PB1-F2 Peptide Derived from Avian Influenza A Virus H7N9 Induces Inflammation via Activation of the NLRP3 Inflammasome*

The emergence of avian H7N9 influenza A virus in humans with associated high mortality has highlighted the threat of a potential pandemic. Fatal H7N9 infections are characterized by hyperinflammation and increased cellular infiltrates in the lung. Currently there are limited therapies to address the pathologies associated with H7N9 infection and the virulence factors that contribute to these pathologies. We have found that PB1-F2 derived from H7N9 activates the NLRP3 inflammasome and induces lung inflammation and cellular recruitment that is NLRP3-dependent. We have also shown that H7N9 and A/Puerto Rico/H1N1 (PR8)PB1-F2 peptide treatment induces significant mitochondrial reactive oxygen production, which contributes to NLRP3 activation. Importantly, treatment of cells or mice with the specific NLRP3 inhibitor MCC950 significantly reduces IL-1β maturation, lung cellular recruitment, and cytokine production. Together, these results suggest that PB1-F2 from H7N9 avian influenza A virus may be a major contributory factor to disease pathophysiology and excessive inflammation characteristic of clinical infections and that targeting the NLRP3 inflammasome may be an effective means to reduce the inflammatory burden associated with H7N9 infections.

Since 2013, novel avian influenza A viruses (IAVs) 5 of the H7N9 subtype have caused sporadic infections in humans, with over 800 laboratory cases now confirmed in China and a mortality rate of ϳ40% (1,2). The potential of H7N9 IAV to cause a pandemic poses a constant and realistic threat to global health (3), particularly as many isolates have been identified to carry genetic modifications that confer antiviral resistance. Understanding the mechanisms involved in the induction of immunopathology and fatal disease is imperative to allow the development of improved and better targeted treatments to reduce the mortality associated with pathogenic IAV.
The NLRP3 inflammasome is an oligomeric innate immune intracellular signaling complex that senses many pathogen-, host-, and environment-derived factors (4). Inflammasome-induced cytokine release requires two signals: up-regulation of components of the NLRP3 inflammasome and synthesis of pro-IL-1␤ through activation of the prototypic inflammatory transcription factor NF-B and inflammasome formation that results in IL-1␤ maturation and secretion. Following activation, NLRP3 binds to the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). ASC further recruits the enzyme caspase-1 to form the inflammasome complex, initiating autocatalytic cleavage of caspase-1. The NLRP3 inflammasome is now recognized as a major route by which the innate immune system recognizes and responds during IAV infection (4). To date, IAV single-stranded RNA and proton flux via the IAV-encoded matrix-2 (M2) ion channel have been shown to activate the NLRP3 inflammasome (5,6), which is important in the development of adaptive immune responses to IAV (7). Studies utilizing mice lacking components of the NLRP3 inflammasome have demonstrated its importance in eliciting rapid protective responses following infection with the mouse-adapted A/Puerto Rico/8/34 strain (PR8, H1N1) (6,8). In these studies, inflammasome-deficient mice were highly susceptible to both low and high inoculum doses of PR8, and the NLRP3 inflammasome was shown to be required for production of pyrogenic IL-1␤ and IL-18 in the airways, cellular infiltration, and lung immunopathology. These findings suggested that NLRP3-induced responses were crucial to host protection via recruitment of effector cells and the induction of tissue repair. Additionally, a lack of the IL-1 receptor has been shown to increase mortality but reduce lung immunopathology following infection with PR8 H1N1 or avian H5N1, suggesting that IL-1 receptor signaling may increase damage to the lung (9,10). Critically, we recently identified that the NLRP3 inflammasome plays both a protective and detrimental role during pathogenic IAV challenge through temporal inhibition of NLRP3 function (11). This therefore suggests that excessive or prolonged NLRP3 inflammasome activation contributes to the hyperinflammatory phenotype associated with pathogenic infections.
