Identification of Aim2 as a Sensor for DNA Vaccines
J Immunol published online 8 December 2014 http://www.jimmunol.org/content/early/2014/12/05/jimmun ol.1402530
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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This information is current as of December 13, 2014.
John J. Suschak, Shixia Wang, Katherine A. Fitzgerald and Shan Lu
Published December 8, 2014, doi:10.4049/jimmunol.1402530 The Journal of Immunology
Identification of Aim2 as a Sensor for DNA Vaccines John J. Suschak,* Shixia Wang,* Katherine A. Fitzgerald,† and Shan Lu*
T
he discovery of DNA vaccine technology in the early 1990s was a major event in the history of vaccinology due to the many unique features of DNA immunization, including its ability to elicit balanced Ab and T cell immunity (1–6). However, in early clinical studies, the immunogenicity of DNA vaccines in humans was low when such vaccines were used alone. More recent human trials have demonstrated that DNA vaccines are actually extremely powerful in priming the host’s immune system to develop high-level protective Ab responses against HIV-1 and pandemic influenza viruses (3, 7–10). Animal studies further demonstrated that DNA immunization is effective in eliciting higher levels of Ag-specific B cell responses (11). One mechanism to achieve such an outcome is that DNA vaccination is effective in eliciting higher germinal center B cell development via enhanced follicular Th cells for the production of high-quality Ab responses (12). DNA vaccines produce immunogens in vivo, which are then presented to the immune system via the endogenous Ag-processing pathway. At the same time, the DNA plasmid itself confers an intrinsic adjuvant effect that enhances the immune response generated toward the vaccine-encoded immunogens (12, 13), but the intracellular processes involved remain to be fully elucidated. Several pattern recognition receptors (PRRs) have been identified that respond to DNA molecules (14, 15). One of the best-characterized DNA-sensing PRRs is the endosomal TLR9, which is essential for *Laboratory of Nucleic Acid Vaccines, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655; and †Program in Innate Immunity, Division of Infectious Diseases, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655 Received for publication October 2, 2014. Accepted for publication November 13, 2014. This study was supported in part by National Institutes of Health Grants 5 U19 AI082676, 5 P01 AI082274, and 5 R33 AI087191 and Bill and Melinda Gates Foundation Grant OPP1033112. Address correspondence and reprint requests to Prof. Shan Lu, University of Massachusetts Medical School, Room LRB-304, 364 Plantation Street, Worcester, MA 01605. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: Aim2, absent in melanoma 2; Aim22/2, Aim2deficient; BMDM, bone marrow–derived macrophage; DAMP, damage-associated molecular pattern; HA, hemagglutinin; LDH, lactate dehydrogenase; poly(dA-dT), poly(deoxyadenylic-deoxythymidylic) acid; PRR, pattern recognition receptor; TBK1, TANK-binding kinase 1; UMMS, University of Massachusetts Medical School. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402530
the recognition of unmethylated CpG containing oligodeoxynucleotide motifs commonly found in bacterial plasmids (16). As DNA vaccine plasmid backbones contain certain CpG/oligodeoxynucleotide motifs, it was initially thought that TLR9 would be critical for DNA vaccine immunogenicity. However, whereas DNA plasmid activates dendritic cells via TLR9, TLR9-deficient mice were able to mount immune responses comparable to wild-type mice (17, 18). Likewise, DNA immunization of MyD88 and TRIF-deficient mice yields robust immune responses, further suggesting that TLR signaling may be dispensable for DNA vaccine-induced immunogenicity (19). In recent years, it has become increasingly evident that the double-stranded nature of DNA itself functions as a potent activator of innate immune signals (20–24). Cytosolic DNA is a powerful initiator of type I IFN (IFN-ab) in both immune and nonimmune cells, functioning through a STING/TANK-binding kinase 1 (TBK1)/IFN regulatory factor 3–dependent pathway that is independent of CpG motifs and TLRs. At the same time, it is not clear whether other components of the innate immune system beyond the STING/TBK1/IFN-ab pathway are involved in the immunogenicity of DNA vaccines (19, 20). This is especially true in the case of inflammasome pathways. Inflammasomes regulate caspase-1 activity, ultimately resulting in cleavage of the proinflammatory cytokines pro–IL-1b and pro–IL-18 into their active forms. Inflammasome activation also results in pyroptotic cell death; a suicidal form of cell death characterized by the release of damage-associated molecular patterns (DAMPs) that further propagates innate immune signaling to surrounding bystander cells (25). One flavor of inflammasome contains absent in melanoma 2 (Aim2), which is a direct sensor of cytosolic DNA and a member of the PYHIN family (22). Aim2 contains a DNA binding HIN200 domain, as well as a pyrin domain. Although Aim2’s role in orchestrating immune responses to both viral and bacterial pathogens is well characterized (26–28), the role of Aim2 in DNA vaccination is unknown. In this study, we found that Aim2 and the adapter molecule Asc were required for the generation of optimal immunogen-specific Ab responses to a DNA vaccine expressing influenza hemagglutinin (HA) immunogen in a mouse model. DNA vaccination leads to transcription of key components of the inflammasome. Importantly, the efficacy of DNA vaccination was independent of IL-1b and IL-18. Surprisingly, Aim2-deficient (Aim22/2) mice were unable to elicit a type I IFN response at the site of injection. Our
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Recent human study data have re-established the value of DNA vaccines, especially in priming high-level Ag-specific Ab responses, but also raised questions about the mechanisms responsible for such effects. Whereas previous reports have shown involvement of downstream signaling molecules in the innate immune system, the current study investigated the role of absent in melanoma 2 (Aim2) as a sensor for DNA vaccines. The Aim2 inflammasome directs maturation of the proinflammatory cytokines IL-1b and IL-18 and an inflammatory form of cell death called pyroptosis. Both the humoral and cellular Ag-specific adaptive responses were significantly reduced in Aim2-deficient mice in an IL-1b/IL-18–independent manner after DNA vaccination. Surprisingly, Aim2-deficient mice also exhibited significantly lower levels of IFN-a/b at the site of injection. These results indicate a previously unreported link between DNA vaccine–induced pyroptotic cell death and vaccine immunogenicity that is instrumental in shaping the Ag-specific immune response to DNA vaccines. The Journal of Immunology, 2015, 194: 000–000.
2 data therefore establish a novel role for Aim2 as a key player in the regulation of DNA vaccination.
Materials and Methods Animals
Gene induction analysis C57BL/6 mice were shaved and immunized with 100 mg H1-TX04-09.tPA DNA vaccine plasmid i.m. injected into the hind quad muscle. Punch biopsies were harvested from the site of immunization at 6, 12, and 24 h postimmunization and snap frozen. RNA was isolated from tissue biopsies using TRIzol Reagent (Life Technologies). We analyzed gene expression using the Nanostring nCounter Analysis system (Nanostring Technologies). Each reaction contained 100 ng RNA in a 5-ml aliquot, plus reporter and capture probes. We also included six pairs of positive control and eight pairs of negative control probes. Gene induction analysis and normalization was conducted using nSolver Analysis Software v1.1. Raw counts were normalized to naive mice using three reference genes: Gapdh, Gusb, and Hprt1.
