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Inflammasome recognition of influenza virus is essential for adaptive immune responses

Inflammasome recognition of influenza virus is essential for adaptive immune responses BRIEF DEFINITIVE REPORT Infl ammasome recognition of infl uenza virus is essential for adaptive immune responses 1 1 1 1,2 Takeshi Ichinohe, Heung Kyu Lee , Yasunori Ogura , Richard Flavell , and Akiko Iwasaki 1 2 Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520 Infl uenza virus infection is recognized by the innate immune system through Toll like receptor (TLR) 7 and retinoic acid inducible gene I. These two recognition pathways lead to the activation of type I interferons and resistance to infection. In addition, TLR signals are required for the CD4 T cell and IgG2a, but not cytotoxic T lymphocyte, responses to infl u- enza virus infection. In contrast, the role of NOD-like receptors (NLRs) in viral recognition and induction of adaptive immunity to infl uenza virus is unknown. We demonstrate that respiratory infection with infl uenza virus results in the activation of NLR infl ammasomes in the lung. Although NLRP3 was required for infl ammasome activation in certain cell types, CD4 and CD8 T cell responses, as well as mucosal IgA secretion and systemic IgG responses, required ASC and caspase-1 but not NLRP3. Consequently, ASC, caspase-1, and IL-1R, but not NLRP3, were required for protective immunity against fl u challenge. Furthermore, we show that caspase-1 infl ammasome activation in the hematopoietic, but not stromal, compartment was required to induce protective antiviral immunity. These results demon- strate that in addition to the TLR pathways, ASC infl ammasomes play a central role in adaptive immunity to infl uenza virus. Infl uenza virus is responsible for annual epi- used by plasmacytoid DCs (pDCs) ( 2 ). Signaling CORRESPONDENCE Akiko Iwasaki: demics that cause severe morbidity and death through both RIG-I and TLR7 results in the [email protected] involving approximately fi ve million people production of type I IFNs, which limit viral rep- worldwide. Lethal pneumonia and encepha- lication and increasing resistance to infection. lopathy caused by infl uenza virus have now In addition to type I IFNs, proinfl ammatory become a serious problem, especially among cytokines such as IL-1 play a crucial role in pro- the elderly and children, respectively ( 1 ). Fur- tection against infl uenza. Infl uenza virus infec- thermore, the H5N1 highly pathogenic avian tion is accompanied by IL-1  production in infl uenza viruses that are associated with a high bronchoalveolar lavage (BAL) of mice ( 5 ). In- fatality rate ( > 60%) have been reported in South- fl uenza virus infection activates IL-1  and IL-18 east Asia, Europe, and Africa. Therefore, there production in human macrophages ( 6 ). IL-1 is is an urgent and important public health need responsible for acute lung immunopathology to develop eff ective vaccines against not only and is required to promote survival of the mice annual seasonal infl uenza viruses but also against after infl uenza virus infection ( 7 ). Infl uenza vi- highly pathogenic H5N1 avian infl uenza vi- rus – specifi c CD4 T cell responses and IgM lev- ruses. Infl uenza virus is recognized through at els were reduced in IL-1R – defi cient mice ( 7 ). least two viral sensors. First, the cytosolic sen- Not surprisingly, infl uenza virus has evolved sor retinoic acid inducible gene I (RIG-I) de- strategies to inhibit the activation of infl amma- tects infl uenza after fusion and replication in somes. NS1 protein of infl uenza virus suppressed infected cells ( 2 ). Second, infl uenza genomic caspase-1 activation, maturation of pro – IL-1 RNA, upon release in late endosomes, is rec- and pro – IL-18, and caspase-1 – dependent apop- ognized by Toll-like receptor (TLR) 7 ( 3, 4 ). tosis in infected primary human macrophages ( 8 ). The RIG-I pathway is used by most cells to respond to virus infection, whereas the latter is © 2009 Ichinohe et al. This article is distributed under the terms of an Attribu- tion–Noncommercial–Share Alike–No Mirror Sites license for the fi rst six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six Y. Ogura ’ s present address is Division of Bacterial Pathogen- months it is available under a Creative Commons License (Attribution–Noncom- esis, Graduate School of Medicine, University of the mercial–Share Alike 3.0 Unported license, as described at http://creativecommons Ryukyus, 207 Uehara, Nishihara-cho Okinawa, Japan. .org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 206 No. 1 79-87 79 www.jem.org/cgi/doi/10.1084/jem.20081667 The Journal of Experimental Medicine However, the mechanism by which IL-1  and IL-18 are ac- RESULTS AND DISCUSSION tivated during infl uenza infection in vivo is unknown. NLRP3 infl ammasome is activated in the lung Infl ammasomes are molecular platforms that allow acti- after respiratory infl uenza infection vation of caspase-1 ( 9 ). Caspase-1 is an essential regulator of Although NLRP3-defi cient macrophages were reported to be unable to activate IL-1  in response to infl uenza virus in infl ammatory response through its capacity to process and activate proIL-1  , proIL-18, and proIL-33 ( 10 ). NOD-like the presence of ATP in vitro ( 21 ), the role of infl ammasomes receptors (NLRs) comprise a large family of intracellular in infl uenza infection in vivo is unknown. In the lung, the PRRs that play an important role in innate immunity in re- primary targets of fl u infection are the respiratory epithelial sponse to recognition of various “ damaged ” self ( 11 ) and cells, which produce large amounts of virus that can subse- nonself molecules ( 9, 12 ). NLR protein (NLRP) 3, also quently infect alveolar macrophages ( 1 ) and DCs ( 22 ). To known as NALP3/Cryopyrin/CIAS1/PYPAF1 ( 13 ), forms begin to dissect the importance of various components of the a caspase-1 – activating infl ammasome. Mature IL-1  se- infl ammasome complex in response to infl uenza virus infec- cretion requires at least two steps: fi rst, transcriptional and tion, we fi rst examined IL-1  secretion in candidate cell translational up-regulation of pro – IL-1  through TLR stim- types including BM – derived macrophages (BMM), BM DCs and lung fi broblasts. Although BMM and BM DC exhibited ulation; and second, the activation of caspase-1 by infl am- masomes ( 9, 12 ). Recent reports indicate that infection by signifi cant reduction in IL-1  secretion in the absence of certain viruses also results in infl ammasome activation ( 14 – 16 ). NLRP3 ( Fig. 1, A and B ), lung fi broblast cells activated in- Kanneganti et al. ( 15 ) showed that Sendai and infl uenza vi- fl ammasomes independently of NLRP3 ( Fig. 1 C ). All cell ruses activated the NLRP3 infl ammasome in macrophages types required caspase-1 for release of IL-1  ( Fig. 1, A – C ). pulsed transiently with ATP for 30 min in vitro. Muruve Next, we examined the target of NLR recognition of infl u- et al. ( 16 ) demonstrated that adenovirus infection activates enza virus. Stimulation of cells with infl uenza virions showed IL-1  processing in NLRP3-, ASC-, and caspase-1 – dependent that attachment (blocked by 65 ° C inactivation), fusion (blocked manners. However, infl ammasomes were not activated by by 56 ° C inactivation), and replication (blocked by UV irra- transfection of RNA, Poly I:C, or infection with reovirus diation) of the virus were required to elicit infl ammasome activation in BM DCs (Fig. S1 A, available at http://www (double-stranded RNA virus) or vesicular stomatitis virus (single-stranded RNA virus). In another seminal study, Johnston .jem.org/cgi/content/full/jem.20081667/DC1) and in lung et al. ( 14 ) reported that Myxoma virus carries a protein that fi broblasts (Fig. S1 C). Purifi ed genomic RNA, upon deliv- inhibits ASC/caspase-1 activation and subsequent cell death ery by DOTAP, induced very low levels of IL-1  secretion after virus infection. Such an evasion mechanism supports in BM DC, which was enhanced by the addition of ATP the idea that infl ammasomes might play a vital role in anti- (Fig. S1 B), indicating that fl u genomic RNA, although in- viral defense. suffi cient to trigger infl ammasomes, could serve as signal 1 for Although the proinfl ammatory role of infl ammasomes NLR-mediated IL-1  secretion. In contrast, fl u genomic RNA is well known, less is understood with respect to the re- and ATP treatment failed to elicit infl ammasome activation quirement for infl ammasomes in the generation of adap- in lung fi broblasts (Fig. S1 D). Collectively, these data indi- cated that infl uenza virus