Fatal IAV infections correlate with production of excessive levels of proinflammatory cytokines and chemokines (termed a "cytokine storm") and excessive cellular inflammation. PB1-F2 protein is encoded by a ϩ1 alternate reading frame of the PB1 gene, and protein expression in cells has been linked to cell death, viral replication, and virulence, although these properties can differ between virus strains (12). Crucially, we have shown that PB1-F2 proteins derived from 20th century pandemic and highly pathogenic IAV strains, but not mildly virulent IAVs, activate the NLRP3 inflammasome, inducing IL-1␤ production and pulmonary inflammation (13). PB1-F2 triggers excessive cellular recruitment and hyperinflammatory responses in the lungs of infected mice (14,15). Specific PB1-F2 amino acids that enhance inflammation and are linked to virulence have been identified at the C terminus and map to residues Leu-62, Arg-75, Arg-79, and Leu-82, termed the four inflammatory residues (16).
In this study, we have identified that PB1-F2 from H7N9 is a potent activator of the NLRP3 inflammasome. We also identified that inflammasome activation was related to PB1-F2-induced mitochondrial reactive oxygen species (mtROS) and that targeting mtROS with the specific inhibitor MitoTempo inhibited PB1-F2 peptide-induced inflammasome activation and IL-1␤ secretion and maturation. PB1-F2 peptide-mediated inflammasome activation and inflammation were phagocytosisdependent and inhibited by the small-molecule NLRP3 inhibitor MCC950 in vitro and in vivo. This study identifies H7N9 PB1-F2 as an activator of the NLRP3 inflammasome and suggests that PB1-F2 may contribute to the hyperinflammation associated with current human H7N9 infections. Our identification of MCC950 as a potent inhibitor of H7N9 PB1-F2-induced lung inflammation and cellular influx suggests that novel therapies that specifically target the NLRP3 inflammasome may therefore provide an effective treatment to reduce the mortality associated with pathogenic H7N9 IAV and offset the ineffectiveness of current antiviral treatments in the later stages of infection.

H7N9-derived PB1-F2 Peptide Induces IL-1␤ in Murine
Macrophages and Human PBMCs-We have previously demonstrated that aggregated PB1-F2 derived from the PR8 strain of IAV (H1N1) activates the NLRP3 inflammasome, contributing to disease pathophysiology (13). We examined the inflammatory potential of aggregated PB1-F2 derived from the recently identified novel avian-origin H7N9 IAV. Using aggregated peptides derived from the C termini of PB1-F2 from both PR8 and H7N9 (the latter being A/Shanghai/02/2013), we show that Ͼ85% of both peptides form similar concentrations of protein aggregates between 0.5 and 2.5 m (supplemental Fig. 1, A-C), a particle size found previously to be optimal for NLRP3 inflammasome activation by other aggregates (17).
The uptake of infection-and disease-related protein aggregates by phagocytes and subsequent lysosomal destabilization are known triggers of inflammasome activation (17). In our experiments, inhibition of phagocytosis with latrunculin A, prevention of phagolysosomal maturation with bafilomycin A, and inhibition of caspase-1 activity with Z-YVAD reduced IL-1␤ secretion in a concentration-dependent manner in both iBMDMs and primary BMDMs stimulated with H7N9 PB1-F2 peptide (Fig. 1, C-E, and supplemental Fig. 2, C and D), respectively.
To evaluate whether H7N9 derived PB1-F2 protein may also enhance inflammatory events in human cells, we examined cellular responses in LPS-primed human peripheral blood mono-nuclear cells (hPBMCs) exposed to H7N9 PB1-F2. H7N9 PB1-F2 peptide induced robust secretion of IL-1␤ from hPB-MCs (Fig. 1F) (17), which was inhibited via treatment with the FIGURE 1. H7N9 PB1-F2 induces caspase-1-dependent release of IL-1␤ in macrophages and PBMCs. iBMDMs or primary BMDMs were primed with LPS (100 ng/ml) for 3 h and then stimulated with a dose range of either PR8 or H7N9 PB1-F2 peptide (5-200 g/ml) or silica (150 g/ml) or left unstimulated (NS) in triplicate for 6 h. A, cellular supernatants from iBMDMs were analyzed for secreted IL-1␤ by ELISA. Results are representative of three independent experiments, and data represent the mean Ϯ S.E. B-E, protein in primary BMDM supernatants were separated by 4 -12% SDS-PAGE and immunoblotted (IB) for the indicated antibodies. The result represents one of three independent experiments. iBMDMs were primed with LPS (100 ng/ml) for 3 h and then pretreated with the inhibitors latrunculin A (Lat A, C), bafilomycin (BAF, D), or YVAD (E) at the indicated doses 40 min prior to treatment with H7N9 PB1-F2 peptide (100 g/ml) or left unstimulated for 6 h. The levels of IL-1␤ in cell supernatants were determined by ELISA. Results are the mean Ϯ S.E. MW, molecular weight. F, PBMCs were primed in LPS (50 pg/ml) for 3 h prior to stimulation with an H7N9 PB1-F2 peptide (10 -200 g/ml), silica (150 g/ml), or nigericin (1 M) or left unstimulated for 6 h. G, primed PBMCs were pretreated with YVAD for 40 min and then stimulated with H7N9 PB1-F2 peptide (100 g/ml) or silica (150 g/ml) or left unstimulated for 6 h. Cultured supernatants were assayed for IL-1␤ by ELISA. Results are the mean Ϯ S.E. of three independent donors. **, p Ͻ 0.01; ***, p Ͻ 0.001; one-way ANOVA.