Cell culture and cytokine ELISA Mouse bone marrow–derived dendritic cells were generated from Aim2+/+ or Aim22/2 mice by culturing fresh bone marrow in R10 medium containing GM-CSF for 8 d at 37˚C. Aim2+/+ and Aim22/2 immortalized bone marrow–derived macrophages (BMDM) were produced in-house. Cells were first primed with 200 ng/ml LPS (Sigma-Aldrich, St. Louis, MO) for 4 to 5 h. prior to treatment with appropriate stimuli. All media was removed from the cells, and the appropriate stimulus was added. Poly (deoxyadenylic-deoxythymidylic) acid [poly (dA-dT)] (Sigma-Aldrich) DNA and H1-TX04-09.tPA DNA vaccine plasmid were transfected using Lipofectamine 2000 at a concentration of 1.5 mg/ml. ATP was added at a concentration of 1.25 mg/ml. Cultures were incubated 16–18 h at 37˚C, and supernatants were harvested. Cell-culture supernatants were assayed for IL-1b (BD Biosciences, Franklin Lakes, NJ) by ELISA. Lactate dehydrogenase (LDH) release was used to measure pyroptotic cell death. LDH assays were performed using the Promega CytoTox96 Nonradioactive Cytotoxicity Kit according to the manufacturer’s directions (Promega, Madison, WI).
ELISA Microtiter plates were coated with transiently expressed H1HA Ag at ∼1 mg/ml in PBS for 1 h at room temperature and assayed as previously described (29). NaSCN displacement was performed at a serum dilution of 1:100. After washing of serum samples, NaSCN was added at various (0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 M) concentrations in PBS for 15 min followed by five washes in ELISA wash buffer. The assay was then completed as above.
ELISPOT assay Splenocyte T and B cell ELISPOT reagents were obtained from Mabtech (Mariemont, OH). H1HA-specific T cells were quantified per the manufacturer’s instructions. Positive controls were stimulated with 20 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (SigmaAldrich). The H1HA relevant peptide used was a CD8+ cell-restricted HA peptide (IYSTVASSL). H1HA-specific Ab-secreting cells were detected by coating of MAIPSWU (Millipore, Billerica, MA) plates with the transiently expressed H1HA Ag used for ELISA (∼1.0 mg/well). Positive spots were visualized on a CTL Imager, and counting was performed with Immunospot software (Cellular Technology, Shaker Heights, OH).
In vivo caspase-1 activation Aim2+/+ and Aim22/2 mice were shaved and immunized with 100 mg H1-TX04-09.tPA DNA vaccine plasmid i.m. injected into the hind quad muscle. Punch biopsies were harvested from the site of immunization at 6, 12, and 24 h postimmunization and snap frozen. Cryopreserved tissue sections were generated and adhered to glass slides. Samples were then stained with a caspase-1 FAM/FLICA kit according to the manufacturer’s instructions (ImmunoChemistry Technologies, Bloomington, MN). Stained slides were visualized on a confocal microscope. Sixteen independent fields were analyzed for fluorescence.
Quantitative real-time PCR Aim2+/+ and Aim22/2 mice were shaved and immunized with 100 mg H1-TX04-09.tPA DNA vaccine plasmid i.m. injected into the hind quad muscle. Punch biopsies were harvested from the site of immunization at 6, 12, and 24 h postimmunization and snap frozen. RNA was isolated from tissues biopsies using TRIzol Reagent (#15596-026; Life Technologies), and cDNA was generated using the Bio-Rad iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). cDNA was then used for RT-PCR reactions on a Bio-Rad CFX-96 cycler (Bio-Rad). Primers sequences are available upon request.
Statistical analysis All data are presented as the mean of individual mice 6 SEM. Statistical analysis was performed using a Student t test, a one-way ANOVA followed by a Tukey posttest, or a two-way ANOVA followed by a Bonferroni posttest.
Results DNA vaccine plasmid induces expression of Aim2, caspase-1, and the inflammasome Although previous studies have mainly used noncoding DNA plasmid or DNA vaccines coding for marker proteins to study DNA-elicited innate immune responses, the current study tested a DNA vaccine (pH1HA) expressing the HA Ag of the type A influenza virus subtype H1N1 virus, which was responsible for a pandemic influenza in 2009. HA is the major protective Ag in clinically licensed inactivated and live-attenuated influenza vaccines. DNA vaccines expressing HA have been shown to be immunogenic in eliciting HA-specific Abs in both animal and human studies (2, 29–33). The expression of HA Ag by pH1HA used in the current study was confirmed by Western blot, and its immunogenicity to elicit HA-specific Ab response was verified in a pilot mouse study (data not shown). We first wanted to profile key immune response genes following DNA vaccine pH1HA using the Nanostring nCounter gene expression system (Nanostring Technologies), which includes a custom array encoding 50 innate immunity targets. Gene induction was quantified from wild-type C57BL/6 mice immunized with the pH1HA DNA vaccine. mRNA was isolated, the expression of innate immune genes profiled using the Nanostring nCounter, and changes in gene induction quantified. Notably, Aim2 was induced ∼6-fold within 12 h of immunization when compared with naive samples. Aim2 is a type I IFN–inducible gene, suggesting a potent ability of cells at the site of vaccination to recognize cytosolic plasmid vaccines (Fig. 1A). In accordance with the induction of Aim2, caspase-1 was also highly upregulated. (Fig. 1B). Most striking were the high levels of the inflammatory cytokines IL-1a and IL-1b (Fig. 1C, 1D). These observations indicate that inflammasome components were present at the site of vaccination. To test if the inflammasome pathway was active at the site of vaccination, we used a caspase1–specific FAM/FLICA fluorescent stain to covalently label catalytically active caspase-1. Mature caspase-1 became apparent within 6 h of immunization and reached a peak at 12 h (Supplemental Fig. 1). Collectively, these results led us to examine the role of the Aim2 inflammasome pathway in Ag-specific immune responses elicited by pH1HA DNA vaccination.
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C57BL/6 mice were obtained from Taconic Laboratories. Aim22/2 mice were generated in house by K. Fitzgerald’s group at the University of Massachusetts Medical School (UMMS) as previously described (26). Aim22/2 mice were on a mixed B6/129 background, and therefore B6 3 129 mice were used as controls. B6.129 (hereafter referred to as Aim2+/+) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred at UMMS. IL-1R, IL-18R, and Asc-deficient mice were produced in house. All mice were maintained in the Department of Animal Medicine at UMMS according to Institutional Animal Care and Use Committee–approved protocols. Mice received 100 mg codon-optimized H1HA DNA vaccine expressing the full-length wild-type HA protein from A/Texas/04/ 09 (pH1HA), divided between quadriceps, at 2 and 4 wk. A third boosting immunization was delivered 1 wk prior to sacrifice.
