Published April 28, 2017, doi:10.4049/jimmunol.1600714 The Journal of Immunology

AIM2 Inflammasome Is Critical for Influenza-Induced Lung Injury and Mortality

Hongbo Zhang,* Jiadi Luo,*,† John F. Alcorn,* Kong Chen,* Songqing Fan,*,† Joseph Pilewski,‡ Aizhong Liu,x Wei Chen,* Jay K. Kolls,* and Jieru Wang*

The absent in 2 (AIM2) inflammasome plays an important role in many viral and bacterial infections, but very little is known about its role in RNA virus infection, including influenza A virus (IAV). In this study, we have designed in vivo and in vitro studies to determine the role of AIM2 in infections with lethal doses of IAVs A/PR8/34 and A/California/07/09. In wild-type mice, IAVinfection enhanced AIM2 expression, induced dsDNA release, and stimulated -1 activation and release of cleaved IL-1b in the lung, which was significantly reduced in AIM2-deficient mice. Interestingly, AIM2 deficiency did not affect the transcription of caspase-1 and IL-1b. In addition, AIM2-deficient mice exhibited attenuated lung injury and significantly improved survival against IAV challenges, but did not alter viral burden in the lung. However, AIM2 deficiency did not seem to affect adaptive immune response against IAV infections. Furthermore, experiments with AIM2-specific small interfering RNA–treated and AIM2-deficient human and mouse lung alveolar macrophages and type II cells indicated a macrophage-specific function of AIM2 in regulation of IAV-stimulated proinflammatory response. Collectively, our results demonstrate that influenza infection activates the AIM2 inflammasome, which plays a critical role in IAV-induced lung injury and mortality. AIM2 might serve as a therapeutic target for combating influenza-associated morbidity and mortality without compromising the host antiviral responses. The Journal of Immunology, 2017, 198: 000–000.

he inflammasome is a multiprotein complex that activates fense against the cytosolic bacterium Francisella and Listeria caspase-1 (Casp1) and results in cleavage of IL-1b (1, 2). monocytogenes and Mycobacterium tuberculosis (12–14). To T Different from other host defense mechanisms, the date, very little is known about whether the AIM2 inflammasome inflammasome uses intracellular pattern recognition receptors to is activated during RNA virus infections, including influenza A sense pathogen and danger-associated molecular patterns to virus (IAV), which can cause life-threatening diseases, especially guard the host. Absent in melanoma 2 (AIM2), a member of in high-risk groups. the pyrin and HIN200 domain-containing family (3–5), IAV, an ssRNA virus, is one of the most important pathogens is an intracellular pattern recognition receptor that can form an for seasonal and pandemic respiratory illness. In severe cases of inflammasome by directly binding to dsDNA from virus, bac- lower respiratory tract infection, IAV infects lung epithelial cells teria, or the host itself (5–7). The role of the AIM2 inflamma- and macrophages, and causes diffuse alveolar damage and ex- some has been reported in many viral infections including cessive inflammatory responses (12, 15, 16). By analyzing our murine CMV, , and HSV (7–11), and is critical in de- previously published microarray data (17, 18), we found that IAV significantly increased expression of AIM2 in both human primary alveolar type II (ATII) cells and alveolar macrophages *Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224; †Department of Pathology, Second Affiliated Xiangya Hospital, Central (AMs), the important targets for influenza infection (12, 15, 16). South University, Changsha 410078, China; ‡Pulmonary, Allergy, and Critical Care This led us to hypothesize that AIM2 might participate in reg- Medicine Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224; and xDepartment of Epidemiology and Biostatistics, ulating the influenza-induced proinflammatory response. In this School of Public Health, Central South University, Changsha 410078, China study, we sought to determine the role of AIM2 in influenza- ORCIDs: 0000-0002-9689-9350 (H.Z.); 0000-0001-6980-5454 (K.C.); 0000-0002- induced disease using in vitro human and mouse lung primary 0893-1155 (J.P.). cells in combination with an in vivo mouse model of influenza A Received for publication April 28, 2016. Accepted for publication March 24, 2017. infections. This work was supported by National Institutes of Health Grants R03AI101953 and Our results indicate that IAVs A/PR8/34 (PR8, a widely used R01HL113655, startup funding from the University of Pittsburgh (to J.W.), and the mouse adaptive strain) and A/California/07/09 (CA07, a clinical Cystic Fibrosis Foundation Research Development Program. isolate) activate the AIM2-dependent inflammasome. AIM2 is Address correspondence and reprint requests to Dr. Jieru Wang, Department of Pe- diatrics, University of Pittsburgh School of Medicine, 4401 Penn Avenue, Rangos critical for virus-induced Casp1 activation and cleavage and release 9126, Pittsburgh, PA 15224. E-mail address: [email protected] of IL-1b from the lung, but not for virus-stimulated increase The online version of this article contains supplemental material. in transcription of these two . Deficiency in AIM2 leads to Abbreviations used in this article: AIM2, absent in melanoma 2; AM, alveolar mac- attenuated lung injury and inflammation, and significantly im- rophage; ATII, alveolar type II; BAL, bronchoalveolar lavage; BALF, BAL fluid; proves survival following lethal IAV infections. AIM2 deficiency CA07, A/California/07/09; Casp1, caspase-1; cCasp1, cleaved Casp1; cIL-1b, cleaved IL-1b; dpi, day postinfection; hpi, hour postinfection; IAV, influenza A virus; appears to be dispensable for host antiviral defense and shaping LDH, lactate dehydrogenase; moi, multiplicity of infection; PR8, A/PR8/34; siAIM2, the adaptive immune response. In addition, AIM2 plays a proin- AIM2-specific siRNA; siRNA, small interfering RNA; WT, wild-type. flammatory role specifically in human and mouse AMs, but not This article is distributed under The American Association of Immunologists, Inc., ATII cells. Our results suggest that the function of AIM2 is fo- Reuse Terms and Conditions for Author Choice articles. cused on the innate immune response, and AIM2 is a detrimental Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 host factor for influenza-induced lung injury and mortality.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600714 2 AIM2 DEFICIENCY CONVEYS PROTECTION AGAINST IAV INFECTION

