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, 9 (5), e1003392

Activation of the NLRP3 Inflammasome by IAV Virulence Protein PB1-F2 Contributes to Severe Pathophysiology and Disease

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Activation of the NLRP3 Inflammasome by IAV Virulence Protein PB1-F2 Contributes to Severe Pathophysiology and Disease

Julie L McAuley et al. PLoS Pathog.

Abstract

The ability for a host to recognize infection is critical for virus clearance and often begins with induction of inflammation. The PB1-F2 of pathogenic influenza A viruses (IAV) contributes to the pathophysiology of infection, although the mechanism for this is unclear. The NLRP3-inflammasome has been implicated in IAV pathogenesis, but whether IAV virulence proteins can be activators of the complex is unknown. We investigated whether PB1-F2-mediated activation of the NLRP3-inflammasome is a mechanism contributing to overt inflammatory responses to IAV infection. We show PB1-F2 induces secretion of pyrogenic cytokine IL-1β by activating the NLRP3-inflammasome, contributing to inflammation triggered by pathogenic IAV. Compared to infection with wild-type virus, mice infected with reverse engineered PB1-F2-deficient IAV resulted in decreased IL-1β secretion and cellular recruitment to the airways. Moreover, mice exposed to PB1-F2 peptide derived from pathogenic IAV had enhanced IL-1β secretion compared to mice exposed to peptide derived from seasonal IAV. Implicating the NLRP3-inflammasome complex specifically, we show PB1-F2 derived from pathogenic IAV induced IL-1β secretion was Caspase-1-dependent in human PBMCs and NLRP3-dependent in mice. Importantly, we demonstrate PB1-F2 is incorporated into the phagolysosomal compartment, and upon acidification, induces ASC speck formation. We also show that high molecular weight aggregated PB1-F2, rather than soluble PB1-F2, induces IL-1β secretion. Furthermore, NLRP3-deficient mice exposed to PB1-F2 peptide or infected with PB1-F2 expressing IAV were unable to efficiently induce the robust inflammatory response as observed in wild-type mice. In addition to viral pore forming toxins, ion channel proteins and RNA, we demonstrate inducers of NLRP3-inflammasome activation may include disordered viral proteins, as exemplified by PB1-F2, acting as host pathogen 'danger' signals. Elucidating immunostimulatory PB1-F2 mediation of NLRP3-inflammasome activation is a major step forward in our understanding of the aetiology of disease attributable to exuberant inflammatory responses to IAV infection.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. PR8 PB1-F2 increases cellularity and IL-1β secretion during infection in the lungs.
Mice (n = 5, 6–8 weeks, C57BL/6) were intranasally infected with 100 PFU X31 or ΔPB1-F2/X31 virus or PBS and BAL-F was collected at 24 hpi. Figure shows A) cellular content of the BAL-F examined by flow cytometry for neutrophils, macrophages and dendritic cells (DCs); B) IL-1β secreted into the BAL-F determined by ELISA. See also Figure S1. Results show the mean ± SEM and are representative of two independent experiments. *** p<0.001, ** p<0.01, * p<0.05 and ns = p>0.05, ANOVA Tukey post hoc, compared to all other groups, or as indicated; C) A representative hematoxylin and eosin stained section of lung tissue obtained from a mouse infected with a sub-lethal dose of X31 at 72 hpi. The section shows perivascular infiltration of lymphocytes, macrophages and neutrophils into interstitial regions, areas of focal necrosis of terminal airways and prominent cellular debris associated with acute hemorrhage into alveoli. The number of foci of inflammatory cells seen in both the interstitium and alveoli was noticeably greater in these mice than in mice infected with D) ΔPB1-F2/X31 at 72 hpi. E) Lung viral titers (PFU/mL) of mice infected with either X31 or ΔPB1-F2/X31 were determined by plaque assay at 24 h and 72 h post-infection (ns: p>0.05 Student's unpaired T-test at each time point). F) The percentage change from the starting weight of mice infected with X31 or ΔPB1-F2/X31 (**p<0.01, ***p<0.001 compared to all other groups on that day, ANOVA Tukey post-hoc).