THE EFFECTS OF Aim2 ON DNA VACCINE IMMUNOGENICITY
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Involvement of Aim2 in cellular responses to DNA vaccine plasmid We first evaluated the ability of macrophages and dendritic cells to recognize pH1HA DNA vaccine by performing in vitro experiments. IL-1b production in response to pH1HA DNA vaccine was evaluated in BMDM collected from either Aim2+/+ or Aim22/2 mice (Fig. 2A). As expected, both the synthetic B-form dsDNA poly(dA-dT) and pH1HA DNA vaccine induced a robust IL-1b response in Aim2+/+ BMDM as measured by ELISA. However, IL-1b production was abolished in Aim22/2 BMDM. Similar results were seen in bone marrow–derived dendritic cells (data not shown). Next, the pan-caspase inhibitor Z-VAD-FMK was included in BMDM cultures prior to adding pH1HA DNA vaccine (Fig. 2B). This resulted in inhibited IL-1b production in Aim2+/+ macrophages, yielding IL-1b levels comparable to Aim22/2 wells, supporting the role of Aim2 in DNA vaccine–mediated IL-1b maturation. Finally, pH1HA DNA vaccine induced an inflammatory form of cell death (pyroptosis) in Aim2+/+ macrophages, as measured by LDH release (Fig. 2C). This response was attenuated in Aim22/2 cells. Collectively, these data indicate that Aim2 acts as a sensor of DNA vaccine plasmid and regulates caspase-1–dependent IL-1b production and pyroptotic cell death in response to pH1HA DNA vaccines in vitro. Effects of Aim2 deletion on pH1HA induced HA-specific immune responses As it is evident that the Aim2 inflammasome recognizes and responds to pH1HA DNA vaccine in cultured cells, the role of Aim2 in pH1HA DNA vaccination was next examined in Aim22/2 and wild-type Aim2+/+ mice. The pH1HA DNA vaccine induced high-level HA-specific Ab responses in Aim2+/+ mice, but significantly lower Ab titers in Aim22/2 mice (Fig. 3A). This reduction is isotype-independent as Aim22/2 mice exhibited significantly lower levels of HA-specific IgG1, IgG2b, and IgG2c responses (data not shown). Likewise, Aim22/2 mice exhibited significantly reduced HA-specific circulating B cells as well as IFN-g–secreting CD8+ T cells in the spleen (Fig. 3C,
FIGURE 2. Aim2 is required for IL-1b production in response to DNA vaccines. LPS-primed (200 ng/ml) Aim2+/+ and Aim22/2 BMDM were transfected with poly(dA-dT) or DNA vaccine plasmid for 18 h. (A) Secreted IL-1b in the culture supernatants was analyzed by ELISA. (B) Aim2+/+ and Aim22/2 BMDM were treated as above with the addition of the pancaspase inhibitor Z-VAD-FMK, and secreted IL-1b was quantified by ELISA. (C) Aim2+/+ and Aim22/2 BMDM were treated as above, and culture LDH amounts were reported as a percentage of lysed cellular controls. Data are presented as mean 6 SEM from three independent experiments. **p , 0.01, ***p , 0.001, ****p , 0.0001 versus control group.
3D). The role of Aim2 in regulating the maturation process of pH1HA-induced Ab responses was further confirmed by measuring the avidity of serum HA-specific Abs in these mice (Fig. 3B). Aim2+/+ mice required high concentrations of the chaotropic agent NaSCN to disrupt Ag/Ab complexes, whereas much lower concentrations of NaSCN were required for disassociation in Aim22/2 mice. To confirm the requirement for inflammasome signaling in DNA vaccine immunogenicity, we also quantified the adaptive response in Asc2/2 mice. Asc deletion similarly inhibited the generation of optimal HA-specific immune responses (Supplemental Fig. 2A–C).
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FIGURE 1. Plasmid DNA vaccination induces the inflammasome. Inflammasome activation at the site of immunization was quantified by Nanostring nCounter analysis 12 h post–DNA immunization. The site of injection was harvested and mRNA was isolated and expression levels were quantified for Aim2 (A), caspase-1 (B), IL-1a (C), and IL-1b (D). Data are the averages 6 SEM of five mice per group. *p , 0.05, **p , 0.01 versus control group.
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THE EFFECTS OF Aim2 ON DNA VACCINE IMMUNOGENICITY
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FIGURE 3. Optimal DNA vaccine immunogenicity requires Aim2. Wild-type Aim2+/+ and Aim22/2 mice were immunized i.m. with a pH1HA encoding DNA vaccine at weeks 2 and 4. (A) HA-specific IgG titers were analyzed 14 d post–second immunization. Anti-HA binding avidity was quantified via ELISA and reported as molar concentration of sodium thiocyanate required to displace anti-HA serum Abs to 23 prebleed levels (B). For ELISPOT, spleens were harvested at termination 7 d following a third boosting immunization. HA-specific Ab-secreting B cells (C) or IFN-g–secreting T cells (D) in mice immunized with either pH1HA or empty vector. Data are the averages 6 SEM of five mice per group. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 versus control group.
Aim22/2 mice fail to cleave caspase-1 into its active form Because DNA vaccination resulted in high levels of caspase-1 activation in Aim2+/+ mice (Supplemental Fig. 1), we analyzed Aim22/2 mice for their ability to generate catalytically active caspase-1 using the FAM/FLICA assay (Fig. 4). Aim22/2 mice demonstrated a clear reduction in caspase-1 activation at the 12-h peak time point when compared with Aim2+/+ controls. Effects of IL-1 and IL-18 deletion on vaccine-induced HAspecific immune responses As inflammasome signaling ultimately results in the downstream cleavage of pro–IL-1b and pro–IL-18 into their respective active forms, the role of IL-1b and IL-18 signaling in DNA vaccine was next investigated (Supplemental Fig. 3). Surprisingly, both of these cytokines were dispensable for the DNA vaccine response as mice lacking the IL-1R or the IL-18R mounted normal vaccine responses. Total serum HA-specific IgG titers were similar to wild-type C57BL/6 mice in both Il-1r2/2 and Il-18r2/2 mice. Likewise, no significant difference was seen in total HA-specific B or CD8+ T cell numbers as measured by ELISPOT. These data are in line with previously published reports demonstrating little impact on DNA vaccination following MyD88 deletion (19). The immune response is lineage dependent Previously published data have demonstrated the requirement for both hematopoietic and non-hematopoietic cell lineages in DNA vaccination (19). To further elucidate the role of Aim2, bone marrow chimeric mice were generated by transferring bone marrow from Aim2+/+ mice into Aim22/2 mice, or vice versa (Fig. 5). Aim2+/+ and Aim22/2 mice reconstituted with Aim22/2 bone marrow exhibited strong defects in both the T cell response and HA-specific IgG production. Interestingly, transfer of Aim2+/+ bone marrow into Aim22/2 rescued the T cell response against
FIGURE 4. Aim22/2 mice exhibit diminished caspase-1 activation at the site of immunization. (A) PBS-injected controls were used for comparison. The site of injection was harvested and cryopreserved for tissue sectioning. Wild-type Aim2+/+ (B) and Aim22/2 mice (C) were immunized i.m. with pH1HA vaccine, and caspase-1 activation was quantified by FAM/FLICA staining 12 h postimmunization. FAM/FLICA staining was visualized by confocal microscopy and is representative of three mice per group. Sections of 10 mm were stained with green fluorescent FLICA caspase-1 inhibitor. Nuclei were counterstained with DAPI. Low-power resolution presented (original magnification 316).
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Discussion
FIGURE 5. Contribution of hematopoietic and nonhematopoietic cells to DNA vaccine–induced immunogenicity. Bone marrow chimeric mice were immunized with a pH1HA vaccine as described in Fig. 2. Fourteen days post–second immunization, sera from chimeric mice were analyzed for HAspecific IgG titers (A). Spleens were harvested at termination 7 d following the third immunization. Frequency of HA-specific B cells (B) or IFN-g+ T cells (C) were reported as spots per million splenocytes in mice immunized with pH1HA vaccine. Data are the averages 6 SEM of five mice per group. *p , 0.05, **p , 0.01, ***p , 0.001 versus control group.