Materials and Methods BAL and lung tissue processing Human lung donors At the indicated time points, mice were euthanized by i.p. injection of Deidentified patient lungs that were not suitable for transplantation and sodium pentobarbital (Henry Schein Animal Health, Lake Forest, IL). donated for medical research were obtained through the International In- The lungs were lavaged with 1 ml of sterile saline solution, and BALF was stitute for the Advancement of Medicine (Edison, NJ) and the Airway collected and centrifuged at 4˚C, 3000 rpm for 10 min. An aliquot of Epithelial Core at the University of Pittsburgh as we described previ- 100 ml of cell-free BALF was snap frozen by dry ice-ethanol bath for ously (17, 19). The Committee for Oversight of Research and Clinical evaluation of viral burden by plaque assay. Another aliquot of 200 mlof Training Involving Decedents and University of Pittsburgh Institutional cell-free BALF was treated with protease inhibitors (Fisher Scientific, Waltham, MA) and stored at 280˚C for analysis of protein expression by Review Board approved use of the human tissues. The donors used in 2 this study included eight male and eight female donors with average age Western blotting. The rest of the BALF was stored at 80˚C for detection of 50.2 years; there were seven current smokers, one ex-smoker, and of albumin, lactate dehydrogenase (LDH), and cytokine by ELISA. BAL eight nonsmokers. cell cytospin slides were stained with a HEMA-3 stain kit (Fisher Scien- tific) for inflammatory cell differential counts. Right superior, middle, and Mice inferior lobes were collected and homogenized in 1 ml of sterile ice-cold PBS at 4˚C using gentleMACS Dissociator (Miltenyi Biotec, San Diego, 2 2 2 2 AIM2 knockout ( / ), NLRP3 / , and wild-type (WT) C57BL/6J mice CA). Tissue-free lung homogenates were collected and stored at 280˚C for 2 2 were purchased from The Jackson Laboratory (Bar Harbor, ME); ASC / use in plaque assay and Western blotting as described earlier. The post- mice were from Genentech (San Francisco, CA). All mice were bred in- caval lobe was saved for RNA assay. Left lobe was fixed with 10% neutral 2 2 house, and AIM2 / mice were further backcrossed with C57BL/6J mice buffered formalin (EMD Millipore, Billerica, MA) and subjected to sub- for two more generations. Mice were maintained under pathogen-free sequent H&E staining. conditions within the animal facilities at the Children’s Hospital of Pittsburgh of University of Pittsburgh Medical Center. All animal studies Real-time RT-PCR were performed on age- and sex-matched mice and conducted with ap- proval from the University of Pittsburgh Institutional Animal Care and Use Lung tissue RNA was extracted from the postcaval lobe using TRIzol re- Committee. agent (Invitrogen, Carlsbad, CA), and cellular RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany). One microgram of RNA was Viruses used as a template to generate cDNA using qScript cDNA Synthesis kit (Quanta Bioscience, Gaithersburg, MD). cDNA was then used in standard Influenza PR8, a laboratory-adapted H1N1 virus, was originally provided by real-time PCR to measure expression using the Applied Biosystems Dr. K. Hartshorn from Boston University and CA07, a 2009 pandemic 7900HT (Life Technologies, Carlsbad, CA). Reaction conditions were 95˚C H1N1 virus, was provided by Dr. T. Ross at University of Georgia. Both for 15 s and 60˚C for 1 min, repeated for 40 cycles, with a 10-min hot start viruses were propagated in MDCK cells as previously described (19); the at 95˚C. Relative mRNA level was quantified using the 2DCt method and concentration of viruses were titrated using plaque assay as previously standardized to the level of GAPDH. The TaqMan real-time PCR probes described (17, 19). In addition, LD50 was determined according to Reed (Thermo Fisher Scientific, Waltham, MA) used in this study are human and Muench’s method (20). For PR8, 1LD50 equals 80 PFU; for CA07, AIM2 (Hs00915710_m1), IL-1b (Hs00174097_m1), and GAPDH 1LD50 equals 10,000 PFU. (Hs02758991_g1), as well as mouse AIM2 (Mm01295719_m1), IL-1b (Mm00434228_m1), and GAPDH (Mm99999915_g1). Infection of primary human and murine ATII cells and AMs with IAVs Western blotting Human ATII cells and AMs were isolated from deidentified donor lungs and Lung tissue and BAL were quantified using BCA protein assay kit cultured as we described previously (17, 19). Mouse ATII cells from age- (Bio-Rad, Hercules, CA). The primary Abs used in this study were rabbit anti- and sex-matched WT and AIM22/2 mice were isolated as previously de- Casp1 (ab108362; Abcam, Cambridge, MA), mouse anti-Casp1 (MAB6215; scribed (21) and plated in DMEM with 5% rat serum in 37˚C incubator R&D Systems), rabbit anti-IL-1b (ab9722; Abcam), mouse anti–IL-1b with 5% CO2 for 2 d, and then cultured with 1% charcoal-stripped FBS (12242S; Cell Signaling, Danvers, MA), rabbit anti–b-actin (A2066; Sigma- plus 10 ng/ml keratinocyte growth factor (R&D Systems, Minneapolis, Aldrich, St. Louis, MO), and goat anti-GAPDH (SAB2500451; Sigma). For MN) for 4 d preinfection. For mouse AM isolation, lungs from WT detection of Casp1 and IL-1b abundance, 30 mg of lung homogenates or and AIM22/2 mice were lavaged with 1 ml of sterile saline 13 times; AMs 5 mg of BAL protein per sample was loaded and separated by 4–15% Mini- were collected by centrifuging the bronchoalveolar lavage (BAL) fluid PROTEAN TGX Precast Gels (Bio-Rad) and further transferred to PVDF (BALF) at 1000 rpm at 4˚C for 10 min and were cultured in DMEM with membranes (Bio-Rad). Blots were then probed with primary Abs against IL- 5% rat serum overnight before viral infection. 1b or Casp1 and subsequently probed with HRP-conjugated secondary Prior to infection, human and mouse cells were washed once with DMEM Abs (Jackson ImmunoResearch Laboratories, West Grove, PA). In the end, and then infected with PR8 or CA07 at multiplicity of infection (moi) of 1 membranes were developed using ECL Western blotting Substrate (Pierce, for 1 h at 37˚C. Cells were washed postinfection and fresh media were Junction City, OH), and images were captured with Fujifilm LAS-3000 added to the culture. At the designated time points postinfection, cells were (Fujifilm Life Science, Stanford, CA) and quantified using ImageJ soft- harvested for evaluation of , viral replication, and cytokine ware (National Institutes of Health, Bethesda, MD). secretion. Analysis of dsDNA in cell-free BALF AIM2 small interfering RNA treatment of human ATII cells and AMs The amount of dsDNA in cell-free BALF from uninfected and infected mice was measured using the QuantiFluor dsDNA kit (Promega, Madison, For knockdown of AIM2 gene expression, 50 nM small interfering RNA WI) following manufacturer’s instructions. The DNA pattern was further (siRNA) against human AIM2 or nontarget control (Dharmacon, Lafayette, evaluated on Agilent High Sensitivity D1000 ScreenTape (Agilent, Santa CO) was transfected into human ATII cells and AMs using GenomONE Clara, CA). HVJ Envelope Vector Kit (Cosmo Bio, Carlsbad, CA) as we described previously (22). Cells were allowed to express transgene for 48 h before Detection of influenza-specific IgG in mouse serum influenza infection, and AIM2 expression level was confirmed by real-time ELISA was used to detect influenza virus–specific IgG in mouse serum RT-PCR with GAPDH normalization. samples as described previously (23). In brief, formalin-inactivated PR8 Mouse influenza infection was used as Ag to coat Nunc 96-well plate (eBioscience) at 4˚C overnight. After washing and blocking with BSA, the plate was incubated with 1:2 Prior to infection, WT and age- and sex-matched AIM22/2, NLRP32/2,or serially diluted serum samples from uninfected and infected mice. Anti- ASC2/2 mice were cohoused (for female) or exchanged bedding (for influenza IgG was detected using HRP-conjugated goat anti-mouse Ab male) for .1 wk. Mice were anesthetized with isoflurane and challenged (Jackson ImmunoResearch Laboratories) and developed using TMB with different doses of PR8 or CA07 in 50 ml of sterile DMEM or DMEM Substrate Set (R&D Systems). The OD was read at wavelength 450 and control intranasally or intratracheally. In designated experiments, AIM22/2 470 nm (as reference). The ratio of OD from infected versus uninfected and their littermate control mice were infected with PR8 or CA07. Mice samples was calculated for each infected sample. If the ratio was .2.1, the were monitored daily postinfection for weight loss and signs of clinical sample was designated as “positive” for influenza-specific IgG, and the illness and were harvested at the indicated time points. dilution factor of this sample was the IgG titer. The Journal of Immunology 3