infection stimulates NLRP3-de- tive immune responses. Uric acid, which triggers NLRP3 infl ammasomes ( 17 ), has been shown to stimulate DC matu- pendent and NLRP-3 independent infl ammasomes in a cell ration and, when coinjected with antigen in vivo, signifi - autonomous and cell type – specifi c manner and that other cantly enhances the generation of responses from CD8 T factors, in addition to viral genomic RNA, are required to cells ( 18 ). NLRP3, as well as its adaptor molecule ASC, are elicit infl ammasome activation. required for contact hypersensitivity responses in vivo ( 19 ). Next, to determine the role of NLRP3 infl ammasomes in More recently, the Th2-inducing adjuvant activity of alum the secretion of IL-1  during physiologically relevant infl u- was shown to be mediated through NLRP3/ASC infl amma- enza virus infection in vivo, mice were challenged intranasally somes ( 20 ). Thus, infl ammasomes appear to play an important with infl uenza strain A/PR8. Active IL-1  was secreted into role in certain types of autoimmune diseases, hyperrespon- the BAL in a dose-dependent manner (Fig. S2 A, available at http://www.jem.org/cgi/content/full/jem.20081667/DC1). siveness, and immunization. However, the role of infl am- masomes in the recognition of viral infection in vivo and Starting at 2 d post infection (p.i.), IL-1  secretion was evi- generation of protective adaptive immunity is unknown. dent in the BAL in WT mice infected with 10 PFU of infl u- In this paper, we examine the role of the NLRP3 infl am- enza virus. However, mice defi cient in NLRP3, ASC, or masomes in the initiation of adaptive immunity after physio- caspase-1, but not NLRC4, failed to secrete IL-1  ( Fig. 1 D ) logical infection with infl uenza virus. We describe requirement or IL-18 (Fig. S2 B) into the alveolar space in response to for caspase-1 infl ammasomes in the hematopoietic, but not infl uenza infection. Infl ammasome-independent cytokines, stromal, cells in the establishment of Th1, CTL, and IgA re- such as IL-6, TNF-  , KC, and MIP-2, were secreted com- sponses to respiratory fl u infection. Collectively, our data parably from NLRP3, ASC, or caspase-1 KO mice (Fig. S2, provide the fi rst evidence of the in vivo requirement for the C – F), indicating that the reduction in IL-1  and IL-18 secre- tion is not caused by a general immune defi ciency in these components of infl ammasomes in adaptive immunity to a vi- rus infection. mice. In contrast to the BAL fl uid, IL-1  secretion in the 80 INFLAMMASOME IN ADAPTIVE IMMUNITY TO INFLUENZA | Ichinohe et al. BRIEF DEFINITIVE REPORT lung tissue was found to be independent of NLRP3 ( Fig. 1 E ). alveolar space after respiratory challenge in a type I IFN-in- To determine the cellular compartment responsible for the dependent manner, whereas the lung tissue resident hemato- secretion of IL-1  in the lung, we generated BM chimeric poietic cells activate caspase-1 through NLRP3-independent mice in which only the hematopoietic (WT → caspase-1 ) pathways (Fig. S4). or the stromal cells (caspase-1 → WT) expressed caspase-1. Measurement of IL-1  in the BAL ( Fig. 1 F ) and lung tissue ASC, caspase-1, and IL-1R, but not NLRP3, is required ( Fig. 1 G ) indicated that infl ammasome activation in vivo for cellular recruitment to the lung after fl u infection occurs within the hematopoietic compartment and the stro- Next, we wished to determine the consequence of infl amma- mal compartment. A previous study demonstrated the im- some activation in cellular recruitment to the infected lungs. portance of type I IFNs in infl ammasome activation after In WT mice infected with A/PR8, accumulation of leuko- Francisella novicida infection ( 23 ). However, in response to in- cytes became apparent starting around day 3 p.i. and peaking fl uenza infection, although partly important in BM DC re- around day 5 p.i. (Fig. S5 A, available at http://www.jem.org/ sponse, responsiveness through type I IFN receptor was cg i/content/full/jem.20081667/DC1). Var ious leukocytes dispensable for IL-1  secretion in the BAL (Fig. S3). Collec- were detected including macrophages, DCs, pDCs, NK cells, tively, these data indicated that NLRP3 infl ammasome is ac- neutrophils, and lymphocytes (Fig. S5 B). These cellular sub- tivated in the alveolar macrophages and DCs within the sets were defi ned by fl ow cytometry as described in Fig. S6. Figure 1. The role of NLRP3, ASC, and caspase-1 in infl ammasome activation and cellular recruitment to the lung after infl uenza infection. (A – G) BM macrophages (A), BMDCs (B), or primary lung fi broblast (C) prepared from WT, NLRP3-, and caspase-1 – defi cient mice were infected with A/PR8 virus at MOI 2.5. Supernatant was collected 12 – 24 h after stimulation and analyzed for IL-1  by ELISA. WT, NLRP3-, NLRC4-, ASC-, and caspase-1 – defi cient mice were infected intranasally with 10 PFU A/PR8 virus. BAL was collected by washing the trachea and lungs twice by injecting a total of 1 ml PBS con- taining 0.1% BSA (D and F). Lung homogenate was prepared in 2 ml PBS containing 0.1% BSA (E and G). IL-1  levels detected from the BAL or lung ho- mogenate at different time points (D) or at 2 d p.i. (E – G) are shown. Horizontal lines show the mean. (H – I) Lung tissue was collected from the indicated groups of mice at 0 and 5 d p.i. Total lung leukocytes were enumerated. The values represent the mean of three mice per group and are expressed as the mean ± SD. Similar results were obtained from at least two separate experiments. *, P < 0.05; **, P < 0.01 versus a group of noninfected mice. JEM VOL. 206, January 19, 2009 81 Figure 2. Infl uenza-specifi c CD4 and CD8 T cell responses depend largely on ASC and Caspase-1 but not NLRP3. WT, NLRP3-, ASC-, and cas- /   /   / pase-1 – defi cient mice (A and B), IL-1R mice (C and D), or WT → caspase-1 and caspase-1 → WT BM chimeric mice (E and F) were infected intra- nasally with a sublethal dose (10 PFU) of A/PR8 virus. 14 d p.i., CD8 and CD4 T cells were isolated from spleen and stimulated with irradiated APCs with 82 INFLAMMASOME IN ADAPTIVE IMMUNITY TO INFLUENZA | Ichinohe et al. BRIEF DEFINITIVE REPORT /   /   / In contrast, mice defi cient in ASC and caspase-1, but not from WT mice, ASC , caspase-1 ( Fig. 2 A ), and IL-1R NLRP3, exhibited signifi cant reduction in cellular recruit- ( Fig. 2 C ) mice failed to mount comparable CD4 T cell ment of all subsets (Fig. S5 C). In addition, IL-1R – defi cient responses. In addition, ASC mice demonstrated a partially mice were also impaired in their ability to induce leukocyte reduced CTL phenotype compared with caspase-1 or /  + recruitment to the lung ( Fig. 1 H ). Furthermore, mice selec- IL-1R mice, in which CD8 T cell responses were barely tively defective in caspase-1 in the hematopoietic compart- detectable after immunization with live fl u infection ( Fig. 2 B ). ment (caspase-1 → WT) were impaired in their ability to Furthermore, comparable reduction in virus-specifi c CD8 T recruit leukocytes to the lung ( Fig. 1 I ). Thus, these data indi- cell numbers in the spleen and lung was observed by intra- cated that although NLRP3 is required for IL-1  secretion cellular IFN-  staining ( Fig. 2 G ) and by tetramer staining into the alveolar space ( Fig. 1 D ), NLRP3-independent ASC/ ( Fig. 2 H ). These data indicated that NLRP3-independent caspase-1 – dependent pathways ( Fig. 1 E ) in the lung tissue ASC/caspase-1 infl ammasomes and responsiveness through hematopoietic cells ( Fig. 1 I ) result in IL-1  secretion leading the IL-1R are required to elicit T cell responses to infl uenza to cellular infi ltration in the lung. virus in vivo. In addition, the intermediate phenotype of the IL-1  is a well known stimulator of neutrophil recruitment, defect in T cell responses in ASC compared with cas- which is triggered by the synthesis and display of chemokines pase-1 mice suggests the presence of an ASC-independent and leukocyte adhesion molecules. Our data demonstrated caspase-1 infl ammasome in the generation of T cell responses that diff erential requirements for NLRP3 – IL-1  secretion after infl uenza infection. into the alveolar space depended on NLRP3 ( Fig. 1 D ), whereas Next, we examined the cellular compartment responsible IL-1  secretion in the lung interstitium ( Fig. 1 E ) and re- for the infl ammasome-dependent generation of CD4 and cruitment of leukocytes to the lung were independent of CD8 T cell responses. T cell responses from fl u-infected cas- /   / NLRP3 ( Fig. 1 H ). Furthermore, lung leukocyte infi ltration pase-1 → WT, but not WT → caspase-1 mice, were depended on ASC, caspase-1, and IL-1R, indicating that diminished ( Fig. 2, E