H7N9 PB1-F2 Induces ASC Speck Formation and IL-1␤ Secretion via the NLRP3 Inflammasome-We next confirmed that phagocytosis of H7N9 PB1-F2 is required for inflammasome activation or ASC speck formation by bioimaging. H7N9 PB1-F2 was labeled with the pH-sensitive dye pHrodo, which dramatically increases in fluorescence intensity in the acidic environment of the phagolysosomal compartment. NLRP3-deficient iBMDMs stably reconstituted with ceruleantagged ASC and NLRP3-FLAG, which obviates the need for priming, were used to visualize inflammasome activation via speck formation. ASC-cerulean macrophages were challenged with labeled H7N9-pHrodo peptide, and ASC speck formation was visualized as a well characterized marker of inflammasome formation and activation of caspase-1 (18).
As observed in Fig. 2 and the live-cell imaging in supplemental Movies 1 and 2, pHrodo-labeled H7N9 PB1-F2 is phagocytosed rapidly into the lysosomal pathway, as indicated by increased PB1-F2 red fluorescence within cellular vesicles (Fig.  2, 120 min). This event is followed by ASC speck formation (Fig.  2, top panels, 200 min). Notably, treatment of ASC-cerulean cells with latrunculin A prior to challenge with pHrodo-labeled H7N9 PB1-F2 blocked cellular uptake of the peptide aggregate, prohibiting ASC speck formation (supplemental Movie 3).
PB1-F2 derived from H1N1 strains has been described previously as containing a mitochondrial targeting sequence (19,20) that disrupts mitochondrial function commensurate with the C-terminal domain that, as we have found, activates the NLRP3 inflammasome. Given that mtROS have been found previously to activate the NLRP3 inflammasome, we examined whether H7N9 and PR8 PB1-F2 peptide could induce mtROS. ASC-cerulean iBMDMs were pretreated with MitoSOX Red, a mitochondrially localized fluorogenic dye selective for mitochondrial superoxide in live cells (21), prior to challenge with H7N9 and PR8 PB1-F2 peptides. As can be observed in supplemental Movies 4 and 5, respectively, live cell imaging of H7N9 and PR8 PB1-F2-treated iBMDMs demonstrated increased mitochondrial fluorescence intensity of MitoSOX rapidly following treatment. Bafilomycin treatment of cells demonstrated that phagolysosomal disruption is required for PB1-F2-induced mtROS production (supplemental Movie 6). To examine whether PB1-F2 peptide-induced mtROS plays a role in PB1-F2-induced inflammasome activation, we next treated both FIGURE 2. The H7N9 PB1-F2 peptide induces ASC speck formation in macrophages. NLRP3-deficient iBMDMs stably reconstituted with ASC-cerulean (blue) and NLRP3-FLAG were stimulated with pHrodo-labeled (red) H7N9 PB1-F2 peptide at 0 min. Live-cell imaging was performed for 200 min to visualize the uptake of pHrodo-labeled H7N9 PB1-F2 peptide (30 g/ml) by the macrophages and subsequent formation of ASC specks. Representative images shown are maximum intensity projections of 3D deconvoluted z stacks using Imaris imaging software. Scale bars ϭ 8 m. BF, Brightfield. JANUARY 20, 2017 • VOLUME 292 • NUMBER 3 iBMDMs (Fig. 4A) and human PBMCs (Fig. 4D) with micromolar concentrations of MitoTempo and examined PB1-F2 peptide-induced IL-1␤ secretion. We found that MitoTempo significantly inhibited PB1-F2-induced secretion in a dose-dependent manner. Furthermore, we found that MitoTempo inhibited the proteolytic maturation of both IL-1␤ and caspase-1 by PB1-F2 H7N9 and PR8 peptides (Fig. 4B) in primary BMDMs, in which secretion of IL-1␤ was also con-firmed (Fig. 4C), and in human PBMCs (Fig. 4D). Taken together, these results suggest that, commensurate with previous studies, PB1-F2 localizes to the mitochondria to induce mtROS, which leads to activation of the inflammasome and IL-1␤ maturation and secretion.