Flow cytometry analyses of infected lung tissues We then evaluated whether influenza infection induces dsDNA release from host because AIM2 has been demonstrated as a critical Flow cytometric analysis was performed as previously described (24). 2/2 Single-cell suspension of homogenized mouse lungs was stained with Abs DNA sensor in host defense. We infected both WT and AIM2 against CD45 (45-0451-80), CD8 (25-0081-81), CD4 (48-0042-80) mice with PR8, collected BALF, and determined the level of (Thermo Fisher Scientific), and TCRb (560656; BD Biosciences, San Jose, dsDNA using QuantiFluor dsDNA kit (Promega). Fig. 1C con- CA). Matched isotype control Abs were used to define specific staining firmed the presence of dsDNA in cell-free BALF as early as 1 day above autofluorescence. Data were acquired using FACS LSR II (BD 2/2 Biosciences) and analyzed by FlowJo software. Percentage of blood postinfection (dpi) in both WT and AIM2 mice, and the mononuclear cells (CD45+), CD4+ T cells (CD45+TCRb+/CD4+), and amount of dsDNA was further increased at 3 dpi. No significant CD8+ T cells (CD45+TCRb+/CD8+) was determined. difference in dsDNA release was detected between WT and 2/2 Statistical analysis AIM2 groups. We further analyzed the DNA pattern from BALF samples. As shown in representative Fig. 1D, most of the All data analyses were performed with Prism 6.0 (GraphPad, La Jolla, CA). DNA fragments peaked at the size around 173 bp, which satisfies Kaplan–Meier survival curve was produced and log-rank (Mantel–Cox) test was used to compare difference between two groups. Mann–Whitney the minimum sequence length of 80 bp required for activation of U test was used for comparison of gene expression and viral replication the AIM2 inflammasome (11). between two groups. For other comparisons between two groups, two- To test whether there is functional inflammasome activation in tailed Student t test was used. influenza-infected mouse lungs, we examined the abundance of pro- and cleaved forms of Casp1 and IL-1b in BAL and lung Results homogenates from uninfected and PR8-infected mice by Western To determine the role of AIM2 in influenza infection, we first in- blotting (Fig. 2). Both pro- and cleaved forms of Casp1 and IL-1b fected primary human ATII cells and AMs from six donors with IAV were present in uninfected and infected lung tissues (Fig. 2A), PR8 and measured expression of AIM2 at 24 h postinfection (hpi) whereas only cleaved Casp1 (cCasp1) and IL-1b were detected in by real-time RT-PCR. The results shown in Fig. 1A confirmed the BAL samples (Fig. 2B). Viral infection significantly increased the findings from our previous microarray experiments that influenza abundance of cCasp1 and cleaved IL-1b (cIL-1b) in BAL and infection significantly increased expression of AIM2 in both cell lung tissues but did not increase the levels of pro-Casp1 and pro– types (17, 18). Compared with ATII cells, the basal expression IL-1b (Fig. 2). These results indicate that influenza infection level of AIM2 is significantly higher in human AMs (Fig. 1A), but activates Casp1 and promotes processing of IL-1b. To further the influenza-induced increase is less than that in ATII cells. To determine whether AIM2 is required for inflammasome activation, analyze whether the findings from human primary cells in vitro we performed additional experiments to compare the levels of represent infection in vivo, we examined the expression of AIM2 Casp1 and IL-1b mRNA and proteins between virus-infected WT and IL-1b in PR8-infected mouse lung tissues. Consistent with the and AIM2-deficient mice (Fig. 3). AIM2 deficiency did not alter results from human lung primary cells, viral infection significantly virus-stimulated increase of the transcription of Casp1 and IL-1b increased AIM2 expression in murine lungs (Fig. 1B). (Fig. 3A). However, AIM2-deficient mice displayed significantly

FIGURE 1. Influenza A infection increases AIM2 and stimulates dsDNA release into BAL. (A) Cultured human primary ATII cells and AMs from six donors were infected with influenza virus PR8 or MOCK control (Ctrl) at moi of 1, and RNA was extracted at 24 hpi for evaluation of AIM2 expression by real-time PCR (17, 18). Data show the relative expression of AIM2 and IL-1b mRNA normalized to the expression of human housekeeping gene GAPDH. 2/2 (B–D) WT and AIM2 mice were challenged with 50LD50 of PR8 virus or DMEM control (Ctrl) intranasally. Lung tissues (B) were harvested at 3 dpi for evaluation of AIM2 and IL-1b expression by real-time RT-PCR. Data illustrate the relative expression of AIM2 normalized to mouse GAPDH. (C and D) Cell-free BALF was collected at 1 and 3 dpi for quantification of dsDNA (C) using QuantiFluor dsDNA kit (Promega) and analysis of DNA pattern (D)on Agilent High Sensitivity D1000 ScreenTape (Agilent). *p , 0.05, **p , 0.01, compared with the Ctrl group. 4 AIM2 DEFICIENCY CONVEYS PROTECTION AGAINST IAV INFECTION less abundance of cCasp1 and cIL-1b in lung tissues (Fig. 3B) and deficiency affects influenza virus-induced acute lung injury, we BAL (Fig. 3C) than WT mice. No difference was present in examined the lung barrier function and inflammation at 3 dpi proforms of these two proteins between the two groups. Further (Fig. 4C–F). BAL samples from PR8-infected AIM22/2 mice quantification of Western blot signal intensity indicated that AIM2 exhibited a significantly lower amount of LDH (Fig. 4C) and al- deficiency reduced around 60% of cIL-1b and cCasp1 (Fig. 3). bumin (Fig. 4D), decreased TNF-a (Fig. 4E), and reduced pro- We further validated these results in additional experiments with portion of neutrophils and increased proportion of macrophages AIM2 littermate controls. As shown in Supplemental Fig. 1, PR8- (Fig. 4F) indicating attenuated lung injury and inflammation when infected AIM2 homozygous knockouts, heterozygous littermates, compared with the samples from WT mice. As expected, high-dose and WT littermate control mice showed similar levels of pro- infection caused more weight loss, higher viral burden, and more Casp1 and pro–IL-1b. However, significant reduction in cCasp1 lung injury and inflammation than low-dose infection (Fig. 4A–F). and cIL-1b was present in AIM2 homozygous knockouts com- Despite the significant weight loss, viral burden, and obvious in- pared with WT controls. The level of cCasp1 and cIL-1b in AIM2 dication of lung injury (Fig. 4A–F), PR8-infected mice did not heterozygous tended to be higher than homozygous knockouts, show significant tissue damage as represented in H&E staining but lower than WT controls, although the difference was not of lung sections from WT or AIM2-deficient mice at low dose of statistically significant due to the variation (Supplemental Fig. 1), infection (data not shown). At early stage of high-dose infection (day suggesting a possible gene dosage effect. These results clearly 3) mild inflammatory cell infiltration around bronchus and edema demonstrate that AIM2 is critical for influenza-stimulated acti- around blood vessels were observed in WT, but not in AIM22/2 vation of Casp1 and processing of IL-1b independent of tran- mice (Fig. 4G). At 9 dpi, when the viral burden had been almost scriptional regulation. cleared (data not shown), infected WT mouse lungs displayed sig- To further determine the role of AIM2 in influenza pathogenesis, nificant lung destruction and inflammatory cell infiltration in pa- 2/2 we challenged WT and AIM2 mice with low (0.5LD50) and renchyma, whereas the cell infiltration was mainly limited in high doses (50LD50) of PR8 virus and evaluated the weight loss, parabronchi in AIM2-deficient mice (Fig. 4G). These results indicate viral replication, and lung damage (Fig. 4). Both WT and AIM22/2 that AIM2 deficiency attenuates influenza-induced lung damage. mice showed significant weight loss and infectious virus release To further validate the findings with contemporary IAV, we ex- with viral infections, but no difference was detected between amined viral infections in WT and AIM22/2 mice with pandemic WT and AIM22/2 groups (Fig. 4A, 4B). To test whether AIM2 influenza CA07 (Figs. 5, 6). Consistent with the findings from PR8