and F ), indicating that activation of infl ammasome-released IL-1  is required to mediate this pro- caspase-1 infl ammasomes by the hematopoietic, but not stro- cess. In contrast, NLRC4-defi cient mice had normal IL-1 mal, cells link NLR recognition of infl uenza virus to the secretion as measured by BAL ( Fig. 1 D ). These data demon- activation of adaptive immunity in vivo. strated a diff erential role of NLRP3; the cell types that secrete IL-1  in the alveolar space (such as alveolar macrophages Differential requirement for infl ammasomes and DCs) require NLRP3 for infl ammasome activation, whereas in immunoglobulin isotype responses to infl uenza virus those that secrete IL-1  in the lung interstitium (interstitial Antibody responses play a critical role in the clearance of macrophages, DCs, and others) can trigger NLRP3-inde- many viral pathogens including infl uenza virus. Previous pendent infl ammasomes. Only the latter source of IL-1  is studies have indicated the role of MyD88 in IgG2a responses required to activate vascular endothelial cell to enable leuko- to infl uenza infection ( 26, 27 ). We determined the contribu- cyte migration into the lung (Fig. S4). Evidence exists for the tions of infl ammasomes in this process. To this end, mice were NLRP3-independent ASC dimer – mediated pyroptosome immunized with a sublethal dose (10 PFU) of live A/PR8 that can activate caspase-1 directly ( 24 ). It is of interest that virus. 2 wk p.i., infl uenza virion – specifi c nasal IgA and serum other studies have described NLRP3/NLRC4-independent titers of IgM and IgG isotype levels were measured by serial ASC/caspase-1 – dependent infl ammasome activation in re- dilution. Nasal IgA responses were found to be completely sponse to LPS ( 25 ) and cytosolic DNA ( 16 ). dependent on ASC, caspase-1, IL-1R, and MyD88 but not on NLRP3 ( Fig. 3 A ). In contrast, serum IgG1 levels were Infl uenza-specifi c CD4 and CD8 T cell responses depend signifi cantly elevated in MyD88-defi cient mice compared largely on ASC and Caspase-1 but not NLRP3 with WT or other infl ammasome KO groups ( Fig. 3 C ). A Next, to decipher the importance of infl ammasomes in adap- slight reduction in the IgG2c response was detected in ASC tive immune responses to infl uenza infection, mice defi cient and caspase-1 mice, which was similar to that seen in IL- /   /   / in NLRP3, ASC, caspase-1, or IL-1R were infected with a 1R and MyD88 mice ( Fig. 3 D ). Only caspase-1 sublethal dose of A/PR8 (10 PFU or 0.4 LD ). 14 d later, mice had a signifi cant reduction in IgG3 responses but not + + CD4 and CD8 T cells were isolated from the spleen of fl u- IL-1R – or MyD88-defi cient mice ( Fig. 3 E ), indicating that primed mice and restimulated with varying concentrations of caspase-1 – dependent IL-1  – , IL-18 – , and IL-33 (all upstream inactivated virion or H-2D – restricted nucleoprotein (NP) of MyD88) – independent factors play a major role in the in- + + peptide, respectively. Although CD4 and CD8 T cells from duction of this isotype response. In contrast, no signifi cant infected NLRP3-defi cient mice responded similarly to those diff erences were found in serum IgM responses ( Fig. 3 F ). + + the indicated amount of heat-inactivated virion or NP peptide for 72 h, respectively. IFN-  production from CD4 T cells (A, C, and E) and CD8 T cells (B, D, and F) was measured by ELISA or by intracellular IFN-  staining (G). (H) Indicated groups of mice were infected with 10 PFU of A/PR8/GP-33 recom- binant infl uenza virus, and GP33-specifi c CD8 T cells were detected in the spleen and the lung at 14 d p.i. using the K -GP33-41 tetramer. These fi gures are representative of three similar experiments. Error bars show SD. *, P < 0.05; **, P < 0.01 versus WT mice. JEM VOL. 206, January 19, 2009 83 Antibody secretion in the BM chimeric mice indicated that the whereas IgG2a/c and CD4 T cell responses depend on MyD88 requirement for caspase-1 was all attributable to its expression ( 26, 27 ). In light of the data we present here, infl ammasome in the hematopoietic compartment alone (Fig. S7, available at recognition of infl uenza virus infection plays a more domi- http://www.jem.org/cgi/content/full/jem.20081667/DC1). nant role in the establishment of CD8 T cell responses com- These data indicated that at least one NLRP3-independent pared with TLR7. In addition, we found that serum IgG2c ASC- and caspase-1 – dependent infl ammasome is required responses were largely dependent on both ASC-dependent for nasal IgA and, to some extent, serum IgG2c responses to caspase-1 infl ammasome signals. These data suggested that infl uenza virus. In addition, IL-1R and MyD88, which is caspase-1 infl ammasomes, as well as TLR7/MyD88 ( 26, 27 ), downstream of TLRs, IL-1R, IL-18R, and ST2, are required play a key role in the formation of IgG2c-secreting B cells for the same process in vivo. In contrast, IgG3 responses appear after fl u infection. A pronounced defect in nasal IgA produc- to depend mainly on caspase-1 but not on IL-1R or MyD88, tion in ASC and caspase-1 – defi cient mice and complete im- whereas MyD88 has a regulatory role for IgG1 responses. pairment in MyD88 mice suggest that infl ammasomes Our results indicated that ASC/caspase-1 infl amma- and IL-1R signals are required to elicit B cell secretion of IgA somes and the secreted IL-1  play a key role in the genera- in the nasal mucosa. tion of Th1, CTL, and IgA responses after infl uenza infection (Table S1, available at http://www.jem.org/cgi/content/full/ ASC, caspase-1, and IL-1R, but not NLRP3, are required jem.20081667/DC1). Previous studies have demonstrated for survival after sublethal infl uenza challenge the requirement for TLR7 and MyD88 in adaptive immu- Finally, we examined the importance of infl ammasomes in nity to infl uenza virus infection with somewhat confl icting immune-mediated protection. To this end, we followed viral results. Lopez et al. ( 28 ) found no requirement for MyD88 titer and survival of mice after intranasal challenge with a sub- on T cells or immunoglobulin responses to aerosolized infl u- lethal dose (10 PFU) of A/PR8. The virus titers in the lung of enza virus challenge. In contrast, Koyama et al. ( 27 ) showed infected mice revealed that all mice sustained similar viral load that TLR7 or MyD88 is required for the induction of B cell at day 5 p.i., indicating that, with the low dose viral chal- secretion of anti-HA IgG2a and IgG2c but not CTL re- lenge, infl ammasomes were not required for innate immune sponses. Heer et al. ( 26 ) demonstrated that IgG2a and IgG2c protection ( Fig. 4, A and B ). In contrast, at later stages of in- responses to fl u were impaired in MyD88 but not in fection, viral clearance was severely impaired in mice defi - TLR7 mice. All studies found IgG1 antibody levels to be cient for ASC, caspase-1, or IL-1R, but not NLRP3 ( Fig. 4 A ). /   / elevated in MyD88 mice ( 26 – 28 ) as well as TLR7 Specifi cally, caspase-1 in the hematopoietic but not stromal mice ( 26 ). Together, these studies indicated that activation of compartment was required for control of virus replication at CD8 T cells during antiinfl uenza immune response relies this stage ( Fig. 4 B ). This eff ect became pronounced on day on mechanisms other than the TLR7 and MyD88 ( 26 – 28 ), 10 p.i., when WT and NLRP3 mice had completely Figure 3. Differential requirement for NLRP3, ASC, and caspase-1 for immunoglobulin isotype responses to infl uenza virus. WT, NLRP3-, ASC-, caspase-1 – , IL-1R – , and MyD88-defi cient mice were infected intranasally with a sublethal dose (10 PFU) of A/PR8 virus. Serum and nasal swab were collected at 2 wk p.i. A/PR8-specifi c nasal IgA levels were measured by ELISA (A). A/PR8-specifi c serum antibody titers were determined by serial dilution of serum total IgG (B), IgG1 (C), IgG2c (D), IgG3 (E), and IgM (F). These fi gures are representative of three similar experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus WT mice. 84 INFLAMMASOME IN ADAPTIVE IMMUNITY TO INFLUENZA | Ichinohe et al. BRIEF DEFINITIVE REPORT cleared the virus and those defi cient in ASC, caspase-1, or indicated that NLRP3-independent ASC-dependent caspase-1 IL-1R sustained high virus burden in the lung ( Fig. 4 A ). infl ammasomes are required to provide IL-1R – dependent Consequently, although WT and NLRP3 mice recovered immune protection against respiratory infl uenza infection. It from sublethal infection with infl uenza virus, the majority is unclear which NLRPs are responsible for the recognition /   /   / of the fl u-challenged ASC , caspase-1 , and IL-1R of infl uenza virus in the initiation of ASC infl ammasome-de- mice