat, J. Immunol. 171, 6154–6163. R.J., Monaco, M., Odoms, K., Sakthivel, B., and Shanley, T.P.; Genomics of Stehlik, C., Krajewska, M., Welsh, K., Krajewski, S., Godzik, A., and Reed, J.C. Pediatric SIRS/Septic Shock Investigators (2009). Genomic expression (2003b). The PAAD/PYRIN-only protein POP1/ASC2 is a modulator of ASC- profiling across the pediatric systemic inflammatory response syndrome, 37 mediated nuclear-factor-kappa B and pro-caspase-1 regulation. Biochem. sepsis, and septic shock spectrum. Crit. Care Med. , 1558–1566. J. 373, 101–113. Yang, H., Antoine, D.J., Andersson, U., and Tracey, K.J. (2013). The many Strowig, T., Henao-Mejia, J., Elinav, E., and Flavell, R. (2012). Inflammasomes faces of HMGB1: molecular structure-functional activity in inflammation, in health and disease. Nature 481, 278–286. apoptosis, and chemotaxis. J. Leukoc. Biol. 93, 865–873.

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