FIGURE 2. Influenza infection activates the inflammasome. Six-week-old C57B/6 mice were infected with PR8 (50LD50) or same volume of DMEM (Ctrl) intranasally. Mice were harvested at 3 dpi, and 5 mg cell-free BALF or 30 mg of lung tissue was analyzed for the expression of Casp1 and IL-1b by Western blotting. (A) Lung homogenate. (B) BAL. Representative images of Western blotting (left panel). Quantification of Western blotting results using ImageJ software (National Institutes of Health) (right panel). Protein abundance was shown as relative abundance to the level of b-actin in the same sample. n =4.**p , 0.01, compared with the Ctrl group. The Journal of Immunology 5

FIGURE 3. AIM2 deficiency reduces PR8-induced activation of the inflammasome. Sex- and age-matched WT and AIM22/2 mice were challenged with

PR8 (50LD50) or DMEM (Ctrl) intranasally. Mice were harvested at 3 dpi for evaluation of expression of Casp1 and IL-1b gene and proteins by real-time RT-PCR and Western blotting, respectively. (A) Gene expression. (B and C) Protein expression. (Left panels) Representative images of Western blotting of lung homogenates (B) and BAL samples (C). (Right panels) Quantification of Western blotting results. n =6.*p , 0.05, **p , 0.01, compared with the Ctrl group; ##p , 0.01, ####p ,0.0001, between AIM22/2 and WT groups. infection, CA07 infection led to inflammasome activation, and To further examine whether AIM2 deficiency provides protec- AIM2 deficiency significantly reduced cleavage of Casp1 and tion against influenza-induced mortality, we compared mouse processing of IL-1b (Fig. 5), but did not show significant changes survival between WT and AIM22/2 groups with lethal doses of in weight loss or viral replication when compared with WT mice PR8 and CA07 challenge. The results in Fig. 7 show that AIM2- (Fig. 6A, 6B). Also, there was less albumin (Fig. 6C), TNF-a and deficient mice display steadily improved survival compared with IL-1b (Fig. 6D, 6E), and neutrophils (Fig. 6F) present in the BAL WT mice after different doses and strains of IAV challenge. The of infected AIM22/2 mice than WT mice at 3 dpi. H&E staining survival rate in AIM22/2 mice doubled with the low dose of in- 2/2 of fixed AIM2 lung sections exhibited significantly less inflam- fection (0.5LD50) and increased to 6-fold with the high dose of matory cell infiltration and tissue damage than WT lungs at 9 dpi PR8 infection (50LD50) at the end of observation (Fig. 7A). These (Fig. 6G). These data support the results observed with PR8 in- results have been confirmed in experiments with AIM2 littermate fection that AIM2 deficiency attenuates inflammasome activation controls (Fig. 7B, left). Both AIM2 knockout homozygous and and lung injury and inflammation after influenza A infection, sug- heterozygous mice showed significantly prolonged survival com- gesting this is not a viral strain-specific phenotype. pared with littermate WT controls post PR8 infection. With CA07 To examine whether AIM2 deficiency alters adaptive immune infection, AIM2-deficient mice also showed significantly im- response against IAV infection, we evaluated the flu-specific IgG proved survival compared with WT mice (Fig. 7B, right). These level in serum samples of survivors from low-dose PR8 infection results demonstrate that AIM2 deficiency provides protection by ELISA (Supplemental Fig. 2). All samples were positive for against IAV-associated mortality. influenza-specific serum IgG at 2 wk postinfection, but no Data in Fig. 1 indicate that influenza infection increases AIM2 difference was present between AIM22 /2 and WT groups expression in primary human AMs and ATII cells, and AMs ex- (Supplemental Fig. 2A). At 8 wk postinfection, the serum IgG titer press a higher level of AIM2 than ATII. To test whether AMs are dropped, but still no difference was shown between the two groups the primary targets for AIM2 function in the lung, we performed (Supplemental Fig. 2B). With CA07 infection, we examined the additional in vitro experiments in primary murine and human lung percentage of CD4+ and CD8+ T lymphocytes in infected lungs cells (Figs. 8, 9). We infected ATII cells and AMs isolated from at 9 dpi by flow analysis (Supplemental Fig. 2C). AIM2-deficient WT and AIM22/2 groups, and compared cellular expression of mice displayed similar amounts of lung CD4+ and CD8+ cells as IL-1b gene and release of IL-1b in culture supernatants. Repre- WT mice. These data suggest that AIM2 deficiency may not affect sentative results in Fig. 8 show that viral infection induced a the adaptive immune response against IAV infections. similar level of IL-1b mRNA (Fig. 8A) and protein release 6 AIM2 DEFICIENCY CONVEYS PROTECTION AGAINST IAV INFECTION

FIGURE 4. AIM2-deficient mice display less lung injury and inflammation, but no difference in viral burden and weight loss compared with WT mice. 2/2 Sex- and age-matched WT and AIM2 mice were challenged with low (0.5LD50) or high (50LD50) dose of PR8 virus intranasally. Mice weight (A) was monitored daily (n = 10 for low-dose infection and n = 6 for high-dose infection), and viral replication (B) lung tissue and BALF were collected at 3 dpi for assays of viral burden (B), LDH (C), albumin (D), release of TNF-a by ELISA (E), and BAL cell distribution by Hema-3 staining of BAL cytospin slides (F). Mouse pathology (G) was examined by H&E staining of fixed lung sections at 3 and 9 dpi. Representative images of H&E slides (original magnification 3100) are shown. *p , 0.05, **p , 0.01, ***p , 0.001, between WT and AIM22/2 groups. The Journal of Immunology 7

FIGURE 5. AIM2 deficiency attenuates contemporary IAV-induced inflammasome activation. Sex- and age-matched WT and AIM22/2 mice were challenged with influenza CA07 (50LD50) or DMEM (Ctrl) intranasally. Mice were harvested at 3 dpi for evaluation of the abundance of Casp1 and IL-1b by Western blotting. (A) Lung homogenate. (B) BAL. Representative images of Western blotting (left panel). Quantification of Western blotting signal intensity (right panels). n =4.*p , 0.05, **p , 0.01, compared with the Ctrl group; #p , 0.05, ##p , 0.01, between AIM22/2 and WT groups.