H7N9 PB1-F2 Activates an NLRP3 Inflammasome
In Vivo H7N9 PB1-F2-induced Inflammation Is NLRP3dependent-We next confirmed the requirement for NLRP3 to elicit H7N9 PB1-F2-induced IL-1␤ secretion by examining inflammatory responses to the peptide in vivo. Neutrophils are known to be recruited in response to NLRP3-inflammasome activation in immune cells (4,22). WT and NLRP3 Ϫ/Ϫ mice were intranasally inoculated with the H7N9 PB1-F2 peptide. At 7 h following treatment, significant neutrophil influx was detected in bronchoalveolar lavage (BAL) of the airways of WT mice (Fig. 5A). Importantly, the cellular infiltrates observed in PB1-F2-treated NLRP3 Ϫ/Ϫ mice were significantly reduced compared with WT mice (Fig. 5A). The difference in cellular infiltration observed between WT and NLRP3 Ϫ/Ϫ mice treated with H7N9 PB1-F2 was reflected in significantly lower concentrations of IL-1␤ in BAL from NLRP3 Ϫ/Ϫ mice compared with WT animals (Fig. 5B). Collectively, these data highlight the role of the NLRP3 inflammasome in the maturation of IL-1␤ in response to H7N9-derived PB1-F2 peptide both in vitro and in vivo.
To explore whether H7N9-derived PB1-F2 protein expressed by virions during infection would induce NLRP3 inflammasome activation similar to that observed for the PR8 PB1-F2 protein, we attempted to use reverse genetics to generate IAVs expressing the PB1 gene derived from A/Anhui/1/ 2013 (H7N9) on the backbone of several well characterized laboratory strains. In parallel, to generate a mutant H7N9, we genetically modified the H7N9 PB1 plasmid to disrupt the PB1-F2 open reading frame and abrogate PB1-F2 expression, as demonstrated previously with PR8 (13). Using well established techniques, we attempted to reverse-engineer IAVs containing a wild-type or mutant H7N9 PB1 gene and the remaining seven viral genes derived from HKx31 (H3N2), A/Udorn/1972 (H3N2), or A/Puerto Rico/8/34 (PR8, H1N1). Despite multiple attempts, the infectious virus could not be rescued, suggesting that the H7N9 PB1 gene is incompatible for incorporation into viruses of either the H3N2 or H1N1 subtypes. The reasons for this unexpected outcome are currently being explored.   activity that acts by preventing ASC complex formation with potent activity in vivo. This compound represents both a potential NLPR3 therapeutic agent and also provides an important tool to dissect the temporal role of NLRP3 during infectious disease pathophysiology, which is not possible using gene-deficient mice (11).
