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DEVELOPMENTAL BIOLOGYCorrection for “Neighbor of Brca1 gene (Nbr1) functions as anegative regulator of postnatal osteoblastic bone formation andp38 MAPK activity,” by Caroline A. Whitehouse, Sarah Waters,Katie Marchbank, Alan Horner, Neil W. A. McGowan, JelenaV. Jovanovic, Guilherme M. Xavier, Takeshi G. Kashima,Martyn T. Cobourne, Gareth O. Richards, Paul T. Sharpe, TimM. Skerry, Agamemnon E. Grigoriadis, and Ellen Solomon,

which appeared in issue 29, July 20, 2010, of Proc Natl Acad SciUSA (107:12913–12918; first published June 29, 2010; 10.1073/pnas.0913058107).The authors note that Figure 4 appeared incorrectly. A well

image was duplicated within panel C. The corrected figure and itslegend appear below. This error does not affect the conclusions ofthe article.

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Fig. 4. p38 MAPK activity is increased in Nbr1tr/tr osteoblasts. (A) p-p38 MAPK interacts with full-length Nbr1 but not trNbr1. COS-7 cells were transfectedwith HA-Nbr1, HA-trNbr1, and p38 MAPKmyc constructs for 24 h and stimulated or not stimulated with 5 ng/mL anisomycin for 15 min; extracts wereprepared, and co-immunoprecipitation of Nbr1 with p38 MAPK was detected by Western blot analysis. Representative blots of two experiments with similarresults are shown. (B) Anisomycin-induced p38 MAPK activation in osteoblasts cultured from bone marrow of 3-mo-old Nbr1tr/tr mice is elevated and pro-longed compared with Wt cells. (C) p38 MAPK inhibition rescues the increased differentiation phenotype in Nbr1tr/tr osteoblasts. Neonatal calvarial-derivedosteoblast cultures from Wt or Nbr1tr/tr mice in osteogenic culture for 18 days in the presence of DMSO (vehicle) or 10 μM SB203580 were stained for alkalinephosphatase only [ALP (Top two rows)] or ALP followed by von Kossa [ALP/von Kossa (Bottom two rows)] and quantified using National Institutes of Health(NIH) Image software. The data represent the mean ± SD of triplicate representative wells normalized to untreated wild-type cells (*P < 0.05). Similar resultswere obtained in the presence of 0.1 μM SB203580.

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BIOPHYSICS AND COMPUTATIONAL BIOLOGY, APPLIED PHYSICALSCIENCESCorrection for “Interpreting the widespread nonlinear forcespectra of intermolecular bonds,” by Raymond W. Friddle,Aleksandr Noy, and James J. De Yoreo, which appeared in issue34, August 21, 2012, of Proc Natl Acad Sci USA (109:13573–13578; first published August 6, 2012; 10.1073/pnas.1202946109).The authors note that on page 13576, right column, first par-

agraph, line 8 “(38 pN/nm)” should instead appear as “(27 pN/nm)”. The authors also note that Fig. 3 appeared incorrectly.The corrected figure and its legend appear below.

www.pnas.org/cgi/doi/10.1073/pnas.1302149110

MEDICAL SCIENCESCorrection for “Inflammasome-independent role of the apoptosis-associated speck-like protein containing CARD (ASC) in theadjuvant effect of MF59,” by Ali H. Ellebedy, Christopher Lupfer,Hazem E. Ghoneim, Jennifer DeBeauchamp, Thirumala-DeviKanneganti, and Richard J. Webby, which appeared in issue 7,February 15, 2011, of Proc Natl Acad Sci USA (108:2927–2932;first published January 26, 2011; 10.1073/pnas.1012455108).The authors wish to note the following: “In Ellebedy et al. we

showed a role for the apoptosis-associated speck-like protein con-taining CARD (ASC) in the adjuvant effect of MF59. However,in pilot studies in an alternative ASC-deficient mouse line gener-ated by Dr. V. Dixit (Genentech, Inc.), this phenotype was notreproduced. A similar observation was recently reported, thushighlighting differences in the available ASC-deficient mouse lines(1). The data in Ellebedy et al. is accurate, and in that line of ASC-deficient mice there is indeed a reduced adjuvant effect of MF59.We stand by that data, but caution should be used in interpretingdata gained from the different ASC-deficient mice.”

1. Ippagunta SK, et al. (2012) Addendum: Defective Dock2 expression in a subset of ASC-deficient mouse lines. Nat Immunol 13(7):701–702.

www.pnas.org/cgi/doi/10.1073/pnas.1301192110

NEUROSCIENCECorrection for “D2 receptor overexpression in the striatum leadsto a deficit in inhibitory transmission and dopamine sensitivity inmouse prefrontal cortex,” by Yan-Chun Li, Christoph Kellendonk,Eleanor H. Simpson, Eric R. Kandel, and Wen-Jun Gao, whichappeared in issue 29, July 19, 2011, of Proc. Natl. Acad. Sci.USA (108:12107-12112; first published July 5, 2011; 10.1073/pnas.1109718108).The authors note that the National Institutes of Health grant

number R01MH232395 should instead appear as R01MH085666.