(Fig. 8B) in both WT and AIM2-deficient ATII cells. However, IL- post IAV infections (Figs. 3B, 5, Supplemental Fig. 1). These results 1b production was impaired in AMs from the AIM2-deficient indicate that AIM2 is critical for IAV-stimulated inflammasome group. We further validated the cell-specific function of AIM2 activation. Interestingly, AIM2-deficient mice do not show differ- in human ATII cells and AMs (Fig. 9) treated with nontarget or ences in Casp1 and IL-1b transcription in the lung (Fig. 3A) and AIM2-specific siRNA (siAIM2). Consistent with the results from dsDNA release (Fig. 1C) from WT mice, suggesting that AIM2 AIM2-deficient mouse cells (Fig. 8A), siAIM2 did not affect the inflammasome does not participate in transcriptional regulation of proinflammatory response in PR8-infected human ATII cells despite proinflammatory genes. There is no difference in levels of pro-Casp1 an 80% reduction in AIM2 expression achieved by the treatment in and pro–IL-1b in the lung or viral replication between WT and five tested donors (Fig. 9A). siAIM2 did not alter viral replication in AIM22/2 groups. These results indicate that the significantly re- these cells either (data not shown). However, siAIM2 significantly duced release of cIL-1b into the BAL from the AIM22/2 group is reduced PR8-stimulated IL-1b,TNF-a, and RANTES release from independent of viral replication and likely the result of significantly human AMs (Fig. 9B). Similar results were observed with CA07 decreased Casp1 activation and impaired processing of IL-1b pre- infection that siAIM2 treatment significantly reduced IL-1b,TNF- cursor because of AIM2 deficiency (Figs. 3, 5, Supplemental Fig. 1). a, and RANTES secretion (Fig. 9C) but did not alter IL-1b gene We speculate that there are two possible mechanisms for the expression (data not shown) in human AMs. These results strongly AIM2 inflammasome activation post influenza infection. One is that support a macrophage-specific role for AIM2 in regulating macrophages uptake the alveolar epithelial cells damaged by IAV influenza-stimulated proinflammatory response. (17, 22) and bring genomic dsDNA from epithelial cells into the of macrophages, which is recognized by intracellular Discussion AIM2. This hypothesis is supported by the fact that AMs from AIM2-deficient mice (Fig. 8B) and siAIM2-treated human AMs AIM2 is critical for IAV-induced inflammasome activation (Fig. 9B, 9C) produce much less IL-1b in response to IAV infec- In this study, we have demonstrated that IAV activates the AIM2 tion. Another possible mechanism is that IAV causes mitochondrial inflammasome. Viral infection increases gene expression of AIM2 damage and release of mitochondrial DNA into the cytosol, which and IL-1b in both human and mouse lung primary cells (Figs. 1A, directly activates the AIM2 inflammasome. ATII cells and AMs are 8A) and mouse lungs (Fig. 1B), which constitutes the first signal, the mitochondria-rich cells (27), and this may help to explain why “priming” signal, required for activation of the inflammasome (25, influenza virus increases AIM2 expression in both primary human 26). Additionally, viral infection stimulates dsDNA release from and mouse ATII cells and AMs (Figs. 1A, 8A). It will be inter- 2/2 infected WT and AIM2 mouse lungs (Fig. 1C), and the size of esting to determine how influenza infection causes mitochondrial dsDNA fragments (Fig. 1D) meets the minimum requirement for the damage (28, 29) in these cells and clarify the mechanism for ac- second signal, the “activation” signal, for the AIM2 inflammasome tivation of the AIM2 inflammasome in the future. (3). Moreover, significantly increased cCasp1 and cIL-1b from the BAL and lung tissue of PR8- or CA07-infected mice (Figs. 2, 3, 5, AIM2 deficiency provides protection against IAV-induced lung Supplemental Fig. 1) provides direct evidence for inflammasome injury and mortality activation by IAV. AIM2-deficient mice display significantly reduced As shown in Fig. 4, infection with PR8 causes dose-dependent Casp1 activation and cleavage of IL-1b compared with WT mice weight loss, viral replication, and lung injury in mice. Infection 8 AIM2 DEFICIENCY CONVEYS PROTECTION AGAINST IAV INFECTION

FIGURE 6. AIM2 deficiency reduces contemporary IAV-induced lung injury and inflammation but does not alter viral burden and weight loss. Sex- and 2/2 age-matched WT and AIM2 mice were challenged with CA07 (50LD50) intranasally. Mice weight (A) was monitored daily (n = 8 per group), and lung tissue and BAL fluid were collected at 3 dpi for assays of viral burden (B), albumin (C), release of TNF-a (D) and IL-1b (E), and BAL cell distribution (F). Mouse pathology (G) was examined at 9 dpi. Representative images of H&E-stained lung sections (original magnification 3200) are shown. This ex- periment was repeated once. *p , 0.05, **p , 0.01, ***p , 0.001, between WT and AIM22/2 groups. with CA07 also leads to significant weight loss and lung injury Supplemental Fig. 1), AIM2-deficient mice exhibit decreased (Figs. 6, 7). We believe that the significant lung damage and death acute lung injury and tissue damage (Figs. 4C–G, 6C–G), and is partly due to the inappropriate recognition of cytoplasmic self- significantly improved survival post IAV infections (Fig. 7). Also, DNA by AIM2; this promotes the cleavage of Casp1 and subse- AIM2-deficient mouse and human AMs show less secretion of IL- quent release of mature IL-1b (Figs. 2, 3, 5) and cytokine storm, 1b, TNF-a, and RANTES stimulated by IAV (Figs. 8, 9). To- which is often observed with severe influenza infections (30–32). gether, these results demonstrate that AIM2 deficiency attenuates IL-1b is a well-known master proinflammatory cytokine and can virus-stimulated proinflammatory response and results in pro- stimulate the secretion of many cytokines and chemokines, as well longed survival against IAV infections. Similar to other inflam- as the subsequent infiltration of immune cells into the infection masome studies (39–41) from the literature, AIM2 deficiency site. In addition, IL-1b is a critical molecule for tissue injury and apparently does not affect viral replication (Figs. 4B, 6B). AIM2 repair (33–35). Several studies have reported a detrimental role for deficiency does not affect the expression of antiviral genes in the IL-1b for severe influenza infections (36–38). We recently also lung either (data not shown). In addition, inhibition of AIM2 reported that IL-1b treatment reduces the transepithelial electrical expression by siAIM2 does not change viral replication in primary resistance of human primary alveolar epithelial cells and leads human ATII cells (data not shown), the main viral replication site to epithelial barrier injury (19). In this study, accompanied by (42, 43) in vivo, for both laboratory-adapted and contemporary the striking reduction in inflammasome activation (Figs. 3, 5, IAVs. Collectively, these results indicate that AIM2 deficiency The Journal of Immunology 9