The innate immune pathways governing DNA vaccination remain to be fully characterized. Recent reports have established the STING/TBK1/IFN-ab axis as required for DNA vaccine immunogenicity (19, 20); however, the PRR(s) required for IFN-ab production remain to be identified in this context. Likewise, the involvement of other innate immune signaling pathways is unclear. In particular, the requirement for the inflammasome signaling machinery in DNA vaccine–elicited Ag-specific immune responses has not been examined. In this study, we identified the Aim2 inflammasome as a DNA vaccine sensor with the ability to regulate the Ag-specific adaptive immune response. Whereas previous reports have focused on downstream signaling mole-
a CD8+ T cell epitope, but only partially rescued the humoral response. Although HA-specific IgG levels were impaired compared with Aim2+/+ mice reconstituted with Aim2+/+ bone marrow, they were significantly higher than Aim22/2 bone marrow– reconstituted mice. This would support that Aim2 is required in both the hematopoietic and nonhematopoietic lineages for optimal humoral responses, but deficiency in nonhematopoietic lineages does not affect CD8+ T cell responses. Aim22/2 mice lack IFN-ab signaling The failure of Aim22/2 mice to generate optimal adaptive immune responses implies a defect in immune priming at the site of injection. Although the major function of the Aim2 inflammasome is to regulate caspase-1 activation, resulting in IL-1b, IL-18, and cell death pathways, our data indicate that IL-1b and IL-18 are not responsible for the Aim2-dependent effects we observed. We therefore endeavored to quantify IFN-a/b, as it has been reported to play a key role in the immune response to B-DNA (24, 34–36). In addition, it has been established that IFN-a/b signaling is required for DNA vaccination (19, 20). Aim2 does not control DNA induced IFN-a/b production directly. Rather, the STING pathway mediates these effects. Because the IFN-a/b response is so critical for DNA vaccination, we performed a detailed kinetic analysis
FIGURE 6. Aim2 deficiency limits IFN-ab production at the site of injection. Aim2+/+ and Aim22/2 mice were immunized i.m. with pH1HA vaccine, and the site of injection was harvested 12 h later. Total RNA was isolated from tissue biopsies and subjected to RT-PCR for IFN-a (A), IFN-b (B), IP-10 (C), and TNF (D). Reported expression levels are relative to naive Aim2 +/+ expression. Data are the averages 6 SEM of five mice per group. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 versus control group.
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measuring IFN-a/b expression in Aim2+/+ and Aim22/2 mice. To ensure we only detect DNA vaccine-induced IFN-a/b, we limited our measurements to the site of immunization. Aim2+/+ and Aim22/2 mice were immunized with the pH1HA DNA vaccine, and punch biopsies were collected from the site of injection. Quantitative RT-PCR analysis of mRNA clearly shows that Aim22/2 mice have reduced IFN-a and IFN-b expression compared with Aim2+/+ controls, with expression peaking at 12 h postimmunization in wild-type mice (Fig. 6). Intriguingly, IFN-ab expression in Aim22/2 mice peaked at 6 h postimmunization and remained static throughout the time course. Consistent with the decrease in IFN-a/b, there was a corresponding decrease in the IFN-stimulated gene IP-10. We also noticed a significant decrease in TNF. Asc2/2 mice had similar levels of IFN-a/b, further confirming the requirement for inflammasome signaling (Supplemental Fig. 2D). These data suggest a previously unreported role for Aim2 in regulating local IFN-a/b levels following DNA vaccination. As Aim2 controls cell death at the site of infection, it is likely that Aim2dependent cell death liberates endogenous DAMP danger signals, which might in turn elicit IFN-a/b via the Aim2-independent STING/ TBK1 pathways. This broad defect in IFN-a/b signaling likely explains the defects we observed in Aim22/2 mice treated with DNA vaccines.
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Disclosures The authors have no financial conflicts of interest.
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Fitzgerald. 2014. Dual
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cules, this is the first report, to our knowledge, to identify a DNA sensor that is required for DNA vaccine immunogenicity. The failure of Aim22/2 mice to generate optimal adaptive immune responses implies a defect in immune priming at the site of injection. Although the function of Aim2 has been well characterized, how and where Aim2 interacts with the vaccine plasmid remains unclear. Interestingly, immunization of bone marrow chimeras revealed varying degrees of necessity for Aim2 signaling in both the hematopoietic and nonhematopoietic lineages. Aim2 is required in both cell lineages for optimal humoral responses, as chimeric mice lacked high levels of anti-HA Abs. This may be attributed to impaired IL-6 production in Aim22/2 mice possibly limiting B cell survival and CD4+ T cell expansion as has been reported in several studies (Supplemental Fig. 4) (37). In addition, recent reports have described the effect of DNA priming on T follicular helper cell generation (12). Even more surprising, Aim2 deletion in nonhematopoietic cells did not impact CD8+ T cell responses, as mice reconstituted with Aim2+/+ bone marrow presented similar levels of IFN-g to Aim2+/+. This provides further evidence for a defect in immune cell priming. Of note, deletion of IL-1b and IL-18 signaling in DNA vaccination did not impact DNA vaccine immunogenicity, confirming previous reports describing minimal effect of MyD88 deletion on DNA vaccine immunogenicity (38). This stands in stark contrast to the importance of IL-1b and IL-18 inflammatory signaling in the early stages of pathogenic infection. Why IL-1b and IL-18 signaling are dispensable remains unclear, but one possible reason is the highly immunostimulatory nature of the DNA vaccine plasmid itself, as DNA is a potent inducer of both IFN-a/b and several NF-kB–regulated immune genes. In addition, the level of several chemokines, such as MIP-1a/b and MCP-1, remained unchanged (data not shown), allowing for recruitment of monocyte and lymphocyte populations. Most intriguingly, the reduction of IFN-a/b levels at the site of immunization in Aim22/2 suggests a previously unknown relationship between Aim2 and local IFN-a/b production. Aim2 is not known to mediate IFN-a/b production directly, hinting at an indirect link between these two divergent pathways. We propose that decreased pyroptotic cell death in Aim22/2 mice results in diminished DAMP release, limiting cellular signaling and bystander cell activation. The release of cellular DNA by pyroptotic cells may augment IFN-a/b production by surrounding cells, possibly through the STING/TBK1 signaling axis, further propagating the immune response. In conclusion, our results indicate that Aim2 plays a significant role in DNA vaccination. Aim22/2 mice failed to generate optimal immune responses upon DNA vaccination, exhibiting decreased serum IgG levels and T cell cytokine production, demonstrating the necessity for Aim2 signaling in DNA vaccine immunogenicity. In addition, we reported a previously unknown function for Aim2 in augmenting IFN-a/b production at the site of immunization. Our results provide a deeper understanding of the cellular mechanisms through which DNA vaccines stimulate both the innate and adaptive immune pathways to enhance the immune responses targeting the encoded Ag.