We investigated the ability of MCC950 to inhibit NLRP3 activation in response to H7N9 PB1-F2 peptide in vitro. Macrophages were primed with LPS and then pretreated with MCC950 prior to challenge with PB1-F2 peptides from PR8 or H7N9 or with silica. Pretreatment with micromolar concentrations of MCC950 potently inhibited secretion of IL-1␤ by iBMDM (Fig. 6, A and B) and primary BMDMs (Fig. 6C) that were subsequently challenged with PR8 PB1-F2, H7N9 PB1-F2 or silica. It was also observed that ASC-cerulean cells pretreated with MCC950 were able to phagocytose pHrodo-labeled H7N9 PB1-F2 peptide but did not undergo ASC speck formation (supplemental Movies 7 and 8), suggesting that MCC950 acts prior to ASC oligomerization. Proteolytic cleavage of caspase-1 was detected in cell supernatants from BMDMs stimulated with either PR8-or H7N9-derived PB1-F2 or silica but not in supernatants from cells treated with MCC950 (Fig. 6D, top panel). Correlative maturation of bioactive IL-1␤ p17 was detected in supernatants of cells treated with either PR8-or H7N9-derived PB1-F2 or silica (Fig. 6D, bottom panel) but not in those treated with MCC950 (Fig. 6D, top panel). This result was reflected in the analysis of IL-1␤ levels in these samples by ELISA (supplemental Fig. 4). Importantly, MCC950 pretreatment of hPBMCs significantly reduced the levels of IL-1␤ in response to both PR8-derived (Fig. 6E) and H7N9-derived (Fig. 6F) PB1-F2 peptide.
MCC950 Blocks PB1-F2-induced Inflammation in Vivo-Having established that MCC950 inhibits PB1-F2-induced IL-1␤ maturation in vitro, we next examined whether targeting inflammasome activation could reduce PB1-F2-induced inflammation in vivo. WT mice were treated intranasally with H7N9 PB1-F2 peptide in combination with MCC950 (5 mg/kg) or vehicle control (PBS). As seen in Fig. 7A, both PR8-and H7N9-derived PB1-F2 peptides induced significant neutrophil influx into the airways, and treatment with MCC950 potently inhibited cellular influx measured at 7 h following challenge. Concomitantly, IL-1␤ levels induced in response to both PR8 and H7N9 PB1-F2 peptides were significantly lower in BAL fluid from mice treated with MCC950 (Fig. 7B) than vehicle control. Together, these results clearly demonstrate that targeting NLRP3 inflammasome activation with a small-molecule inhibitor such as MCC950 ablates H7N9 PB1-F2-induced IL-1␤ and leads to reduced lung inflammation.

Discussion
The strength of the innate immune response to IAV infection is a key determinant in clinical outcome. Excessive inflammation can cause death, particularly in the case of highly pathogenic IAV infections. It is well known that the three pandemics of the 20th century caused millions of deaths worldwide. Although a large proportion of deaths have been attributed to complications arising from secondary bacterial infections (24), the initial infection by the novel H1N1, H2N2, and H3N2 viruses caused remarkable inflammatory disease and contributed significantly to the hospitalization of patients presenting with pneumonia-like illness.
Common to 20th century pandemic IAVs is the direct reassortment of the HA and PB1 gene segments from avian IAV. The avian-derived PB1 gene segments in pandemic viruses all encoded a full-length PB1-F2 protein and contained the four proinflammatory amino acids identified previously (16). Conversely, the pandemic H1N1 IAV that emerged in 2009 contained a PB1 gene that had been circulating in swine IAV lineages and encoded a truncated and presumably non-functional PB1-F2 protein. Interestingly, in otherwise healthy individuals, infection with the 2009 pandemic IAV caused illness symptoms considered to be milder than those induced by the previous pandemic viruses and that were similar to those typically observed during a seasonal IAV infection, in which inflammatory disease is limited (25). Therefore, the contribution of the PB1-F2 protein toward enhancing the pathophysiology of IAV infections may have a significant impact on disease outcomes.
In this study, we have demonstrated that PB1-F2 peptide from H7N9 IAV activates an NLRP3-dependent inflammasome to induce the secretion of IL-1␤. Analysis of PB1-F2 sequences from isolated human and avian H7N9 IAVs has identified only two minor amino acid changes from the avian to human isolate: G70E and S77L (3), both of which are found within the H7N9 peptide used in our study. Importantly, neither of these changes corresponds to the four amino acids that have been identified to be important for the pro-inflammatory properties of PB1-F2 (i.e. Leu-62, Arg-75, Arg-79, and Leu-82), nor are they expected to affect its aggregating potential (data not shown). Indeed, we compared the sequences of 89 human H7N9 virus isolates and found PB1-F2 to be highly conserved among all isolates (supplemental Fig. S5), particularly in the C-terminally aggregating peptide used in this study, where all four inflammatory amino acids are conserved throughout. Therefore, we would predict that, if H7N9 was to cause a pandemic, then it would likely contain a conserved, highly virulent PB1-F2 gene, further supporting that inhibition of the inflammasome is a potential strategy to reduce inflammation during infection.