succumbed to death by 18 d p.i. ( Fig. 4 C ). These data pendent immune responses. Future identifi cation of NLRP responsible for infl uenza recognition will provide important insights into the molecular mechanism. /   /   / Mortality of ASC , caspase-1 , and IL-1R mice after a low-dose infl uenza challenge was associated with re- duced CD4 and CD8 T cell responses ( Fig. 2 ) as well as im- paired mucosal IgA and systemic IgG2 responses ( Fig. 3 ) to infl uenza viral antigens (Table S1). It is well known that re- covery from infl uenza infection requires CD8 T cells. The antiviral capacity of fl u-specifi c CD8 T cells is dependent on their ability to migrate to the lungs and come in contact with infected airway epithelium ( 29 ). Eff ector CD8 T cells begin to appear in the lung  5 – 7 d after infection ( 30 ). Thus, the inability of ASC- and caspase-1 – defi cient mice to clear virus in the lung at 8 and 10 d after infl uenza infection ( Fig. 4 A ) is likely the result of a combined eff ect of their failure to de- velop robust systemic CD8 T cell responses and to recruit ef- fector CD8 T cells to the lung ( Fig. 2 and Fig. S5 C). This recruitment and eff ector T cell induction are downstream of IL-1R signaling, as IL-1R mice failed to elicit lung cellu- lar infi ltration ( Fig. 1 ) or T cell responses ( Fig. 2 ). These data provide important evidence for the requirements for infl am- masomes in multiple stages of antiviral immune defense. In conclusion, our study demonstrated the requirement for ASC/caspase-1 infl ammasomes in the development of adap- tive immune responses to respiratory infl uenza virus infection. Our data support the notion that infl uenza virus is recognized through an ASC-dependent pathway in addition to TLR7 and RIG-I. Both caspase-1 (this paper) and TLR7 ( 26, 27 ) recog- nition pathways, but not RIG-I ( 27 ), appear to be required for adaptive immune responses to infl uenza virus. These data have signifi cant implications in the design of prophylactic vaccines and management of infl uenza infection in general. Stimulants of infl ammasomes may provide an ideal adjuvant candidate in fl u vaccines that will confer protective immune responses. Finally, clinical management of severe pathogenic infl uenza strains, such as H5N1 avian strains, which often cause immuno- pathology, may require blockade of infl ammasome to atten- uate pathology in addition to antiviral therapy. MATERIALS AND METHODS Animals. The generation of mice defi cient in NLRP3, ASC ( 19 ), NLRC4 Figure 4. ASC-1 – , caspase-1 – , and IL-1R – defi cient mice, but not ( 31 ), caspase-1 ( 10 ), and MyD88 ( 32 ) has been reported previously. All NLRP3-defi cient mice, are more susceptible to infl uenza infection KO mice have been backcrossed at least nine generations onto the C57BL/6 and disease. WT, NLRP3-, ASC-, caspase-1 – , and IL-1R – defi cient mice background. Age- and sex-matched C57BL/6 mice from National Cancer /   / (A) and WT → caspase-1 and caspase-1 → WT BM chimeric mice Institute (Frederick, MD) were used as WT controls. IL-1R mice were (B) were infected intranasally with a sublethal dose (10 PFU) of A/PR8 obtained from The Jackson Laboratory. All procedures used in this study virus. The lungs of infl uenza-infected mice were harvested at 5, 8, and 10 complied with federal guidelines and were approved by the Yale Animal Care d p.i., and viral titers were determined by plaque assay (A and B). The val- and Use Committee. ues are representative of three mice per group and are expressed as the mean ± SD. *, P < 0.05; **, P < 0.01 versus WT mice. (C) Survival of mice Infl uenza virus infection in vitro . Lung fi broblasts were prepared accord- after infection is depicted. Similar results were obtained from at least two ing to published procedures ( 33 ). BMM, DCs (8 × 10 cells/24 wells), or lung separate experiments. fi broblasts (2 × 10 cells/96 wells) were stimulated with live or inactivated JEM VOL. 206, January 19, 2009 85 A/PR8 virus. 1 h after stimulation, cells were washed thoroughly and incu- The serum titers of IgM and IgG isotypes against A/PR8 viruses were de- bated in complete media for 24 h. Cell-free supernatant was collected and termined by ELISA. In brief, a 96-well plate (EIA plates; Costar; Corning) was analyzed for IL-1  by ELISA. coated with formalin-inactivated A/PR8 virion with carbonate buff er. The re- actions were detected by goat anti – mouse IgA (Invitrogen), goat anti – mouse IgG1 (Bethyl laboratories, Inc.), goat anti – mouse IgG2c (Bethyl laboratories, Infl uenza virus infection in vivo. A/PR8 virus (H1N1) used for all ex- Inc.), goat anti – mouse IgG3 (SouthernBiotech), goat anti – mouse IgG (Jackson periments was grown in allantoic cavities from 10 – 11-d-old fertile chicken ImmunoResearch Laboratories), or goat anti – mouse IgM (SouthernBiotech) eggs for 2 d at 37 ° C. This virus was stored at  80 ° C, and viral was quanti- antibodies conjugated to horseradish peroxidase. Endpoint titers were consid- fi ed by a standard plaque assay using Madin-Darby canine kidney cells. For ered positive for dilutions with OD values that were twofold higher than the intranasal infection, mice were fully anesthetized by i.p. injection of ket- background level (nonimmune serum). amine and xylazine and then infected by intranasal application of 20 μ l of vi- rus suspension (10 – 1,000 PFU in PBS). This procedure leads to the upper Online supplemental material. Fig. S1 shows the viral and cellular determi- and lower respiratory tract infection. Recombinant PR8 virus expressing the nant for infl uenza recognition. Fig. S2 shows infl ammasome-dependent and in- LCMV glycoprotein epitope GP33-41 (PR8-GP33) was a gift from S. dependent cytokine secretion in the BAL of infl uenza-infected mice. Fig. S3 Kaech (Yale University, New Haven, CT). examines the role of type I IFNs in infl ammasome activation by infl uenza virus. Fig. S4 is a schematic representation of the role of infl ammasomes in the genera- Preparation of lung single-cell suspensions. To obtain single lung cell tion of adaptive immunity against fl u infection. Fig. S5 depicts leukocyte re- suspensions, lungs were perfused with 10 ml PBS through the right ventri- cruitment to the lung of infl uenza-infected mice. Fig. S6 describes the strategy cle, minced using razor blades, and incubated in HBSS containing 2.5 mM for cell type identifi cation by fl ow cytometry. Fig. S7 shows infl uenza virus Hepes and 1.3 mM EDTA at 37 ° C for 30 min. The cells were resuspended specifi c antibody production in BM chimeric mice. Online supplemental mate- in RPMI containing 5% FBS, 1 mM CaCl , 1 mM MgCl , 2.5 mM Hepes, 2 2 rial is available at http://www.jem.org/cgi/content/full/jem.20081667/DC1. and 0.5 mg/ml collagenase d (Roche) and incubated at 37 ° C for 60 min. A single-cell suspension was prepared after RBC lysis. The resulting cells were We thank Drs. S. Kaech and A. Chandele for critical reagents and technical support. fi ltered through a 70- μ m cell strainer (BD) and used for FACS analysis. This study was supported by grants from the National Institutes of Health (AI054359, AI062428, and AI064705). T. Ichinohe is a recipient of the Japan Society for Flow cytometry. The single-cell suspensions of lung samples were stained the Promotion of Science Postdoctoral Fellow for Research Abroad. H.K. Lee was with anti-B220, anti-CD3, anti-CD4, anti – CD8-  , anti – MHC class II, supported by the Ministry of Science and Technology of Korea and is a recipient of anti-CD19, anti-DX5, anti-F4/80, anti-CD11c, anti – mPDCA-1, anti – Si- Richard K. Gershon fellowship. A. Iwasaki is a recipient of the Burroughs Wellcome glec-H, anti-CD11b, or anti-CD45.2 antibodies. Leukocytes were gated Investigators in Pathogenesis of Infectious Disease. based on forward and side scatter properties, and live cells were gated based The authors have no confl icting fi nancial interests. on 7-aminoactinomycin d exclusion. Acquisition of eight color samples was Submitted: 29 July 2008 performed on a cytometer (LSR II; BD). Leukocytes from spleen or lung of Accepted: 11 December 2008 immunized mice were cultured in the presence of 10 μ g/ml NP peptide (ASNENMETM; H-2D ) for 6 h, and 10 μ g/ml of brefeldin A (Sigma-Al- REFERENCES drich) was added for an additional 12 h. 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Inflammasome recognition of influenza virus is essential for adaptive immune responses

Ichinohe, Takeshi; Lee, Heung Kyu; Ogura, Yasunori; Flavell, Richard; Iwasaki, Akiko
The Journal of Experimental Medicine , Volume 206 (1) – Jan 19, 2009
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© 2009 Ichinohe et al.