THE EFFECTS OF Aim2 ON DNA VACCINE IMMUNOGENICITY
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engagement of the NLRP3 and AIM2 inflammasomes by plasmodium-derived hemozoin and DNA during malaria. Cell Reports 6: 196–210. Hanamsagar, R., A. Aldrich, and T. Kielian. 2014. Critical role for the AIM2 inflammasome during acute CNS bacterial infection. J. Neurochem. 129: 704–711. Wang, S., J. Taaffe, C. Parker, A. Solo´rzano, H. Cao, A. Garcı´a-Sastre, and S. Lu. 2006. Hemagglutinin (HA) proteins from H1 and H3 serotypes of influenza A viruses require different antigen designs for the induction of optimal protective antibody responses as studied by codon-optimized HA DNA vaccines. J. Virol. 80: 11628–11637. Wang, S., C. Parker, J. Taaffe, A. Solo´rzano, A. Garcı´a-Sastre, and S. Lu. 2008. Heterologous HA DNA vaccine prime—inactivated influenza vaccine boost is more effective than using DNA or inactivated vaccine alone in eliciting antibody responses against H1 or H3 serotype influenza viruses. Vaccine 26: 3626–3633. Suguitan, A. L., Jr., X. Cheng, W. Wang, S. Wang, H. Jin, and S. Lu. 2011. Influenza H5 hemagglutinin DNA primes the antibody response elicited by the live attenuated influenza A/Vietnam/1203/2004 vaccine in ferrets. PLoS ONE 6: e21942. Zhang, L., N. Jia, J. Li, Y. Han, W. Cao, S. Wang, Z. Huang, and S. Lu. 2014. Optimal designs of an HA-based DNA vaccine against H7 subtype influenza viruses. Hum. Vaccin. Immunother. 10: 10.
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This information is current as of December 13, 2014.
John J. Suschak, Shixia Wang, Katherine A. Fitzgerald and Shan Lu
Published December 8, 2014, doi:10.4049/jimmunol.1402530 The Journal of Immunology
Identification of Aim2 as a Sensor for DNA Vaccines John J. Suschak,* Shixia Wang,* Katherine A. Fitzgerald,† and Shan Lu*
T
he discovery of DNA vaccine technology in the early 1990s was a major event in the history of vaccinology due to the many unique features of DNA immunization, including its ability to elicit balanced Ab and T cell immunity (1–6). However, in early clinical studies, the immunogenicity of DNA vaccines in humans was low when such vaccines were used alone. More recent human trials have demonstrated that DNA vaccines are actually extremely powerful in priming the host’s immune system to develop high-level protective Ab responses against HIV-1 and pandemic influenza viruses (3, 7–10). Animal studies further demonstrated that DNA immunization is effective in eliciting higher levels of Ag-specific B cell responses (11). One mechanism to achieve such an outcome is that DNA vaccination is effective in eliciting higher germinal center B cell development via enhanced follicular Th cells for the production of high-quality Ab responses (12). DNA vaccines produce immunogens in vivo, which are then presented to the immune system via the endogenous Ag-processing pathway. At the same time, the DNA plasmid itself confers an intrinsic adjuvant effect that enhances the immune response generated toward the vaccine-encoded immunogens (12, 13), but the intracellular processes involved remain to be fully elucidated. Several pattern recognition receptors (PRRs) have been identified that respond to DNA molecules (14, 15). One of the best-characterized DNA-sensing PRRs is the endosomal TLR9, which is essential for *Laboratory of Nucleic Acid Vaccines, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655; and †Program in Innate Immunity, Division of Infectious Diseases, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655 Received for publication October 2, 2014. Accepted for publication November 13, 2014. This study was supported in part by National Institutes of Health Grants 5 U19 AI082676, 5 P01 AI082274, and 5 R33 AI087191 and Bill and Melinda Gates Foundation Grant OPP1033112. Address correspondence and reprint requests to Prof. Shan Lu, University of Massachusetts Medical School, Room LRB-304, 364 Plantation Street, Worcester, MA 01605. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: Aim2, absent in melanoma 2; Aim22/2, Aim2deficient; BMDM, bone marrow–derived macrophage; DAMP, damage-associated molecular pattern; HA, hemagglutinin; LDH, lactate dehydrogenase; poly(dA-dT), poly(deoxyadenylic-deoxythymidylic) acid; PRR, pattern recognition receptor; TBK1, TANK-binding kinase 1; UMMS, University of Massachusetts Medical School. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402530
the recognition of unmethylated CpG containing oligodeoxynucleotide motifs commonly found in bacterial plasmids (16). As DNA vaccine plasmid backbones contain certain CpG/oligodeoxynucleotide motifs, it was initially thought that TLR9 would be critical for DNA vaccine immunogenicity. However, whereas DNA plasmid activates dendritic cells via TLR9, TLR9-deficient mice were able to mount immune responses comparable to wild-type mice (17, 18). Likewise, DNA immunization of MyD88 and TRIF-deficient mice yields robust immune responses, further suggesting that TLR signaling may be dispensable for DNA vaccine-induced immunogenicity (19). In recent years, it has become increasingly evident that the double-stranded nature of DNA itself functions as a potent activator of innate immune signals (20–24). Cytosolic DNA is a powerful initiator of type I IFN (IFN-ab) in both immune and nonimmune cells, functioning through a STING/TANK-binding kinase 1 (TBK1)/IFN regulatory factor 3–dependent pathway that is independent of CpG motifs and TLRs. At the same time, it is not clear whether other components of the innate immune system beyond the STING/TBK1/IFN-ab pathway are involved in the immunogenicity of DNA vaccines (19, 20). This is especially true in the case of inflammasome pathways. Inflammasomes regulate caspase-1 activity, ultimately resulting in cleavage of the proinflammatory cytokines pro–IL-1b and pro–IL-18 into their active forms. Inflammasome activation also results in pyroptotic cell death; a suicidal form of cell death characterized by the release of damage-associated molecular patterns (DAMPs) that further propagates innate immune signaling to surrounding bystander cells (25). One flavor of inflammasome contains absent in melanoma 2 (Aim2), which is a direct sensor of cytosolic DNA and a member of the PYHIN family (22). Aim2 contains a DNA binding HIN200 domain, as well as a pyrin domain. Although Aim2’s role in orchestrating immune responses to both viral and bacterial pathogens is well characterized (26–28), the role of Aim2 in DNA vaccination is unknown. In this study, we found that Aim2 and the adapter molecule Asc were required for the generation of optimal immunogen-specific Ab responses to a DNA vaccine expressing influenza hemagglutinin (HA) immunogen in a mouse model. DNA vaccination leads to transcription of key components of the inflammasome. Importantly, the efficacy of DNA vaccination was independent of IL-1b and IL-18. Surprisingly, Aim2-deficient (Aim22/2) mice were unable to elicit a type I IFN response at the site of injection. Our
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Recent human study data have re-established the value of DNA vaccines, especially in priming high-level Ag-specific Ab responses, but also raised questions about the mechanisms responsible for such effects. Whereas previous reports have shown involvement of downstream signaling molecules in the innate immune system, the current study investigated the role of absent in melanoma 2 (Aim2) as a sensor for DNA vaccines. The Aim2 inflammasome directs maturation of the proinflammatory cytokines IL-1b and IL-18 and an inflammatory form of cell death called pyroptosis. Both the humoral and cellular Ag-specific adaptive responses were significantly reduced in Aim2-deficient mice in an IL-1b/IL-18–independent manner after DNA vaccination. Surprisingly, Aim2-deficient mice also exhibited significantly lower levels of IFN-a/b at the site of injection. These results indicate a previously unreported link between DNA vaccine–induced pyroptotic cell death and vaccine immunogenicity that is instrumental in shaping the Ag-specific immune response to DNA vaccines. The Journal of Immunology, 2015, 194: 000–000.
2 data therefore establish a novel role for Aim2 as a key player in the regulation of DNA vaccination.