In contrast to our findings demonstrating that IAV PB1-F2 peptide activates the NLRP3 inflammasome (Figs. 1-4 and Ref. 13), it was recently reported that PB1-F2 derived from PR8 and A/California(CA)/04/09 (H1N1) translocates to mitochondria, accelerating mitochondrial fragmentation to suppress inflammasome activation (26). That study found that reconstituting NLRP3 inflammasome activation in HEK293 cells by ectopically expressing NLRP3, ASC, caspase-1, and pro-IL-1␤ in the presence of PB1-F2 reduced IL-1␤ secretion. By contrast, in our previous study, we utilized reverse-engineered IAV in which PB1-F2 was absent to demonstrate the direct role of PB1-F2 in activating the inflammasome and inducing IL-1␤ secretion in vitro and in vivo in an NLRP3-dependent manner (13). Interestingly, in this study, we did observe that PB1-F2 peptide induced robust mtROS production, inhibition of which with the specific mtROS inhibitor MitoTempo reduced ROS production and inflammasome activation. These findings would therefore be consistent with PB1-F2-inducing dysregulation of the mitochondria, the result of which is the induction of the inflammasome and inflammation.
Two recent breakthrough studies also identified "prion-like" activities for ASC-containing inflammasomes whereby oligomeric inflammasome complexes released from cells following pyroptosis are engulfed by phagocytic cells to initiate inflammasome activation independent of stimuli, provided the cell had been already stimulated with signal 1 (18,27). Within the confined lung environment, excessive ASC oligomerization and inflammasome activation may therefore induce a cellular hyperinflammatory state contributing to the cytokine storm and morbidity in the later stages of infection. Secondary bacterial infection is a leading cause of death during severe IAV infection and has been reported in human cases of H7N9 (28). Induction of inflammasome-driven pyroptosis by PB1-F2 may also lead to removal of alveolar macrophages and epithelial cell barriers, which may result in increased susceptibility to secondary bacterial infections causing pneumonia (29). We have demonstrated previously that PR8 or 1918 pandemic PB1-F2 proteins induce inflammatory disease that predisposes mice to secondary bacterial pneumonia (15). Indeed, IAV RNA and the IAV M2 ion channel protein are potential NLRP3 inflammasome activators that, during infection with IAV strains lacking full-length PB1-F2, may be responsible for initiating "protective" inflammasome activation, leading to protective immunity, increased disease tolerance through cellular recruitment, and induction of tissue repair (4,5,7).
The emergence of H7N9 avian IAV and the high levels of mortality associated with infections pose a considerable risk to human health. Recently, genetic analysis of H7N9 strains from human isolates and those isolated from poultry found considerable reassortment among viruses in the influenza ecosystem of China and concluded that it is reasonable to consider H7N9 a major candidate to emerge as a pandemic strain in humans and cause a substantial number of severe human infections (3). Given the potentially dire consequences of a virulent pandemic IAV outbreak, the emergence of viral resistance to existing antiviral therapies and the requirement to produce and disseminate a vaccine, there is a clear need to identify the molecular mechanisms of IAV-induced hyperinflammation and therapeutic strategies to reduce the inflammatory burden associated with pathogenic IAV infections.
Overall, our results suggest that H7N9 PB1-F2 represents a major virulence factor and mediator of excessive inflammation and that therapeutic targeting of the NLRP3 inflammasome may constitute a viable treatment strategy to decrease the excessive inflammation associated with pathogenic IAV, such as the H5N1 and H7N9 avian strains. Recently, we demonstrated that temporal administration of MCC950 to inhibit NLRP3 inflammasome activation delayed lethality associated with highly virulent PR8 challenge and reduced lung inflammation and cellular influx (11).