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10.1084/jem.20081667
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Abstract

BRIEF DEFINITIVE REPORT Infl ammasome recognition of infl uenza virus is essential for adaptive immune responses 1 1 1 1,2 Takeshi Ichinohe, Heung Kyu Lee , Yasunori Ogura , Richard Flavell , and Akiko Iwasaki 1 2 Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520 Infl uenza virus infection is recognized by the innate immune system through Toll like receptor (TLR) 7 and retinoic acid inducible gene I. These two recognition pathways lead to the activation of type I interferons and resistance to infection. In addition, TLR signals are required for the CD4 T cell and IgG2a, but not cytotoxic T lymphocyte, responses to infl u- enza virus infection. In contrast, the role of NOD-like receptors (NLRs) in viral recognition and induction of adaptive immunity to infl uenza virus is unknown. We demonstrate that respiratory infection with infl uenza virus results in the activation of NLR infl ammasomes in the lung. Although NLRP3 was required for infl ammasome activation in certain cell types, CD4 and CD8 T cell responses, as well as mucosal IgA secretion and systemic IgG responses, required ASC and caspase-1 but not NLRP3. Consequently, ASC, caspase-1, and IL-1R, but not NLRP3, were required for protective immunity against fl u challenge. Furthermore, we show that caspase-1 infl ammasome activation in the hematopoietic, but not stromal, compartment was required to induce protective antiviral immunity. These results demon- strate that in addition to the TLR pathways, ASC infl ammasomes play a central role in adaptive immunity to infl uenza virus. Infl uenza virus is responsible for annual epi- used by plasmacytoid DCs (pDCs) ( 2 ). Signaling CORRESPONDENCE Akiko Iwasaki: demics that cause severe morbidity and death through both RIG-I and TLR7 results in the [email protected] involving approximately fi ve million people production of type I IFNs, which limit viral rep- worldwide. Lethal pneumonia and encepha- lication and increasing resistance to infection. lopathy caused by infl uenza virus have now In addition to type I IFNs, proinfl ammatory become a serious problem, especially among cytokines such as IL-1 play a crucial role in pro- the elderly and children, respectively ( 1 ). Fur- tection against infl uenza. Infl uenza virus infec- thermore, the H5N1 highly pathogenic avian tion is accompanied by IL-1  production in infl uenza viruses that are associated with a high bronchoalveolar lavage (BAL) of mice ( 5 ). In- fatality rate ( > 60%) have been reported in South- fl uenza virus infection activates IL-1  and IL-18 east Asia, Europe, and Africa. Therefore, there production in human macrophages ( 6 ). IL-1 is is an urgent and important public health need responsible for acute lung immunopathology to develop eff ective vaccines against not only and is required to promote survival of the mice annual seasonal infl uenza viruses but also against after infl uenza virus infection ( 7 ). Infl uenza vi- highly pathogenic H5N1 avian infl uenza vi- rus – specifi c CD4 T cell responses and IgM lev- ruses. Infl uenza virus is recognized through at els were reduced in IL-1R – defi cient mice ( 7 ). least two viral sensors. First, the cytosolic sen- Not surprisingly, infl uenza virus has evolved sor retinoic acid inducible gene I (RIG-I) de- strategies to inhibit the activation of infl amma- tects infl uenza after fusion and replication in somes. NS1 protein of infl uenza virus suppressed infected cells ( 2 ). Second, infl uenza genomic caspase-1 activation, maturation of pro – IL-1 RNA, upon release in late endosomes, is rec- and pro – IL-18, and caspase-1 – dependent apop- ognized by Toll-like receptor (TLR) 7 ( 3, 4 ). tosis in infected primary human macrophages ( 8 ). The RIG-I pathway is used by most cells to respond to virus infection, whereas the latter is © 2009 Ichinohe et al. This article is distributed under the terms of an Attribu- tion–Noncommercial–Share Alike–No Mirror Sites license for the fi rst six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six Y. Ogura ’ s present address is Division of Bacterial Pathogen- months it is available under a Creative Commons License (Attribution–Noncom- esis, Graduate School of Medicine, University of the mercial–Share Alike 3.0 Unported license, as described at http://creativecommons Ryukyus, 207 Uehara, Nishihara-cho Okinawa, Japan. .org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 206 No. 1 79-87 79 www.jem.org/cgi/doi/10.1084/jem.20081667 The Journal of Experimental Medicine However, the mechanism by which IL-1  and IL-18 are ac- RESULTS AND DISCUSSION tivated during infl uenza infection in vivo is unknown. NLRP3 infl ammasome is activated in the lung Infl ammasomes are molecular platforms that allow acti- after respiratory infl uenza infection vation of caspase-1 ( 9 ). Caspase-1 is an essential regulator of Although NLRP3-defi cient macrophages were reported to be unable to activate IL-1  in response to infl uenza virus in infl ammatory response through its capacity to process and activate proIL-1  , proIL-18, and proIL-33 ( 10 ). NOD-like the presence of ATP in vitro ( 21 ), the role of infl ammasomes receptors (NLRs) comprise a large family of intracellular in infl uenza infection in vivo is unknown. In the lung, the PRRs that play an important role in innate immunity in re- primary targets of fl u infection are the respiratory epithelial sponse to recognition of various “ damaged ” self ( 11 ) and cells, which produce large amounts of virus that can subse- nonself molecules ( 9, 12 ). NLR protein (NLRP) 3, also quently infect alveolar macrophages ( 1 ) and DCs ( 22 ). To known as NALP3/Cryopyrin/CIAS1/PYPAF1 ( 13 ), forms begin to dissect the importance of various components of the a caspase-1 – activating infl ammasome. Mature IL-1  se- infl ammasome complex in response to infl uenza virus infec- cretion requires at least two steps: fi rst, transcriptional and tion, we fi rst examined IL-1  secretion in candidate cell translational up-regulation of pro – IL-1  through TLR stim- types including BM – derived macrophages (BMM), BM DCs and lung fi broblasts. Although BMM and BM DC exhibited ulation; and second, the activation of caspase-1 by infl am- masomes ( 9, 12 ). Recent reports indicate that infection by signifi cant reduction in IL-1  secretion in the absence of certain viruses also results in infl ammasome activation ( 14 – 16 ). NLRP3 ( Fig. 1, A and B ), lung fi broblast cells activated in- Kanneganti et al. ( 15 ) showed that Sendai and infl uenza vi- fl ammasomes independently of NLRP3 ( Fig. 1 C ). All cell ruses activated the NLRP3 infl ammasome in macrophages types required caspase-1 for release of IL-1  ( Fig. 1, A – C ). pulsed transiently with ATP for 30 min in vitro. Muruve Next, we examined the target of NLR recognition of infl u- et al. ( 16 ) demonstrated that adenovirus infection activates enza virus. Stimulation of cells with infl uenza virions showed IL-1  processing in NLRP3-, ASC-, and caspase-1 – dependent that attachment (blocked by 65 ° C inactivation), fusion (blocked manners. However, infl ammasomes were not activated by by 56 ° C inactivation), and replication (blocked by UV irra- transfection of RNA, Poly I:C, or infection with reovirus diation) of the virus were required to elicit infl ammasome activation in BM DCs (Fig. S1 A, available at http://www (double-stranded RNA virus) or vesicular stomatitis virus (single-stranded RNA virus). In another seminal study, Johnston .jem.org/cgi/content/full/jem.20081667/DC1) and in lung et al. ( 14 ) reported that Myxoma virus carries a protein that fi broblasts (Fig. S1 C). Purifi ed genomic RNA, upon deliv- inhibits ASC/caspase-1 activation and subsequent cell death ery by DOTAP, induced very low levels of IL-1  secretion after virus infection. Such an evasion mechanism supports in BM DC, which was enhanced by the addition of ATP the idea that infl ammasomes might play a vital role in anti- (Fig. S1 B), indicating that fl u genomic RNA, although in- viral defense. suffi cient to trigger infl ammasomes, could serve as signal 1 for Although the proinfl ammatory role of infl ammasomes NLR-mediated IL-1  secretion. In contrast, fl u genomic RNA is well known, less is understood with respect to the re- and ATP treatment failed to elicit infl ammasome activation quirement for infl ammasomes in the generation of adap- in lung fi broblasts (Fig. S1 D). Collectively, these data indi- cated that infl uenza virus infection stimulates