Materials and Methods Animals
Gene induction analysis C57BL/6 mice were shaved and immunized with 100 mg H1-TX04-09.tPA DNA vaccine plasmid i.m. injected into the hind quad muscle. Punch biopsies were harvested from the site of immunization at 6, 12, and 24 h postimmunization and snap frozen. RNA was isolated from tissue biopsies using TRIzol Reagent (Life Technologies). We analyzed gene expression using the Nanostring nCounter Analysis system (Nanostring Technologies). Each reaction contained 100 ng RNA in a 5-ml aliquot, plus reporter and capture probes. We also included six pairs of positive control and eight pairs of negative control probes. Gene induction analysis and normalization was conducted using nSolver Analysis Software v1.1. Raw counts were normalized to naive mice using three reference genes: Gapdh, Gusb, and Hprt1.
Cell culture and cytokine ELISA Mouse bone marrow–derived dendritic cells were generated from Aim2+/+ or Aim22/2 mice by culturing fresh bone marrow in R10 medium containing GM-CSF for 8 d at 37˚C. Aim2+/+ and Aim22/2 immortalized bone marrow–derived macrophages (BMDM) were produced in-house. Cells were first primed with 200 ng/ml LPS (Sigma-Aldrich, St. Louis, MO) for 4 to 5 h. prior to treatment with appropriate stimuli. All media was removed from the cells, and the appropriate stimulus was added. Poly (deoxyadenylic-deoxythymidylic) acid [poly (dA-dT)] (Sigma-Aldrich) DNA and H1-TX04-09.tPA DNA vaccine plasmid were transfected using Lipofectamine 2000 at a concentration of 1.5 mg/ml. ATP was added at a concentration of 1.25 mg/ml. Cultures were incubated 16–18 h at 37˚C, and supernatants were harvested. Cell-culture supernatants were assayed for IL-1b (BD Biosciences, Franklin Lakes, NJ) by ELISA. Lactate dehydrogenase (LDH) release was used to measure pyroptotic cell death. LDH assays were performed using the Promega CytoTox96 Nonradioactive Cytotoxicity Kit according to the manufacturer’s directions (Promega, Madison, WI).
ELISA Microtiter plates were coated with transiently expressed H1HA Ag at ∼1 mg/ml in PBS for 1 h at room temperature and assayed as previously described (29). NaSCN displacement was performed at a serum dilution of 1:100. After washing of serum samples, NaSCN was added at various (0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 M) concentrations in PBS for 15 min followed by five washes in ELISA wash buffer. The assay was then completed as above.
ELISPOT assay Splenocyte T and B cell ELISPOT reagents were obtained from Mabtech (Mariemont, OH). H1HA-specific T cells were quantified per the manufacturer’s instructions. Positive controls were stimulated with 20 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (SigmaAldrich). The H1HA relevant peptide used was a CD8+ cell-restricted HA peptide (IYSTVASSL). H1HA-specific Ab-secreting cells were detected by coating of MAIPSWU (Millipore, Billerica, MA) plates with the transiently expressed H1HA Ag used for ELISA (∼1.0 mg/well). Positive spots were visualized on a CTL Imager, and counting was performed with Immunospot software (Cellular Technology, Shaker Heights, OH).
In vivo caspase-1 activation Aim2+/+ and Aim22/2 mice were shaved and immunized with 100 mg H1-TX04-09.tPA DNA vaccine plasmid i.m. injected into the hind quad muscle. Punch biopsies were harvested from the site of immunization at 6, 12, and 24 h postimmunization and snap frozen. Cryopreserved tissue sections were generated and adhered to glass slides. Samples were then stained with a caspase-1 FAM/FLICA kit according to the manufacturer’s instructions (ImmunoChemistry Technologies, Bloomington, MN). Stained slides were visualized on a confocal microscope. Sixteen independent fields were analyzed for fluorescence.
Quantitative real-time PCR Aim2+/+ and Aim22/2 mice were shaved and immunized with 100 mg H1-TX04-09.tPA DNA vaccine plasmid i.m. injected into the hind quad muscle. Punch biopsies were harvested from the site of immunization at 6, 12, and 24 h postimmunization and snap frozen. RNA was isolated from tissues biopsies using TRIzol Reagent (#15596-026; Life Technologies), and cDNA was generated using the Bio-Rad iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). cDNA was then used for RT-PCR reactions on a Bio-Rad CFX-96 cycler (Bio-Rad). Primers sequences are available upon request.
Statistical analysis All data are presented as the mean of individual mice 6 SEM. Statistical analysis was performed using a Student t test, a one-way ANOVA followed by a Tukey posttest, or a two-way ANOVA followed by a Bonferroni posttest.
Results DNA vaccine plasmid induces expression of Aim2, caspase-1, and the inflammasome Although previous studies have mainly used noncoding DNA plasmid or DNA vaccines coding for marker proteins to study DNA-elicited innate immune responses, the current study tested a DNA vaccine (pH1HA) expressing the HA Ag of the type A influenza virus subtype H1N1 virus, which was responsible for a pandemic influenza in 2009. HA is the major protective Ag in clinically licensed inactivated and live-attenuated influenza vaccines. DNA vaccines expressing HA have been shown to be immunogenic in eliciting HA-specific Abs in both animal and human studies (2, 29–33). The expression of HA Ag by pH1HA used in the current study was confirmed by Western blot, and its immunogenicity to elicit HA-specific Ab response was verified in a pilot mouse study (data not shown). We first wanted to profile key immune response genes following DNA vaccine pH1HA using the Nanostring nCounter gene expression system (Nanostring Technologies), which includes a custom array encoding 50 innate immunity targets. Gene induction was quantified from wild-type C57BL/6 mice immunized with the pH1HA DNA vaccine. mRNA was isolated, the expression of innate immune genes profiled using the Nanostring nCounter, and changes in gene induction quantified. Notably, Aim2 was induced ∼6-fold within 12 h of immunization when compared with naive samples. Aim2 is a type I IFN–inducible gene, suggesting a potent ability of cells at the site of vaccination to recognize cytosolic plasmid vaccines (Fig. 1A). In accordance with the induction of Aim2, caspase-1 was also highly upregulated. (Fig. 1B). Most striking were the high levels of the inflammatory cytokines IL-1a and IL-1b (Fig. 1C, 1D). These observations indicate that inflammasome components were present at the site of vaccination. To test if the inflammasome pathway was active at the site of vaccination, we used a caspase1–specific FAM/FLICA fluorescent stain to covalently label catalytically active caspase-1. Mature caspase-1 became apparent within 6 h of immunization and reached a peak at 12 h (Supplemental Fig. 1). Collectively, these results led us to examine the role of the Aim2 inflammasome pathway in Ag-specific immune responses elicited by pH1HA DNA vaccination.
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C57BL/6 mice were obtained from Taconic Laboratories. Aim22/2 mice were generated in house by K. Fitzgerald’s group at the University of Massachusetts Medical School (UMMS) as previously described (26). Aim22/2 mice were on a mixed B6/129 background, and therefore B6 3 129 mice were used as controls. B6.129 (hereafter referred to as Aim2+/+) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred at UMMS. IL-1R, IL-18R, and Asc-deficient mice were produced in house. All mice were maintained in the Department of Animal Medicine at UMMS according to Institutional Animal Care and Use Committee–approved protocols. Mice received 100 mg codon-optimized H1HA DNA vaccine expressing the full-length wild-type HA protein from A/Texas/04/ 09 (pH1HA), divided between quadriceps, at 2 and 4 wk. A third boosting immunization was delivered 1 wk prior to sacrifice.