FIGURE 8. Ams, but not ATII cells, from AIM2-deficient mice exhibit an impairment in IL-1b response post IAV infection. ATII cells and AMs isolated from WT and AIM22/2 mice were infected with PR8 at an moi of 1. At 24 hpi, RNA was harvested from cells for evaluation of IL-1b gene expression by real-time RT-PCR, and culture supernatants were collected for detection of IL-1b using Bio-Plex IL-1b singleplex set (BioRad) fol- FIGURE 7. AIM2 deficiency conveys protection against IAV-induced lowing manufacturer’s protocol. (A) ATII cells. (B) AMs. Data represent 2/2 mortality. (A) Sex- and age-matched WT and AIM2 mice were infected results from one of three experiments. with PR8 intranasally. (B) AIM22/2 and littermate AIM2+/2 and AIM2+/+ mice were infected with PR8 (100LD50 intratracheally) or CA07 (50LD50 intranasally). Mice survival was monitored daily. The low-dose and high- through increased cytokine production and lung cellular infiltrates dose survival experiment (A) was repeated once. *p , 0.05, **p , 0.01, 2/2 (39). The NLRP3 inflammasome can be activated by many con- between AIM2 and WT groups. ditions including a variety of pathogen- and damage-associated molecular patterns and different stresses (45–47). Therefore, a conveys protection to influenza-infected mice, and this protection difference in viral dose, virus production, course of infection, an- likely results from attenuation of proinflammatory response through imal facility, and the sex of mice could lead to different results. The impaired inflammasome activation. Targeting AIM2 might be an clarification of NLRP3 on influenza infection is beyond the scope effective approach to limit IAV-induced mortality or morbidity of this study and requires carefully designed future experiments. without compromising the host antiviral responses. We notice that there is still partial release of cIL-1b and cleavage AMs are the likely target cells for AIM2 function in regulating of Casp1 in PR8- or CA07-infected AIM22/2 mice (Figs. 3, 5, inflammation Supplemental Fig. 1). This might be because of the activation of Consistent with findings with NLRP3 inflammasome (23, 48), AIM2 other inflammasomes such as the NLRP3 inflammasome (6, 9, 23, deficiency seems to be dispensable for regulating the adaptive immune 39–41, 44), the well-studied inflammasome in influenza infection. response in influenza infection. AIM2-deficient mice display similar AIM2 deficiency did not alter the expression of NLRP3 or ASC, levels of influenza-specific IgG and similar amounts of CD4+ and the common adaptor for AIM2 and NLRP3 inflammasomes, in CD8+ T cell influx into the lung (Supplemental Fig. 2) post IAV in- infected lung tissue or primary cells (data not shown), suggesting fections. These results suggest that AIM2 mainly functions in innate that virus could still activate the NLRP3 and other inflammasomes immunity against influenza infection. Both ATII cells and macro- in the absence of AIM2. We examined the survival of ASC and phages are critically important sentinel cells in lung innate immunity. NLRP3 knockout mice after lethal doses of PR8 or CA07 infection In an effort to determine which target cells are critical for AIM2 func- (Supplemental Fig. 3). We did not evaluate Casp1 knockout mice tion, ATII or AM, we examined the effect of AIM2 knockdown on because the commercial Casp1-deficient mice also exhibit a defi- IAV-stimulated proinflammatory response (Figs. 8, 9). Despite the ciency in Casp11 (http://www.jax.org). Consistent with the reports fact that IAV increases AIM2 expression in both cell types (Fig. 1A), from other groups (23, 40), we confirmed a protective role for siAIM2-treated human AMs, but not ATII cells, exhibit significantly ASC in influenza-associated mortality. ASC-deficient mice dis- reduced release of the proinflammatory cytokine IL-1b,TNF-a,and play further reduced survival against PR8 and CA07 infections RANTES (Fig. 9). These results are consistent with the findings (Supplemental Fig. 3, top). However, NLRP3-deficient mice do with primary lung cells from AIM2-deficient mice (Fig. 8), in which not exhibit different survival when compared with WT mice AIM2-deficient AMs fail to produce IL-1b in response to IAV in- (Supplemental Fig. 3, bottom). There are controversial reports in fection. Although in vitro inflammasome studies have shown that the literature about the role of NLRP3 in influenza infection. Some influenza infection can activate inflammasome in respiratory epi- studies report a positive role for NLRP3 in host defense against thelial cells (23, 49, 50) and immune cells (51–53), our results reveal influenza infection. NLRP3-deficient mice have been shown to a central role for AMs in AIM2-related inflammasome regulation. have worsened mortality and delayed viral clearance (40, 41). Our Interestingly, our results are partially in conflict with the recent results are consistent with the results from Iwasaki’s group (23) report of an anti-inflammatory function for AIM2 (54). Although that NLRP3 deficiency does not alter mouse susceptibility to both studies have shown that PR8 infection induces dsDNA re- influenza-induced mortality. Also, a recent study using NLRP3 lease into the BAL, the published study reported that AIM2- inhibitor reported that NLRP3 plays a protective role in the early deficient mice displayed decreased survival compared with WT stage of influenza infection, but a detrimental role in the late stage mice, and AIM2 played an anti-inflammatory role. However, our 10 AIM2 DEFICIENCY CONVEYS PROTECTION AGAINST IAV INFECTION

FIGURE 9. siAIM2 treatment significantly decreases AIM2 expression and release of proinflammatory cytokines from AMs after IAV infections. Primary human ATII cells and AMs were treated with siAIM2 or nontarget control siRNA (NT) 2 d before infections with PR8 or CA07 (moi = 1). At 24 hpi, RNA was extracted for examining AIM2 gene expression, and culture supernatants were collected for detection of IL-1b, TNF-a, and RANTES using DuoSet ELISA kits from R&D Systems. (A) ATII cells with PR8 infection. n =5.(B) AMs with PR8 infection. n =5.(C) AMs with CA07 infection. n = 3. Data represent % expression or secretion to NT siRNA-treated condition. **p , 0.01, ***p , 0.001, between NT and AIM2 siRNA-treated conditions. study indicates a proinflammatory role for AIM2 in influenza in- critical for influenza-induced acute lung injury and mortality. Our fection. We noticed several significant differences in the two results revealed that AIM2 has the potential to serve as a thera- studies. First, in our study, we used in vitro primary cell culture as peutic target for limiting influenza-associated mortality and well as an in vivo mouse model of IAV infections, whereas the morbidity. other study was focused solely on a mouse study. Second, the two studies used a different source of PR8 virus, and viral doses seem Acknowledgments significantly different. The published study used PR8 produced in We thank William Horne, Kevin J. McHugh, Jennifer Profozich, and Ye eggs, whereas our PR8 virus was propagated from mammalian Liu for technical help. cells. Our PR8 study is mainly lethal dose infections, whereas the other study seems to be sublethal dose infection, although they Disclosures used 40,000 PFU of virus (54), reflecting variable infectivity of The authors have no financial conflicts of interest. viruses propagated from different sources. When we performed the survival experiment using the 40,000 PFU of PR8 propagated in mammalian cells, all mice died, but AIM22/2 mice survived References longer than WT mice in response to PR8 or CA07 infection (p , 1. Lamkanfi, M., and V. M. Dixit. 2009. Inflammasomes: guardians of cytosolic 0.01). We believe the apparent discrepancy between the two studies sanctity. Immunol. Rev. 227: 95–105. 2. Franchi, L., T. Eigenbrod, R. Mun˜oz-Planillo, and G. Nun˜ez. 2009. The is mainly due to the different doses, which may cause a different inflammasome: a caspase-1-activation platform that regulates immune responses immune response. Also, the different animal facility may affect the and disease pathogenesis. Nat. Immunol. 10: 241–247. 3. Burckst€ ummer,€ T., C. Baumann, S. Bluml,€ E. Dixit, G. Durnberger,€ H. Jahn, microbiome of the animals and result in different host innate and M. Planyavsky, M. Bilban, J. Colinge, K. L. Bennett, and G. Superti-Furga. adaptive responses against influenza (55, 56). Our primary cell 2009. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplas- system data (Figs. 8, 9) support the proinflammatory role of AIM2 mic DNA sensor for the inflammasome. Nat. Immunol. 10: 266–272. 4. Fernandes-Alnemri, T., J. W. Yu, P. Datta, J. Wu, and E. S. Alnemri. 2009. AIM2 in influenza infection in the absence of microbiome effects. activates the inflammasome and cell death in response to cytoplasmic DNA. In summary, we have performed in vitro and in vivo studies Nature 458: 509–513. using laboratory and contemporary strains of IAVs to determine 5. Hornung, V., A. Ablasser, M. Charrel-Dennis, F. Bauernfeind, G. Horvath, D. R. Caffrey, E. Latz, and K. A. Fitzgerald. 2009. AIM2 recognizes cytosolic the role of AIM2 in influenza infection. The results from this study dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458: demonstrate that IAVactivates the AIM2 inflammasome, which is 514–518. The Journal of Immunology 11