Figure 2
Figure 2. PB1-F2 peptide derived from pathogenic IAV increases cellularity and IL-1β secretion in the lungs.
Mice (n = 5, 6–8 weeks, C57BL/6) were intranasally exposed to 100 µg PR8 or Wuhan peptide or PBS and BAL-F was collected at 24 hpi. Figure shows A) cellular content of the BAL-F examined by flow cytometry for neutrophils, macrophages and dendritic cells (DCs); B) IL-1β secreted into the BAL-F determined by ELISA. See also Figure S1. Results show the mean ± SEM and are representative of two independent experiments. *** p<0.001, ** p<0.01, * p<0.05 and ns = p>0.05, ANOVA Tukey post hoc, compared to all other groups, or as indicated; C) A representative hematoxylin and eosin stained section of lung tissue obtained from a mouse 24 h after exposure to PR8 peptide. Analysis revealed similar cellular responses compared to mice infected with X31 at the same time point (not shown). The section shows diffuse infiltration of inflammatory cells into the interstitium and alveoli compared to D) minimal rare, perivascular infiltration of lymphocytes in mice exposed to peptide derived from the H3N2 seasonal strain A/Wuhan/359/1995.
Figure 3
Figure 3. PR8 PB1-F2 aggregates activate the NLRP3 inflammasome.
A) Wild-type immortalized bone marrow derived macrophages (BMMs) were either primed or not with 100 ng/mL of LPS for 3 h prior to stimulation with a range of doses (10–50 µg/mL) of PR8 PB1-F2 or Wuhan PB1-F2 peptide as indicated for a further 6 h. Results show LPS priming of BMMs was required to induce IL-1β secretion in wild-type cells by PR8 PB1-F2 peptide. * p<0.05 and ** p<0.01 compared to negative (non-stimulated, -) control, ns = not significant, ANOVA Dunnett's Multiple Comparison test. B) Immortalized BMMs were primed with LPS and treated with indicated doses of molecular weight fractionated PR8 PB1-F2 for 6 h. Cellular supernatants were collected and analyzed for IL-1β secretion by ELISA according to manufacturer's instructions. Results are representative of three independent experiments and are represented as mean ± SEM. ND = not detectable. * p<0.05 and ** p<0.01 compared to NS (non-stimulated) control, ANOVA Dunnett's Multiple Comparison test.
Figure 4
Figure 4. PR8 PB1-F2-mediated inflammation is NLRP3-, Caspase 1- and phagocytosis- dependent and induces ASC speck formation in macrophages.
A) Immortalized NLRP3-deficient macrophages stably reconstituted with ASC-cerulean (blue) and NLRP3 were seeded at 105/mL in 35 mm glass bottom dishes 24 h prior to stimulation with pHrodo-labeled (red) PB1-F2 peptide. Time-lapse imaging was performed for 3.5 h. Representative images shown are flattened maximum intensity projections of 3D deconvolved z-stacks using Imaris. Scale bar is 3 µm. See Video S1 and S2. Immortalized BMMs were primed with LPS, then B) pre-treated with 20 µM Latrunculin A (Lat A) for 45 min to inhibit phagocytosis, prior to stimulation with PR8 PB1-F2, nigericin (10 µM), silica (125 µg/mL), or unstimulated (NS) for 6 h; C) pre-treated with 10 µM caspase-1 inhibitor z-YVAD for 45 min, prior to stimulation with PR8 PB1-F2 (50 µg/mL), nigericin (10 µM), silica (125 µg/mL), or unstimulated (NS) for 6 h. Cellular supernatants were collected and analyzed