THE EFFECTS OF Aim2 ON DNA VACCINE IMMUNOGENICITY
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Involvement of Aim2 in cellular responses to DNA vaccine plasmid We first evaluated the ability of macrophages and dendritic cells to recognize pH1HA DNA vaccine by performing in vitro experiments. IL-1b production in response to pH1HA DNA vaccine was evaluated in BMDM collected from either Aim2+/+ or Aim22/2 mice (Fig. 2A). As expected, both the synthetic B-form dsDNA poly(dA-dT) and pH1HA DNA vaccine induced a robust IL-1b response in Aim2+/+ BMDM as measured by ELISA. However, IL-1b production was abolished in Aim22/2 BMDM. Similar results were seen in bone marrow–derived dendritic cells (data not shown). Next, the pan-caspase inhibitor Z-VAD-FMK was included in BMDM cultures prior to adding pH1HA DNA vaccine (Fig. 2B). This resulted in inhibited IL-1b production in Aim2+/+ macrophages, yielding IL-1b levels comparable to Aim22/2 wells, supporting the role of Aim2 in DNA vaccine–mediated IL-1b maturation. Finally, pH1HA DNA vaccine induced an inflammatory form of cell death (pyroptosis) in Aim2+/+ macrophages, as measured by LDH release (Fig. 2C). This response was attenuated in Aim22/2 cells. Collectively, these data indicate that Aim2 acts as a sensor of DNA vaccine plasmid and regulates caspase-1–dependent IL-1b production and pyroptotic cell death in response to pH1HA DNA vaccines in vitro. Effects of Aim2 deletion on pH1HA induced HA-specific immune responses As it is evident that the Aim2 inflammasome recognizes and responds to pH1HA DNA vaccine in cultured cells, the role of Aim2 in pH1HA DNA vaccination was next examined in Aim22/2 and wild-type Aim2+/+ mice. The pH1HA DNA vaccine induced high-level HA-specific Ab responses in Aim2+/+ mice, but significantly lower Ab titers in Aim22/2 mice (Fig. 3A). This reduction is isotype-independent as Aim22/2 mice exhibited significantly lower levels of HA-specific IgG1, IgG2b, and IgG2c responses (data not shown). Likewise, Aim22/2 mice exhibited significantly reduced HA-specific circulating B cells as well as IFN-g–secreting CD8+ T cells in the spleen (Fig. 3C,
FIGURE 2. Aim2 is required for IL-1b production in response to DNA vaccines. LPS-primed (200 ng/ml) Aim2+/+ and Aim22/2 BMDM were transfected with poly(dA-dT) or DNA vaccine plasmid for 18 h. (A) Secreted IL-1b in the culture supernatants was analyzed by ELISA. (B) Aim2+/+ and Aim22/2 BMDM were treated as above with the addition of the pancaspase inhibitor Z-VAD-FMK, and secreted IL-1b was quantified by ELISA. (C) Aim2+/+ and Aim22/2 BMDM were treated as above, and culture LDH amounts were reported as a percentage of lysed cellular controls. Data are presented as mean 6 SEM from three independent experiments. **p , 0.01, ***p , 0.001, ****p , 0.0001 versus control group.
3D). The role of Aim2 in regulating the maturation process of pH1HA-induced Ab responses was further confirmed by measuring the avidity of serum HA-specific Abs in these mice (Fig. 3B). Aim2+/+ mice required high concentrations of the chaotropic agent NaSCN to disrupt Ag/Ab complexes, whereas much lower concentrations of NaSCN were required for disassociation in Aim22/2 mice. To confirm the requirement for inflammasome signaling in DNA vaccine immunogenicity, we also quantified the adaptive response in Asc2/2 mice. Asc deletion similarly inhibited the generation of optimal HA-specific immune responses (Supplemental Fig. 2A–C).
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FIGURE 1. Plasmid DNA vaccination induces the inflammasome. Inflammasome activation at the site of immunization was quantified by Nanostring nCounter analysis 12 h post–DNA immunization. The site of injection was harvested and mRNA was isolated and expression levels were quantified for Aim2 (A), caspase-1 (B), IL-1a (C), and IL-1b (D). Data are the averages 6 SEM of five mice per group. *p , 0.05, **p , 0.01 versus control group.
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FIGURE 3. Optimal DNA vaccine immunogenicity requires Aim2. Wild-type Aim2+/+ and Aim22/2 mice were immunized i.m. with a pH1HA encoding DNA vaccine at weeks 2 and 4. (A) HA-specific IgG titers were analyzed 14 d post–second immunization. Anti-HA binding avidity was quantified via ELISA and reported as molar concentration of sodium thiocyanate required to displace anti-HA serum Abs to 23 prebleed levels (B). For ELISPOT, spleens were harvested at termination 7 d following a third boosting immunization. HA-specific Ab-secreting B cells (C) or IFN-g–secreting T cells (D) in mice immunized with either pH1HA or empty vector. Data are the averages 6 SEM of five mice per group. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 versus control group.
Aim22/2 mice fail to cleave caspase-1 into its active form Because DNA vaccination resulted in high levels of caspase-1 activation in Aim2+/+ mice (Supplemental Fig. 1), we analyzed Aim22/2 mice for their ability to generate catalytically active caspase-1 using the FAM/FLICA assay (Fig. 4). Aim22/2 mice demonstrated a clear reduction in caspase-1 activation at the 12-h peak time point when compared with Aim2+/+ controls. Effects of IL-1 and IL-18 deletion on vaccine-induced HAspecific immune responses As inflammasome signaling ultimately results in the downstream cleavage of pro–IL-1b and pro–IL-18 into their respective active forms, the role of IL-1b and IL-18 signaling in DNA vaccine was next investigated (Supplemental Fig. 3). Surprisingly, both of these cytokines were dispensable for the DNA vaccine response as mice lacking the IL-1R or the IL-18R mounted normal vaccine responses. Total serum HA-specific IgG titers were similar to wild-type C57BL/6 mice in both Il-1r2/2 and Il-18r2/2 mice. Likewise, no significant difference was seen in total HA-specific B or CD8+ T cell numbers as measured by ELISPOT. These data are in line with previously published reports demonstrating little impact on DNA vaccination following MyD88 deletion (19). The immune response is lineage dependent Previously published data have demonstrated the requirement for both hematopoietic and non-hematopoietic cell lineages in DNA vaccination (19). To further elucidate the role of Aim2, bone marrow chimeric mice were generated by transferring bone marrow from Aim2+/+ mice into Aim22/2 mice, or vice versa (Fig. 5). Aim2+/+ and Aim22/2 mice reconstituted with Aim22/2 bone marrow exhibited strong defects in both the T cell response and HA-specific IgG production. Interestingly, transfer of Aim2+/+ bone marrow into Aim22/2 rescued the T cell response against
FIGURE 4. Aim22/2 mice exhibit diminished caspase-1 activation at the site of immunization. (A) PBS-injected controls were used for comparison. The site of injection was harvested and cryopreserved for tissue sectioning. Wild-type Aim2+/+ (B) and Aim22/2 mice (C) were immunized i.m. with pH1HA vaccine, and caspase-1 activation was quantified by FAM/FLICA staining 12 h postimmunization. FAM/FLICA staining was visualized by confocal microscopy and is representative of three mice per group. Sections of 10 mm were stained with green fluorescent FLICA caspase-1 inhibitor. Nuclei were counterstained with DAPI. Low-power resolution presented (original magnification 316).