NLRP3-de- tive immune responses. Uric acid, which triggers NLRP3 infl ammasomes ( 17 ), has been shown to stimulate DC matu- pendent and NLRP-3 independent infl ammasomes in a cell ration and, when coinjected with antigen in vivo, signifi - autonomous and cell type – specifi c manner and that other cantly enhances the generation of responses from CD8 T factors, in addition to viral genomic RNA, are required to cells ( 18 ). NLRP3, as well as its adaptor molecule ASC, are elicit infl ammasome activation. required for contact hypersensitivity responses in vivo ( 19 ). Next, to determine the role of NLRP3 infl ammasomes in More recently, the Th2-inducing adjuvant activity of alum the secretion of IL-1  during physiologically relevant infl u- was shown to be mediated through NLRP3/ASC infl amma- enza virus infection in vivo, mice were challenged intranasally somes ( 20 ). Thus, infl ammasomes appear to play an important with infl uenza strain A/PR8. Active IL-1  was secreted into role in certain types of autoimmune diseases, hyperrespon- the BAL in a dose-dependent manner (Fig. S2 A, available at http://www.jem.org/cgi/content/full/jem.20081667/DC1). siveness, and immunization. However, the role of infl am- masomes in the recognition of viral infection in vivo and Starting at 2 d post infection (p.i.), IL-1  secretion was evi- generation of protective adaptive immunity is unknown. dent in the BAL in WT mice infected with 10 PFU of infl u- In this paper, we examine the role of the NLRP3 infl am- enza virus. However, mice defi cient in NLRP3, ASC, or masomes in the initiation of adaptive immunity after physio- caspase-1, but not NLRC4, failed to secrete IL-1  ( Fig. 1 D ) logical infection with infl uenza virus. We describe requirement or IL-18 (Fig. S2 B) into the alveolar space in response to for caspase-1 infl ammasomes in the hematopoietic, but not infl uenza infection. Infl ammasome-independent cytokines, stromal, cells in the establishment of Th1, CTL, and IgA re- such as IL-6, TNF-  , KC, and MIP-2, were secreted com- sponses to respiratory fl u infection. Collectively, our data parably from NLRP3, ASC, or caspase-1 KO mice (Fig. S2, provide the fi rst evidence of the in vivo requirement for the C – F), indicating that the reduction in IL-1  and IL-18 secre- tion is not caused by a general immune defi ciency in these components of infl ammasomes in adaptive immunity to a vi- rus infection. mice. In contrast to the BAL fl uid, IL-1  secretion in the 80 INFLAMMASOME IN ADAPTIVE IMMUNITY TO INFLUENZA | Ichinohe et al. BRIEF DEFINITIVE REPORT lung tissue was found to be independent of NLRP3 ( Fig. 1 E ). alveolar space after respiratory challenge in a type I IFN-in- To determine the cellular compartment responsible for the dependent manner, whereas the lung tissue resident hemato- secretion of IL-1  in the lung, we generated BM chimeric poietic cells activate caspase-1 through NLRP3-independent mice in which only the hematopoietic (WT → caspase-1 ) pathways (Fig. S4). or the stromal cells (caspase-1 → WT) expressed caspase-1. Measurement of IL-1  in the BAL ( Fig. 1 F ) and lung tissue ASC, caspase-1, and IL-1R, but not NLRP3, is required ( Fig. 1 G ) indicated that infl ammasome activation in vivo for cellular recruitment to the lung after fl u infection occurs within the hematopoietic compartment and the stro- Next, we wished to determine the consequence of infl amma- mal compartment. A previous study demonstrated the im- some activation in cellular recruitment to the infected lungs. portance of type I IFNs in infl ammasome activation after In WT mice infected with A/PR8, accumulation of leuko- Francisella novicida infection ( 23 ). However, in response to in- cytes became apparent starting around day 3 p.i. and peaking fl uenza infection, although partly important in BM DC re- around day 5 p.i. (Fig. S5 A, available at http://www.jem.org/ sponse, responsiveness through type I IFN receptor was cg i/content/full/jem.20081667/DC1). Var ious leukocytes dispensable for IL-1  secretion in the BAL (Fig. S3). Collec- were detected including macrophages, DCs, pDCs, NK cells, tively, these data indicated that NLRP3 infl ammasome is ac- neutrophils, and lymphocytes (Fig. S5 B). These cellular sub- tivated in the alveolar macrophages and DCs within the sets were defi ned by fl ow cytometry as described in Fig. S6. Figure 1. The role of NLRP3, ASC, and caspase-1 in infl ammasome activation and cellular recruitment to the lung after infl uenza infection. (A – G) BM macrophages (A), BMDCs (B), or primary lung fi broblast (C) prepared from WT, NLRP3-, and caspase-1 – defi cient mice were infected with A/PR8 virus at MOI 2.5. Supernatant was collected 12 – 24 h after stimulation and analyzed for IL-1  by ELISA. WT, NLRP3-, NLRC4-, ASC-, and caspase-1 – defi cient mice were infected intranasally with 10 PFU A/PR8 virus. BAL was collected by washing the trachea and lungs twice by injecting a total of 1 ml PBS con- taining 0.1% BSA (D and F). Lung homogenate was prepared in 2 ml PBS containing 0.1% BSA (E and G). IL-1  levels detected from the BAL or lung ho- mogenate at different time points (D) or at 2 d p.i. (E – G) are shown. Horizontal lines show the mean. (H – I) Lung tissue was collected from the indicated groups of mice at 0 and 5 d p.i. Total lung leukocytes were enumerated. The values represent the mean of three mice per group and are expressed as the mean ± SD. Similar results were obtained from at least two separate experiments. *, P < 0.05; **, P < 0.01 versus a group of noninfected mice. JEM VOL. 206, January 19, 2009 81 Figure 2. Infl uenza-specifi c CD4 and CD8 T cell responses depend largely on ASC and Caspase-1 but not NLRP3. WT, NLRP3-, ASC-, and cas- /   /   / pase-1 – defi cient mice (A and B), IL-1R mice (C and D), or WT → caspase-1 and caspase-1 → WT BM chimeric mice (E and F) were infected intra- nasally with a sublethal dose (10 PFU) of A/PR8 virus. 14 d p.i., CD8 and CD4 T cells were isolated from spleen and stimulated with irradiated APCs with 82 INFLAMMASOME IN ADAPTIVE IMMUNITY TO INFLUENZA | Ichinohe et al. BRIEF DEFINITIVE REPORT /   /   / In contrast, mice defi cient in ASC and caspase-1, but not from WT mice, ASC , caspase-1 ( Fig. 2 A ), and IL-1R NLRP3, exhibited signifi cant reduction in cellular recruit- ( Fig. 2 C ) mice failed to mount comparable CD4 T cell ment of all subsets (Fig. S5 C). In addition, IL-1R – defi cient responses. In addition, ASC mice demonstrated a partially mice were also impaired in their ability to induce leukocyte reduced CTL phenotype compared with caspase-1 or /  + recruitment to the lung ( Fig. 1 H ). Furthermore, mice selec- IL-1R mice, in which CD8 T cell responses were barely tively defective in caspase-1 in the hematopoietic compart- detectable after immunization with live fl u infection ( Fig. 2 B ). ment (caspase-1 → WT) were impaired in their ability to Furthermore, comparable reduction in virus-specifi c CD8 T recruit leukocytes to the lung ( Fig. 1 I ). Thus, these data indi- cell numbers in the spleen and lung was observed by intra- cated that although NLRP3 is required for IL-1  secretion cellular IFN-  staining ( Fig. 2 G ) and by tetramer staining into the alveolar space ( Fig. 1 D ), NLRP3-independent ASC/ ( Fig. 2 H ). These data indicated that NLRP3-independent caspase-1 – dependent pathways ( Fig. 1 E ) in the lung tissue ASC/caspase-1 infl ammasomes and responsiveness through hematopoietic cells ( Fig. 1 I ) result in IL-1  secretion leading the IL-1R are required to elicit T cell responses to infl uenza to cellular infi ltration in the lung. virus in vivo. In addition, the intermediate phenotype of the IL-1  is a well known stimulator of neutrophil recruitment, defect in T cell responses in ASC compared with cas- which is triggered by the synthesis and display of chemokines pase-1 mice suggests the presence of an ASC-independent and leukocyte adhesion molecules. Our data demonstrated caspase-1 infl ammasome in the generation of T cell responses that diff erential requirements for NLRP3 – IL-1  secretion after infl uenza infection. into the alveolar space depended on NLRP3 ( Fig. 1 D ), whereas Next, we examined the cellular compartment responsible IL-1  secretion in the lung interstitium ( Fig. 1 E ) and re- for the infl ammasome-dependent generation of CD4 and cruitment of leukocytes to the lung were independent of CD8 T cell responses. T cell responses from fl u-infected cas- /   / NLRP3 ( Fig. 1 H ). Furthermore, lung leukocyte infi ltration pase-1 → WT, but not WT → caspase-1 mice, were depended on ASC, caspase-1, and