6. Martinon, F. 2012. Dangerous liaisons: mitochondrial DNA meets the NLRP3 32. Short, K. R., E. J. Kroeze, R. A. Fouchier, and T. Kuiken. 2014. Pathogenesis of inflammasome. Immunity 36: 313–315. influenza-induced acute respiratory distress syndrome. Lancet Infect. Dis. 14: 7. Rathinam, V. A., Z. Jiang, S. N. Waggoner, S. Sharma, L. E. Cole, L. Waggoner, 57–69. S. K. Vanaja, B. G. Monks, S. Ganesan, E. Latz, et al. 2010. The AIM2 33. Capaldo, C. T., and A. Nusrat. 2009. Cytokine regulation of tight junctions. inflammasome is essential for host defense against cytosolic bacteria and DNA Biochim. Biophys. Acta 1788: 864–871. viruses. Nat. Immunol. 11: 395–402. 34. Al-Sadi, R. M., and T. Y. Ma. 2007. IL-1beta causes an increase in intestinal 8. Strittmatter, G. E., J. Sand, M. Sauter, M. Seyffert, R. Steigerwald, C. Fraefel, epithelial tight junction permeability. J. Immunol. 178: 4641–4649. S. Smola, L. E. French, and H. D. Beer. 2016. IFN-g primes keratinocytes for 35. Borthwick, L. A., E. I. McIlroy, M. R. Gorowiec, M. Brodlie, G. E. Johnson, HSV-1-induced inflammasome activation. J. Invest. Dermatol. 136: 610–620. C. Ward, J. L. Lordan, P. A. Corris, J. A. Kirby, and A. J. Fisher. 2010. Inflam- 9. Pang, I. K., and A. Iwasaki. 2011. Inflammasomes as mediators of immunity mation and epithelial to mesenchymal transition in lung transplant recipients: role against influenza virus. Trends Immunol. 32: 34–41. in dysregulated epithelial wound repair. Am. J. Transplant. 10: 498–509. 10. Man, S. M., R. Karki, and T. D. Kanneganti. 2016. AIM2 inflammasome in 36. Palomo, J., D. Dietrich, P. Martin, G. Palmer, and C. Gabay. 2015. The inter- infection, , and autoimmunity: role in DNA sensing, inflammation, and leukin (IL)-1 cytokine family–balance between agonists and antagonists in in- innate immunity. Eur. J. Immunol. 46: 269–280. flammatory diseases. Cytokine 76: 25–37. 11. Rathinam, V. A., and K. A. Fitzgerald. 2010. Inflammasomes and anti-viral 37. Roux, J., H. Kawakatsu, B. Gartland, M. Pespeni, D. Sheppard, M. A. Matthay, immunity. J. Clin. Immunol. 30: 632–637. C. M. Canessa, and J. F. Pittet. 2005. Interleukin-1beta decreases expression of 12. Shieh, W. J., D. M. Blau, A. M. Denison, M. Deleon-Carnes, P. Adem, the epithelial sodium channel alpha-subunit in alveolar epithelial cells via a p38 J. Bhatnagar, J. Sumner, L. Liu, M. Patel, B. Batten, et al. 2010. 2009 pandemic MAPK-dependent signaling pathway. J. Biol. Chem. 280: 18579–18589. influenza A (H1N1): pathology and pathogenesis of 100 fatal cases in the United 38. Dolinay, T., Y. S. Kim, J. Howrylak, G. M. Hunninghake, C. H. An, States. Am. J. Pathol. 177: 166–175. L. Fredenburgh, A. F. Massaro, A. Rogers, L. Gazourian, K. Nakahira, et al. 13. Fernandes-Alnemri, T., J. W. Yu, C. Juliana, L. Solorzano, S. Kang, J. Wu, 2012. Inflammasome-regulated cytokines are critical mediators of acute lung P. Datta, M. McCormick, L. Huang, E. McDermott, et al. 2010. The AIM2 injury. Am. J. Respir. Crit. Care Med. 185: 1225–1234. inflammasome is critical for innate immunity to Francisella tularensis. Nat. 39. Tate, M. D., J. D. Ong, J. K. Dowling, J. L. McAuley, A. B. Robertson, E. Latz, Immunol. 11: 385–393. G. R. Drummond, M. A. Cooper, P. J. Hertzog, and A. Mansell. 2016. Reas- 14. Saiga, H., S. Kitada, Y. Shimada, N. Kamiyama, M. Okuyama, M. Makino, sessing the role of the NLRP3 inflammasome during pathogenic influenza A M. Yamamoto, and K. Takeda. 2012. Critical role of AIM2 in Mycobacterium virus infection via temporal inhibition. Sci. Rep. 6: 27912. tuberculosis infection. Int. Immunol. 24: 637–644. 40. Allen, I. C., M. A. Scull, C. B. Moore, E. K. Holl, E. McElvania-TeKippe, 15. Wonderlich, E. R., Z. D. Swan, S. J. Bissel, A. L. Hartman, J. P. Carney, D. J. Taxman, E. H. Guthrie, R. J. Pickles, and J. P. Ting. 2009. The NLRP3 K. J. O’Malley, A. O. Obadan, J. Santos, R. Walker, T. J. Sturgeon, et al. 2017. inflammasome mediates in vivo innate immunity to influenza A virus through Widespread virus replication in alveoli drives acute respiratory distress syn- recognition of viral RNA. Immunity 30: 556–565. drome in aerosolized H5N1 influenza infection of macaques. J. Immunol. 198: 41. Thomas, P. G., P. Dash, J. R. Aldridge, Jr., A. H. Ellebedy, C. Reynolds, 1616–1626. A. J. Funk, W. J. Martin, M. Lamkanfi, R. J. Webby, K. L. Boyd, et al. 2009. The 16. Mauad, T., L. A. Hajjar, G. D. Callegari, L. F. da Silva, D. Schout, F. R. Galas, intracellular sensor NLRP3 mediates key innate and healing responses to in- V. A. Alves, D. M. Malheiros, J. O. Auler, Jr., A. F. Ferreira, et al. 2010. Lung fluenza A virus via the regulation of caspase-1. Immunity 30: 566–575. pathology in fatal novel human influenza A (H1N1) infection. Am. J. Respir. 42. Wang, J., R. Oberley-Deegan, S. Wang, M. Nikrad, C. J. Funk, K. L. Hartshorn, Crit. Care Med. 181: 72–79. and R. J. Mason. 2009. Differentiated human alveolar type II cells secrete an- 17. Wang, J., M. P. Nikrad, T. Phang, B. Gao, T. Alford, Y. Ito, K. Edeen, tiviral IL-29 (IFN-lambda 1) in response to influenza A infection. J. Immunol. E. A. Travanty, B. Kosmider, K. Hartshorn, and R. J. Mason. 2011. Innate im- 182: 1296–1304. mune response to influenza A virus in differentiated human alveolar type II cells. 43. Yu, W. C., R. W. Chan, J. Wang, E. A. Travanty, J. M. Nicholls, J. S. Peiris, Am. J. Respir. Cell Mol. Biol. 45: 582–591. R. J. Mason, and M. C. Chan. 2011. Viral replication and innate host responses in 18. Wang, J., M. P. Nikrad, E. A. Travanty, B. Zhou, T. Phang, B. Gao, T. Alford, primary human alveolar epithelial cells and alveolar macrophages infected with Y. Ito, P. Nahreini, K. Hartshorn, et al. 2012. Innate immune response of human influenza H5N1 and H1N1 viruses. J. Virol. 