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Discussion
FIGURE 5. Contribution of hematopoietic and nonhematopoietic cells to DNA vaccine–induced immunogenicity. Bone marrow chimeric mice were immunized with a pH1HA vaccine as described in Fig. 2. Fourteen days post–second immunization, sera from chimeric mice were analyzed for HAspecific IgG titers (A). Spleens were harvested at termination 7 d following the third immunization. Frequency of HA-specific B cells (B) or IFN-g+ T cells (C) were reported as spots per million splenocytes in mice immunized with pH1HA vaccine. Data are the averages 6 SEM of five mice per group. *p , 0.05, **p , 0.01, ***p , 0.001 versus control group.
The innate immune pathways governing DNA vaccination remain to be fully characterized. Recent reports have established the STING/TBK1/IFN-ab axis as required for DNA vaccine immunogenicity (19, 20); however, the PRR(s) required for IFN-ab production remain to be identified in this context. Likewise, the involvement of other innate immune signaling pathways is unclear. In particular, the requirement for the inflammasome signaling machinery in DNA vaccine–elicited Ag-specific immune responses has not been examined. In this study, we identified the Aim2 inflammasome as a DNA vaccine sensor with the ability to regulate the Ag-specific adaptive immune response. Whereas previous reports have focused on downstream signaling mole-
a CD8+ T cell epitope, but only partially rescued the humoral response. Although HA-specific IgG levels were impaired compared with Aim2+/+ mice reconstituted with Aim2+/+ bone marrow, they were significantly higher than Aim22/2 bone marrow– reconstituted mice. This would support that Aim2 is required in both the hematopoietic and nonhematopoietic lineages for optimal humoral responses, but deficiency in nonhematopoietic lineages does not affect CD8+ T cell responses. Aim22/2 mice lack IFN-ab signaling The failure of Aim22/2 mice to generate optimal adaptive immune responses implies a defect in immune priming at the site of injection. Although the major function of the Aim2 inflammasome is to regulate caspase-1 activation, resulting in IL-1b, IL-18, and cell death pathways, our data indicate that IL-1b and IL-18 are not responsible for the Aim2-dependent effects we observed. We therefore endeavored to quantify IFN-a/b, as it has been reported to play a key role in the immune response to B-DNA (24, 34–36). In addition, it has been established that IFN-a/b signaling is required for DNA vaccination (19, 20). Aim2 does not control DNA induced IFN-a/b production directly. Rather, the STING pathway mediates these effects. Because the IFN-a/b response is so critical for DNA vaccination, we performed a detailed kinetic analysis
FIGURE 6. Aim2 deficiency limits IFN-ab production at the site of injection. Aim2+/+ and Aim22/2 mice were immunized i.m. with pH1HA vaccine, and the site of injection was harvested 12 h later. Total RNA was isolated from tissue biopsies and subjected to RT-PCR for IFN-a (A), IFN-b (B), IP-10 (C), and TNF (D). Reported expression levels are relative to naive Aim2 +/+ expression. Data are the averages 6 SEM of five mice per group. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 versus control group.
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measuring IFN-a/b expression in Aim2+/+ and Aim22/2 mice. To ensure we only detect DNA vaccine-induced IFN-a/b, we limited our measurements to the site of immunization. Aim2+/+ and Aim22/2 mice were immunized with the pH1HA DNA vaccine, and punch biopsies were collected from the site of injection. Quantitative RT-PCR analysis of mRNA clearly shows that Aim22/2 mice have reduced IFN-a and IFN-b expression compared with Aim2+/+ controls, with expression peaking at 12 h postimmunization in wild-type mice (Fig. 6). Intriguingly, IFN-ab expression in Aim22/2 mice peaked at 6 h postimmunization and remained static throughout the time course. Consistent with the decrease in IFN-a/b, there was a corresponding decrease in the IFN-stimulated gene IP-10. We also noticed a significant decrease in TNF. Asc2/2 mice had similar levels of IFN-a/b, further confirming the requirement for inflammasome signaling (Supplemental Fig. 2D). These data suggest a previously unreported role for Aim2 in regulating local IFN-a/b levels following DNA vaccination. As Aim2 controls cell death at the site of infection, it is likely that Aim2dependent cell death liberates endogenous DAMP danger signals, which might in turn elicit IFN-a/b via the Aim2-independent STING/ TBK1 pathways. This broad defect in IFN-a/b signaling likely explains the defects we observed in Aim22/2 mice treated with DNA vaccines.
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Disclosures The authors have no financial conflicts of interest.
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cules, this is the first report, to our knowledge, to identify a DNA sensor that is required for DNA vaccine immunogenicity. The failure of Aim22/2 mice to generate optimal adaptive immune responses implies a defect in immune priming at the site of injection. Although the function of Aim2 has been well characterized, how and where Aim2 interacts with the vaccine plasmid remains unclear. Interestingly, immunization of bone marrow chimeras revealed varying degrees of necessity for Aim2 signaling in both the hematopoietic and nonhematopoietic lineages. Aim2 is required in both cell lineages for optimal humoral responses, as chimeric mice lacked high levels of anti-HA Abs. This may be attributed to impaired IL-6 production in Aim22/2 mice possibly limiting B cell survival and CD4+ T cell expansion as has been reported in several studies (Supplemental Fig. 4) (37). In addition, recent reports have described the effect of DNA priming on T follicular helper cell generation (12). Even more surprising, Aim2 deletion in nonhematopoietic cells did not impact CD8+ T cell responses, as mice reconstituted with Aim2+/+ bone marrow presented similar levels of IFN-g to Aim2+/+. This provides further evidence for a defect in immune cell priming. Of note, deletion of IL-1b and IL-18 signaling in DNA vaccination did not impact DNA vaccine immunogenicity, confirming previous reports describing minimal effect of MyD88 deletion on DNA vaccine immunogenicity (38). This stands in stark contrast to the importance of IL-1b and IL-18 inflammatory signaling in the early stages of pathogenic infection. Why IL-1b and IL-18 signaling are dispensable remains unclear, but one possible reason is the highly immunostimulatory nature of the DNA vaccine plasmid itself, as DNA is a potent inducer of both IFN-a/b and several NF-kB–regulated immune genes. In addition, the level of several chemokines, such as MIP-1a/b and MCP-1, remained unchanged (data not shown), allowing for recruitment of monocyte and lymphocyte populations. Most intriguingly, the reduction of IFN-a/b levels at the site of immunization in Aim22/2 suggests a previously unknown relationship between Aim2 and local IFN-a/b production. Aim2 is not known to mediate IFN-a/b production directly, hinting at an indirect link between these two divergent pathways. We propose that decreased pyroptotic cell death in Aim22/2 mice results in diminished DAMP release, limiting cellular signaling and bystander cell activation. The release of cellular DNA by pyroptotic cells may augment IFN-a/b production by surrounding cells, possibly through the STING/TBK1 signaling axis, further propagating the immune response. In conclusion, our results indicate that Aim2 plays a significant role in DNA vaccination. Aim22/2 mice failed to generate optimal immune responses upon DNA vaccination, exhibiting decreased serum IgG levels and T cell cytokine production, demonstrating the necessity for Aim2 signaling in DNA vaccine immunogenicity. In addition, we reported a previously unknown function for Aim2 in augmenting IFN-a/b production at the site of immunization. Our results provide a deeper understanding of the cellular mechanisms through which DNA vaccines stimulate both the innate and adaptive immune pathways to enhance the immune responses targeting the encoded Ag.
THE EFFECTS OF Aim2 ON DNA VACCINE IMMUNOGENICITY
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