Given that we have now demonstrated the capacity of the human isolate H7N9 PB1-F2 peptide to induce NLRP3 inflammasome activation (Fig. 1) and amelioration of inflammation with MCC950 (Figs. 5 and 7), interventions with small-molecule NLRP3 inhibitors could be used therapeutically to augment current antiviral and vaccination strategies to potentially treat IAV outbreaks such as H7N9. Highly specific small-molecule inhibitors such as MCC950 have a relatively short half-life in vivo, and, therefore, the timing and dosing of treatments could be cost-effective and easily controlled (23). Given the need to stockpile antivirals for prophylactic protection and the limited therapeutic options available to infected individuals, our study not only identifies PB1-F2 as a major virulence factor in pandemic IAV infections but acknowledges that therapeutically targeting activation of the inflammasome may be an effective strategy to reduce IL-1␤ maturation in the lung to protect against the damaging hyperinflammatory effects characteristic of lethal infections.
Quantification of Cytokines-For the detection of IL-1␤, cultured supernatants were collected and stored at Ϫ80°C. Mouse IL-1␤ and human IL-1␤ were quantified by ELISA according to the instructions of the manufacturer (BD Biosciences). Levels of IL-1␤ in mouse BAL fluid were quantified by ELISA according to the instructions of the manufacturer.
Live-cell Bioimaging-NLRP3-deficient immortalized macrophages stably expressing ASC-cerulean and NLRP3 were seeded in 8-well Ibidi chamber slides 24 h prior to stimulation. Cells were pretreated with MitoTempo (20 M, Sigma-Aldrich) for 10 min prior to challenge or stimulated with pHrodo-labeled H7N9 PB1-F2 peptide (100 g/ml) where indicated at 37°C. Imaging was performed on a Leica SP5 multichannel Acousto Optical Beam Splitter confocal laser-scanning microscope equipped with a temperature-and CO 2 -controlled sample chamber for live-cell imaging. Images are deconvoluted z stacks by overlapping tile scanning processed using Imaris software.
Caspase-1 and IL-1␤ Immunoblot-Primary BMDMs were plated into 6-well plates 24 h prior to priming with LPS (200 ng/ml, Enzo Life Sciences) for 3 h in serum-free medium. Macrophages were pretreated with inhibitors (as indicated) 40 min prior to stimulation with the indicated ligands or left unstimulated for a further 6 h. Protein lysates were boiled in SDS-PAGE sample buffer proteins while cultured supernatants were concentrated using Strataclean resin (Agilent). Concen-trated supernatants were separated by 4 -12% SDS-PAGE and transferred to PVDF membranes. Caspase-1 and IL-1␤ were imaged by immunoblotting with anti-mouse caspase-1 monoclonal antibody (AdipoGen Life Sciences) and anti-mouse IL-1␤ detection biotinylated IgG antibody (R&D Systems), respectively. Caspase-1 was imaged using chemiluminescence, and IL-1␤ was imaged using anti-streptavidin Alexa Fluor 680 conjugate (Life Technologies).
PB1-F2 Peptide Challenge and Influenza Virus Infection of Mice-6-to 8-week-old male or female C57BL/6 and NLRP3 knockout mice were maintained in the specific pathogen-free physical containment level 2 (PC2) animal research facility in the Monash Medical Centre animal facility. All experimental procedures were approved by the Monash Medical Centre Animal Ethics Committee.
Mice were anesthetized and inoculated with 50 g of PR8 or H7N9 PB1-F2 peptides alone or in combination with MCC950 intranasally (5 mg/kg). Mice were euthanized 7 h following treatment. BAL was obtained from euthanized mice by flushing the lungs three times with 1 ml of PBS.
Recovery and Characterization of Leukocytes from Mice-For flow cytometric analysis, BAL cells were treated with red blood cell lysis buffer (Sigma-Aldrich), and cell numbers and viability were assessed via trypan blue exclusion using a hemacytometer. BAL cells were incubated with supernatants from hybridoma 2.4G2 to block Fc receptors. Neutrophils (Ly6G ϩ ) were quantified by flow cytometry as described previously (30). Living cells (propidium iodine-negative) were analyzed using a BD FACSCanto II flow cytometer (BD Biosciences), and total cell counts were calculated from viable cell counts performed via trypan blue exclusion.
Statistical Analysis-When comparing three or more sets of values, a one-way analysis of variance (ANOVA) was used with Tukey's post hoc analysis. Student's t test was used when comparing two values (two-tailed, two-sample equal variance). p Ͻ 0.05 was considered statistically significant. Survival proportions were compared using the Mantel-Cox log-rank test. p Ͻ 0.05 was considered statistically significant.