for IL-1β secretion by ELISA according to manufacturer's instructions. Results are representative of three independent experiments and are represented as mean ± SEM. ** p<0.01 and *** p<0.001, Unpaired t-test. D) Immortalized wild-type macrophages were seeded at 2×106 cells in 6 well plates, 24 h prior to 3 h priming with LPS (200 ng/ml) in serum free media. Cells were pretreated (where indicated) with the caspase-1 inhibitor z-YVAD (20 µM) for 40 mins and then exposed to either PR8 or Wuhan PB1-F2 (50 µg/mL), or silica (125 µg/ml) for 6 h. Proteins were concentrated from cultured supernatants and separated by 4–12% SDS-PAGE, transferred to nitrocellulose and immunoblotted for caspase-1 cleavage. Result represents one of three independent experiments.
Figure 5
Figure 5. Murine cells unable to express NLRP3-inflammasome complex activators are not stimulated by PR8 PB1-F2 peptide.
Bone marrow derived macrophages derived from A) wild-type, B) Caspase-1, C) ASC, and D) NLRP3-deficient mice were primed with LPS (100 ng/mL) for 3 h and then stimulated with PR8 PB1-F2 peptide, nigericin (10 µM), silica (125 µg/mL), or transfected with poly (dA∶dT) (250 ng/mL), or left unstimulated (NS) for a further 6 h. Priming of cells was confirmed by disrupting cellular membranes of unstimulated cells by repeated freezing and analysis of cellular lysate IL-1β concentrations. Cellular supernatants were collected and analyzed for IL-1β secretion by ELISA according to manufacturer's instructions. Results are representative of three independent experiments and are represented as mean ± SEM. **p<0.01 compared to NS, ANOVA Dunnett's Multiple Comparison Test. E) Wild type and NLRP3-deficient macrophages were primed with 100 ng/mL of LPS for 3 h prior to infection with 10MOI X31, ΔPB1-F2/X31 or infection media (vehicle only). Cellular supernatants were collected and analyzed for IL-1β secretion by ELISA. * p<0.05, *** p<0.001 ANOVA, Tukey post-hoc.
Figure 6
Figure 6. Induction of inflammasome activation by PR8 PB1-F2 peptide occurs rapidly in vivo.
Wild-type and NLRP3-deficient (NLRP3−/−) mice (n = 4–5 per group) were intranasally infected with 100 PFU X31 or ΔPB1-F2/X31 virus, or treated with PBS or 100 µg PR8 PB1-F2 peptide. BAL-F was assessed 2 d later for virus infected and 5 h later for peptide challenged mice, by flow cytometry for detection of A) and E) leukocytes (CD45+) and B) and F) neutrophils; and via ELISA for C) and G) IL-1β and D) and H) TNFα levels. These data are representative of two independent experiments. Error bars represent mean ± SEM. ΦΦΦ p = 0.0004, ΦΦ p = 0.0069, Φ p = 0.0243 Student's unpaired T-test; ns: p>0.05, *p<0.05, **p<0.01 and ***p<0.001 one-way ANOVA Tukey post-hoc.
Figure 7
Figure 7. PB1-F2 induction of IL-1β secretion of IL-1β by human PBMCs is caspase-1 dependent.
Human PBMCs from three individuals were primed with LPS (50 pg/mL) for 3 h or not, then stimulated with A) PR8 PB1-F2 or B) PR8 PB1-F2, nigericin (10 µM), silica (125 µg/mL), or ATP (100 mM) for a further 6 h. Cultured supernatants were assayed for IL-1β by ELISA. C) LPS-primed hPBMCs were stimulated with PR8 PB1-F2 or silica (125 µg/mL) in the presence or absence of the caspase-1 inhibitor z-YVAD for 6 h. NS = not-stimulated. Results are the mean ± SEM of three independent donors. **p<0.01, ***p<0.001 compared to NS, ANOVA Dunnett's Multiple Comparison Test.

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Funding was from National Health and Medical Research Council (NHMRC) grants 566780 and 606976, and the Victorian Government's Operational Infrastructure Support Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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