IL-1R, indicating that diminished ( Fig. 2, E and F ), indicating that activation of infl ammasome-released IL-1  is required to mediate this pro- caspase-1 infl ammasomes by the hematopoietic, but not stro- cess. In contrast, NLRC4-defi cient mice had normal IL-1 mal, cells link NLR recognition of infl uenza virus to the secretion as measured by BAL ( Fig. 1 D ). These data demon- activation of adaptive immunity in vivo. strated a diff erential role of NLRP3; the cell types that secrete IL-1  in the alveolar space (such as alveolar macrophages Differential requirement for infl ammasomes and DCs) require NLRP3 for infl ammasome activation, whereas in immunoglobulin isotype responses to infl uenza virus those that secrete IL-1  in the lung interstitium (interstitial Antibody responses play a critical role in the clearance of macrophages, DCs, and others) can trigger NLRP3-inde- many viral pathogens including infl uenza virus. Previous pendent infl ammasomes. Only the latter source of IL-1  is studies have indicated the role of MyD88 in IgG2a responses required to activate vascular endothelial cell to enable leuko- to infl uenza infection ( 26, 27 ). We determined the contribu- cyte migration into the lung (Fig. S4). Evidence exists for the tions of infl ammasomes in this process. To this end, mice were NLRP3-independent ASC dimer – mediated pyroptosome immunized with a sublethal dose (10 PFU) of live A/PR8 that can activate caspase-1 directly ( 24 ). It is of interest that virus. 2 wk p.i., infl uenza virion – specifi c nasal IgA and serum other studies have described NLRP3/NLRC4-independent titers of IgM and IgG isotype levels were measured by serial ASC/caspase-1 – dependent infl ammasome activation in re- dilution. Nasal IgA responses were found to be completely sponse to LPS ( 25 ) and cytosolic DNA ( 16 ). dependent on ASC, caspase-1, IL-1R, and MyD88 but not on NLRP3 ( Fig. 3 A ). In contrast, serum IgG1 levels were Infl uenza-specifi c CD4 and CD8 T cell responses depend signifi cantly elevated in MyD88-defi cient mice compared largely on ASC and Caspase-1 but not NLRP3 with WT or other infl ammasome KO groups ( Fig. 3 C ). A Next, to decipher the importance of infl ammasomes in adap- slight reduction in the IgG2c response was detected in ASC tive immune responses to infl uenza infection, mice defi cient and caspase-1 mice, which was similar to that seen in IL- /   /   / in NLRP3, ASC, caspase-1, or IL-1R were infected with a 1R and MyD88 mice ( Fig. 3 D ). Only caspase-1 sublethal dose of A/PR8 (10 PFU or 0.4 LD ). 14 d later, mice had a signifi cant reduction in IgG3 responses but not + + CD4 and CD8 T cells were isolated from the spleen of fl u- IL-1R – or MyD88-defi cient mice ( Fig. 3 E ), indicating that primed mice and restimulated with varying concentrations of caspase-1 – dependent IL-1  – , IL-18 – , and IL-33 (all upstream inactivated virion or H-2D – restricted nucleoprotein (NP) of MyD88) – independent factors play a major role in the in- + + peptide, respectively. Although CD4 and CD8 T cells from duction of this isotype response. In contrast, no signifi cant infected NLRP3-defi cient mice responded similarly to those diff erences were found in serum IgM responses ( Fig. 3 F ). + + the indicated amount of heat-inactivated virion or NP peptide for 72 h, respectively. IFN-  production from CD4 T cells (A, C, and E) and CD8 T cells (B, D, and F) was measured by ELISA or by intracellular IFN-  staining (G). (H) Indicated groups of mice were infected with 10 PFU of A/PR8/GP-33 recom- binant infl uenza virus, and GP33-specifi c CD8 T cells were detected in the spleen and the lung at 14 d p.i. using the K -GP33-41 tetramer. These fi gures are representative of three similar experiments. Error bars show SD. *, P < 0.05; **, P < 0.01 versus WT mice. JEM VOL. 206, January 19, 2009 83 Antibody secretion in the BM chimeric mice indicated that the whereas IgG2a/c and CD4 T cell responses depend on MyD88 requirement for caspase-1 was all attributable to its expression ( 26, 27 ). In light of the data we present here, infl ammasome in the hematopoietic compartment alone (Fig. S7, available at recognition of infl uenza virus infection plays a more domi- http://www.jem.org/cgi/content/full/jem.20081667/DC1). nant role in the establishment of CD8 T cell responses com- These data indicated that at least one NLRP3-independent pared with TLR7. In addition, we found that serum IgG2c ASC- and caspase-1 – dependent infl ammasome is required responses were largely dependent on both ASC-dependent for nasal IgA and, to some extent, serum IgG2c responses to caspase-1 infl ammasome signals. These data suggested that infl uenza virus. In addition, IL-1R and MyD88, which is caspase-1 infl ammasomes, as well as TLR7/MyD88 ( 26, 27 ), downstream of TLRs, IL-1R, IL-18R, and ST2, are required play a key role in the formation of IgG2c-secreting B cells for the same process in vivo. In contrast, IgG3 responses appear after fl u infection. A pronounced defect in nasal IgA produc- to depend mainly on caspase-1 but not on IL-1R or MyD88, tion in ASC and caspase-1 – defi cient mice and complete im- whereas MyD88 has a regulatory role for IgG1 responses. pairment in MyD88 mice suggest that infl ammasomes Our results indicated that ASC/caspase-1 infl amma- and IL-1R signals are required to elicit B cell secretion of IgA somes and the secreted IL-1  play a key role in the genera- in the nasal mucosa. tion of Th1, CTL, and IgA responses after infl uenza infection (Table S1, available at http://www.jem.org/cgi/content/full/ ASC, caspase-1, and IL-1R, but not NLRP3, are required jem.20081667/DC1). Previous studies have demonstrated for survival after sublethal infl uenza challenge the requirement for TLR7 and MyD88 in adaptive immu- Finally, we examined the importance of infl ammasomes in nity to infl uenza virus infection with somewhat confl icting immune-mediated protection. To this end, we followed viral results. Lopez et al. ( 28 ) found no requirement for MyD88 titer and survival of mice after intranasal challenge with a sub- on T cells or immunoglobulin responses to aerosolized infl u- lethal dose (10 PFU) of A/PR8. The virus titers in the lung of enza virus challenge. In contrast, Koyama et al. ( 27 ) showed infected mice revealed that all mice sustained similar viral load that TLR7 or MyD88 is required for the induction of B cell at day 5 p.i., indicating that, with the low dose viral chal- secretion of anti-HA IgG2a and IgG2c but not CTL re- lenge, infl ammasomes were not required for innate immune sponses. Heer et al. ( 26 ) demonstrated that IgG2a and IgG2c protection ( Fig. 4, A and B ). In contrast, at later stages of in- responses to fl u were impaired in MyD88 but not in fection, viral clearance was severely impaired in mice defi - TLR7 mice. All studies found IgG1 antibody levels to be cient for ASC, caspase-1, or IL-1R, but not NLRP3 ( Fig. 4 A ). /   / elevated in MyD88 mice ( 26 – 28 ) as well as TLR7 Specifi cally, caspase-1 in the hematopoietic but not stromal mice ( 26 ). Together, these studies indicated that activation of compartment was required for control of virus replication at CD8 T cells during antiinfl uenza immune response relies this stage ( Fig. 4 B ). This eff ect became pronounced on day on mechanisms other than the TLR7 and MyD88 ( 26 – 28 ), 10 p.i., when WT and NLRP3 mice had completely Figure 3. Differential requirement for NLRP3, ASC, and caspase-1 for immunoglobulin isotype responses to infl uenza virus. WT, NLRP3-, ASC-, caspase-1 – , IL-1R – , and MyD88-defi cient mice were infected intranasally with a sublethal dose (10 PFU) of A/PR8 virus. Serum and nasal swab were collected at 2 wk p.i. A/PR8-specifi c nasal IgA levels were measured by ELISA (A). A/PR8-specifi c serum antibody titers were determined by serial dilution of serum total IgG (B), IgG1 (C), IgG2c (D), IgG3 (E), and IgM (F). These fi gures are representative of three similar experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus WT mice. 84 INFLAMMASOME IN ADAPTIVE IMMUNITY TO INFLUENZA | Ichinohe et al. BRIEF DEFINITIVE REPORT cleared the virus and those defi cient in ASC, caspase-1, or indicated that NLRP3-independent ASC-dependent caspase-1 IL-1R sustained high virus burden in the lung ( Fig. 4 A ). infl ammasomes are required to provide IL-1R – dependent Consequently, although WT and NLRP3 mice recovered immune protection against respiratory infl uenza infection. It from sublethal infection with infl uenza virus, the majority is unclear which NLRPs are responsible for the recognition /   /   / of the fl u-challenged ASC , caspase-1 , and IL-1R of infl uenza virus in the initiation of ASC infl ammasome-de- mice succumbed to death by 18 d p.i. ( Fig. 4 C ). These data pendent immune responses. Future identifi cation of NLRP responsible for infl uenza recognition will provide important insights into the molecular mechanism. /   /   / Mortality of ASC , caspase-1 , and IL-1R mice after a low-dose infl uenza challenge was associated with re- duced CD4 and CD8 T cell responses ( Fig. 2 ) as well as im- paired mucosal IgA and systemic IgG2 responses ( Fig. 3 ) to infl uenza viral antigens (Table S1). It is well known that re- covery from infl uenza infection requires CD8 T cells. The antiviral capacity of fl u-specifi c CD8 T cells is dependent on their ability to migrate to the lungs and come in contact with infected airway epithelium ( 29 ). Eff ector CD8 T cells begin to appear in the lung  5 – 7 d after infection ( 30 ). Thus, the inability of ASC- and caspase-1 – defi cient mice to clear virus in the lung at 8 and 10 d after infl uenza infection ( Fig. 4 A ) is likely the result of a combined eff ect of their failure to de- velop robust systemic CD8 T cell responses and to recruit ef- fector CD8 T cells to the lung ( Fig. 2 and Fig. S5 C). This recruitment and eff ector T cell induction are downstream of IL-1R signaling, as IL-1R mice failed to elicit lung cellu- lar infi ltration ( Fig. 1 ) or T cell responses ( Fig. 2 ). These data provide important evidence for the requirements for infl am- masomes in multiple stages of antiviral immune defense. In conclusion, our study demonstrated the requirement for ASC/caspase-1 infl ammasomes in the development of adap- tive immune responses to respiratory infl uenza virus infection. Our data support the notion that infl uenza virus is recognized through an ASC-dependent pathway in addition to TLR7 and RIG-I. Both caspase-1 (this paper) and TLR7 ( 26, 27 ) recog- nition pathways, but not RIG-I ( 27 ), appear to be required for adaptive immune responses to infl uenza virus. These data have signifi cant implications in the design of prophylactic vaccines and management of infl uenza infection in general. Stimulants of infl ammasomes may provide an ideal adjuvant candidate in fl u vaccines that will confer protective immune responses. Finally, clinical management of severe pathogenic infl uenza strains, such as H5N1 avian strains, which often cause immuno- pathology, may require blockade of infl ammasome to atten- uate pathology in addition to antiviral therapy. MATERIALS AND METHODS Animals. The generation of mice defi cient in NLRP3, ASC ( 19 ), NLRC4 Figure 4. ASC-1 – , caspase-1 – , and IL-1R – defi cient mice, but not ( 31 ), caspase-1 ( 10 ), and MyD88 ( 32 ) has been reported previously. All NLRP3-defi cient mice, are more susceptible to infl uenza infection KO mice have been backcrossed at least nine generations onto the C57BL/6 and disease. WT, NLRP3-, ASC-, caspase-1 – , and IL-1R – defi cient mice background. Age- and sex-matched C57BL/6 mice from National Cancer /   / (A) and WT → caspase-1 and caspase-1 → WT BM chimeric mice Institute (Frederick, MD) were used as WT controls. IL-1R mice were (B) were infected intranasally with a sublethal dose (10 PFU) of A/PR8 obtained from The Jackson Laboratory. All procedures used in this study virus. The lungs of infl uenza-infected mice were harvested at 5, 8, and 10 complied with federal guidelines and were approved by the Yale Animal Care d p.i., and viral titers were determined by plaque assay (A and B). The val- and Use Committee. ues are representative of three mice per group and are expressed as the mean ± SD. *, P < 0.05; **, P < 0.01 versus WT mice. (C) Survival of mice Infl uenza virus infection in vitro . Lung fi broblasts were prepared accord- after infection is depicted. Similar results were obtained from at least two ing to published procedures ( 33 ). BMM, DCs (8 × 10 cells/24 wells), or lung separate experiments. fi broblasts (2 × 10 cells/96 wells) were stimulated with live or inactivated JEM VOL. 206, January 19, 2009 85 A/PR8 virus. 1 h after stimulation, cells were washed thoroughly and incu- The serum titers of IgM and IgG isotypes against A/PR8 viruses were de- bated in complete media for 24 h. Cell-free supernatant was collected and termined by ELISA. In brief, a 96-well plate (EIA plates; Costar; Corning) was analyzed for IL-1  by ELISA. coated with formalin-inactivated A/PR8 virion with carbonate buff er. The re- actions were detected by goat anti – mouse IgA (Invitrogen), goat anti – mouse IgG1 (Bethyl laboratories, Inc.), goat anti – mouse IgG2c (Bethyl laboratories, Infl uenza virus infection in vivo. A/PR8 virus (H1N1) used for all ex- Inc.), goat anti – mouse IgG3 (SouthernBiotech), goat anti – mouse IgG (Jackson periments was grown in allantoic cavities from 10 – 11-d-old fertile chicken ImmunoResearch Laboratories), or goat anti – mouse IgM (SouthernBiotech) eggs for 2 d at 37 ° C. This virus was stored at  80 ° C, and viral was quanti- antibodies conjugated to horseradish peroxidase. Endpoint titers were consid- fi ed by a standard plaque assay using Madin-Darby canine kidney cells. For ered positive for dilutions with OD values that were twofold higher than the intranasal infection, mice were fully anesthetized by i.p. injection of ket- background level (nonimmune serum). amine and xylazine and then infected by intranasal application of 20 μ l of vi- rus suspension (10 – 1,000 PFU in PBS). This procedure leads to the upper Online supplemental material. Fig. S1 shows the viral and cellular determi- and lower respiratory tract infection. Recombinant PR8 virus expressing the nant for infl uenza recognition. Fig. S2 shows infl ammasome-dependent and in- LCMV glycoprotein epitope GP33-41 (PR8-GP33) was a gift from S. dependent cytokine secretion in the BAL of infl uenza-infected mice. Fig. S3 Kaech (Yale University, New Haven, CT). examines the role of type I IFNs in infl ammasome activation by infl uenza virus. Fig. S4 is a schematic representation of the role of infl ammasomes in the genera- Preparation of lung single-cell suspensions. To obtain single lung cell tion of adaptive immunity against fl u infection. Fig. S5 depicts leukocyte re- suspensions, lungs were perfused with 10 ml PBS through the right ventri- cruitment to the lung of infl uenza-infected mice. Fig. S6 describes the strategy cle, minced using razor blades, and incubated in HBSS containing 2.5 mM for cell type identifi cation by fl ow cytometry. Fig. S7 shows infl uenza virus Hepes and 1.3 mM EDTA at 37 ° C for 30 min. The cells were resuspended specifi c antibody production in BM chimeric mice. Online supplemental mate- in RPMI containing 5% FBS, 1 mM CaCl , 1 mM MgCl , 2.5 mM Hepes, 2 2 rial is available at http://www.jem.org/cgi/content/full/jem.20081667/DC1. and 0.5 mg/ml collagenase d (Roche) and incubated at 37 ° C for 60 min. A single-cell suspension was prepared after RBC lysis. The resulting cells were We thank Drs. S. Kaech and A. Chandele for critical reagents and technical support. fi ltered through a 70- μ m cell strainer (BD) and used for FACS analysis. This study was supported by grants from the National Institutes of Health (AI054359, AI062428, and AI064705). T. Ichinohe is a recipient of the Japan Society for Flow cytometry. The single-cell suspensions of lung samples were stained the Promotion of Science Postdoctoral Fellow for Research Abroad. H.K. Lee was with anti-B220, anti-CD3, anti-CD4, anti – CD8-  , anti – MHC class II, supported by the Ministry of Science and Technology of Korea and is a recipient of anti-CD19, anti-DX5, anti-F4/80, anti-CD11c, anti – mPDCA-1, anti – Si- Richard K. Gershon fellowship. A. Iwasaki is a recipient of the Burroughs Wellcome glec-H, anti-CD11b, or anti-CD45.2 antibodies. Leukocytes were gated Investigators in Pathogenesis of Infectious Disease. based on forward and side scatter properties, and live cells were gated based The authors have no confl icting fi nancial interests. on 7-aminoactinomycin d exclusion. Acquisition of eight color samples was Submitted: 29 July 2008 performed on a cytometer (LSR II; BD). Leukocytes from spleen or lung of Accepted: 11 December 2008 immunized mice were cultured in the presence of 10 μ g/ml NP peptide (ASNENMETM; H-2D ) for 6 h, and 10 μ g/ml of brefeldin A (Sigma-Al- REFERENCES drich) was added for an additional 12 h. 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Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Jan 19, 2009

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