85: 6844–6855. alveolar macrophages during influenza A infection. PLoS One 7: e29879. 44. Ong, J. D., A. Mansell, and M. D. Tate. 2017. Hero turned villain: NLRP3 19. Travanty, E., B. Zhou, H. Zhang, Y. P. Di, J. F. Alcorn, D. E. Wentworth, inflammasome-induced inflammation during influenza A virus infection. J. R. Mason, and J. Wang. 2015. Differential susceptibility of human lung primary Leukoc. Biol. 101: 863–874. cells to H1N1 influenza viruses. J. Virol. 89: 11935–11944. 45. Ting, J. P., and J. A. Harton. 2015. NLRP3 moonlights in TH2 polarization. Nat. 20. Reed, L. J, and H. Muench. 1938. A simple method of estimating fifty percent Immunol. endpoints. Am. J. Hyg. 27: 493–497. 16: 794–796. 21. Hidvegi, T., D. B. Stolz, J. F. Alcorn, S. A. Yousem, J. Wang, A. S. Leme, 46. Kuriakose, T., and T. D. Kanneganti. 2017. Regulation and functions of NLRP3 A. M. Houghton, P. Hale, M. Ewing, H. Cai, et al. 2015. Enhancing autophagy inflammasome during influenza virus infection. Mol. Immunol. DOI: 10.1016/j. with drugs or lung-directed gene therapy reverses the pathological effects of molimm.2017.01.023 respiratory epithelial cell proteinopathy. J. Biol. Chem. 290: 29742–29757. 47. Jo, E. K., J. K. Kim, D. M. Shin, and C. Sasakawa. 2016. Molecular mechanisms 22. Kosmider, B., E. M. Messier, W. J. Janssen, P. Nahreini, J. Wang, regulating NLRP3 inflammasome activation. Cell. Mol. Immunol. 13: 148–159. K. L. Hartshorn, and R. J. Mason. 2012. Nrf2 protects human alveolar epithelial 48. Ellebedy, A. H., C. Lupfer, H. E. Ghoneim, J. DeBeauchamp, T. D. Kanneganti, cells against injury induced by influenza A virus. Respir. Res. 13: 43. and R. J. Webby. 2011. Inflammasome-independent role of the apoptosis- 23. Ichinohe, T., H. K. Lee, Y. Ogura, R. Flavell, and A. Iwasaki. 2009. Inflam- associated speck-like protein containing CARD (ASC) in the adjuvant effect masome recognition of influenza virus is essential for adaptive immune re- of MF59. [Published erratum appears in 2013 Proc. Natl. Acad. Sci. USA 110: sponses. J. Exp. Med. 206: 79–87. 4429.] Proc. Natl. Acad. Sci. USA 108: 2927–2932. 24. Chen, K., J. P. McAleer, Y. Lin, D. L. Paterson, M. Zheng, J. F. Alcorn, 49. Bauer, R. N., L. E. Brighton, L. Mueller, Z. Xiang, J. E. Rager, R. C. Fry, C. T. Weaver, and J. K. Kolls. 2011. Th17 cells mediate clade-specific, serotype- D. B. Peden, and I. Jaspers. 2012. Influenza enhances caspase-1 in bronchial independent mucosal immunity. Immunity 35: 997–1009. epithelial cells from asthmatic volunteers and is associated with pathogenesis. J 25. McAuley, J. L., M. D. Tate, C. J. MacKenzie-Kludas, A. Pinar, W. Zeng, Allergy Clin. Immunol. 130: 958–967.e914. A. Stutz, E. Latz, L. E. Brown, and A. Mansell. 2013. Activation of the NLRP3 50. Pothlichet, J., I. Meunier, B. K. Davis, J. P. Ting, E. Skamene, V. von Messling, and inflammasome by IAV virulence protein PB1-F2 contributes to severe patho- S. M. Vidal. 2013. Type I IFN triggers RIG-I/TLR3/NLRP3-dependent inflam- physiology and disease. PLoS Pathog. 9: e1003392. masome activation in influenza A virus infected cells. PLoS Pathog. 9: e1003256. 26. Opitz, B., V. van Laak, J. Eitel, and N. Suttorp. 2010. Innate immune recognition 51. Tate, M. D., D. L. Pickett, N. van Rooijen, A. G. Brooks, and P. C. Reading. in infectious and noninfectious diseases of the lung. Am. J. Respir. Crit. Care 2010. Critical role of airway macrophages in modulating disease severity during Med. 181: 1294–1309. influenza virus infection of mice. J. Virol. 84: 7569–7580. 27. Miller, M. L., A. Andringa, and L. Hastings. 1995. Relationships between the 52. Tate, M. D., H. C. Schilter, A. G. Brooks, and P. C. Reading. 2011. Responses of nuclear membrane, nuclear pore complexes, and organelles in the type II mouse airway epithelial cells and alveolar macrophages to virulent and avirulent pneumocyte. Tissue Cell 27: 613–619. strains of influenza A virus. Viral Immunol. 24: 77–88. 28. Tran, A. T., J. P. Cortens, Q. Du, J. A. Wilkins, and K. M. Coombs. 2013. In- 53. Ioannidis, L. J., E. E. Verity, S. Crawford, S. P. Rockman, and L. E. Brown. 2012. fluenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation. Abortive replication of influenza virus in mouse dendritic cells. J. Virol. 86: J. Virol. 87: 1049–1060. 5922–5925. 29. Ichinohe, T., T. Yamazaki, T. Koshiba, and Y. Yanagi. 2013. Mitochondrial 54. Schattgen, S. A., G. Gao, E. A. Kurt-Jones, and K. A. Fitzgerald. 2016. Cutting protein mitofusin 2 is required for NLRP3 inflammasome activation after RNA edge: DNA in the lung microenvironment during influenza virus infection tempers virus infection. Proc. Natl. Acad. Sci. USA 110: 17963–17968. inflammation by engaging the DNA sensor AIM2. J. Immunol. 196: 29–33. 30. Beigel, J. H., J. Farrar, A. M. Han, F. G. Hayden, R. Hyer, M. D. de Jong, 55. Ichinohe, T., I. K. Pang, Y. Kumamoto, D. R. Peaper, J. H. Ho, T. S. Murray, and S. Lochindarat, T. K. Nguyen, T. H. Nguyen, T. H. Tran, et al; Writing Com- A. Iwasaki. 2011. Microbiota regulates immune defense against respiratory tract mittee of the World Health Organization (WHO) Consultation on Human In- influenza A virus infection. Proc. Natl. Acad. Sci. USA 108: 5354–5359. fluenza A/H5. 2005. Avian influenza A (H5N1) infection in humans. N. Engl. J. 56. Salk, H. M., W. L. Simon, N. D. Lambert, R. B. Kennedy, D. E. Grill, Med. 353: 1374–1385. B. F. Kabat, and G. A. Poland. 2016. Taxa of the nasal microbiome are asso- 31. Peiris, J. S., C. Y. Cheung, C. Y. Leung, and J. M. Nicholls. 2009. Innate immune ciated with influenza-specific IgA response to live attenuated influenza vaccine. responses to influenza A H5N1: friend or foe? Trends Immunol. 30: 574–584. PLoS One 11: e0162803.