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Fig. 3. Model fits to the nonlinear force spectra of intermolecular bonds. (A)Force spectrum of the Ni-NTA/His6 bond measured in this work along with thedata of Verbelen et al. (34). Measurements made without Ni2+ demonstratethe specificity of the interaction (open circles). Solid lines represent fits to Eq. 6using identical parameters for both data sets except for the respective springconstants. (B) Force spectra of 10 data sets taken from the literature are fit toEq. 9 assuming a generic equilibrium force and apparent transition state dis-tance. Data are exploded along the loading rate axis for clarity. Inset: The sameforce spectra in their raw form illustrating the theoretical range of equilibriumforce and cross-over loading rate span (shaded regions). The upward inflectionat high forces in the Biotin/Avidin data of ref. 2 may be due to limited samplingrate effects (41) and enhanced force error at very fast loading rates (42). (C)The same data in B plotted in the natural coordinates of Eq. 9 (see Eq. S3) showthat all spectra collapse onto a single line. (D) Biotin-avidin bond rupture dataof Teulon et al. (35) are globally fitted to Eq. 9 assuming N = 1, 2, and 3 parallelbonds. Only feq is independently fit, while xt and k0u are shared. Fitted valuesare xt = 0.78 Å, k0u ¼ 6:75  s-1, and feq = 24.6 pN (N = 1), 58.5 pN (N = 2), 142.3 pN(N = 3). Legend in (B) refers to references and corresponding bonds as follows:Biotin/Avidin (2); LFA-1/ICAM-1 [rest 3A9] (10); Aβ-40/Aβ-40 (11); N,C,N-pincer/pyridine (12); Si3N4/Mica in Ethanol (14); peptide/steel (43); Integrin/Fibronectin(44; Lysozyme/Anti-Lysozyme (45); Dig/Anti-Dig (46); Actomyosin/ADP (47).

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Inflammasome-independent role of the apoptosis-associated speck-like protein containing CARD (ASC)in the adjuvant effect of MF59Ali H. Ellebedya, Christopher Lupferb, Hazem E. Ghoneima, Jennifer DeBeauchampa, Thirumala-Devi Kannegantib,1,2,and Richard J. Webbya,1,2

Departments of aInfectious Diseases and bImmunology, St. Jude Children’s Research Hospital, Memphis, TN 38105

Edited* by Robert G. Webster, St. Jude Children’s Research Hospital, Memphis, TN, and approved December 21, 2010 (received for review August 23, 2010)

Clinical studies have indicated that subvirion inactivated vaccinesagainst avian influenza viruses, particularly H5N1, are poorly im-munogenic in humans. As a consequence, the use of adjuvantshas been championed for the efficient vaccination of a naïvepopulation against avian influenza. Aluminum salts (alum) andthe oil-in-water emulsion MF59 are safe and effective adjuvantsthat are being used with influenza vaccines, but the mechanismunderlying their stimulation of the immune system remainspoorly understood. It was shown recently that activation of a cy-tosolic innate immune-sensing complex known as “NLR-Pyrin do-main containing 3” (NLRP3) inflammasome, also known as“cryopyrin,” “cold-induced autoinflammatory syndrome 1” (CIAS1),or nacht domain-, leucine-rich repeat-, and PYD-containing protein3 (Nalp3), is essential for the adjuvant effect of alum. Here weshow that the inflammasome component apoptosis-associatedspeck-like protein containing a caspase recruitment domain(ASC), an adapter protein within the NLRP3 inflammasome, is a cru-cial element in the adjuvant effect of MF59 when combined withH5N1 subunit vaccines. In the absence of ASC, H5-specific IgG an-tibody responses are significantly reduced, whereas the responsesare intact in NLRP3−/− and caspase-1−/− mice. This defect is causedmainly by the failure of antigen-specific B cells to switch from IgMto IgG production. We conclude that ASC plays an inflammasome-independent role in the induction of antigen-specific humoral im-munity after vaccination with MF59-adjuvanted influenza vac-cines. These findings have important implications for the rationaldesign of next-generation adjuvants.

The primary means of infection- and vaccine-mediated im-munologic protection against influenza A viruses is the

development of antibodies that bind and neutralize the re-ceptor-binding function of HA. A number of avian influenza Aviruses, such as H5N1, have infected humans during the pastdecade. In response to the recent H5N1 outbreak, several gov-ernments, including that of the United States, produced and stock-piled H5N1 subvirion inactivated vaccines (http://www.who.int/csr/resources/publications/WHO_HSE_EPR_GIP_2008_1d.pdf). Theprimary purpose of these stockpiles is for deployment during theearly phases of a pandemic while a matching vaccine is developed.The results from the first clinical trials of unadjuvanted H5N1vaccines were disappointing in terms of immunogenicity (1).However, substantial improvements have been seen in trials withthe adjuvanted formulations specifically using the new-generationoil-in-water emulsion-based adjuvants, which have induced sig-nificantly higher antibody responses to low antigen-content vac-cines (2, 3).The most common adjuvants in clinical use with influenza

vaccines are the insoluble aluminum salts, generically referred toas “alum,” and the oil-in-water emulsion adjuvant, MF59 (4).MF59 has been tested with H5 and H9 influenza vaccines andshowed promising results; an ability to potentiate and increase thebreadth of the immune response induced by low-antigen-contentvaccines (2, 5). It has been shown that alum and MF59 induce the

secretion of a range of cytokines associated with recruitment ofinnate immune cells to the injection site (6). Among the lattercells are dendritic cells (DCs), which are essential for antigenpresentation and the subsequent activation of naïve T cells. Theimmunostimulatory activity of alum has been attributed recentlyto a family of innate immune sensors known as “NOD-like re-ceptors” (NLRs) (7). NLRs are a large family of intracellularproteins that are believed to be involved primarily in the innateimmune response to microbial pathogens through the recognitionof a conserved pathogen-associated molecular pattern (8–10).However, they also contribute by sensing “danger signals,” i.e.,endogenous molecules that are produced during tissue damage orinflammation (9, 11). Specifically, in mice deficient in NLR-Pyrindomain containing 3 (NLRP3), decreased IL-1β secretion andantigen-specific humoral immune responses to immunization withalum-adsorbed antigens have been observed (7, 12).NLRP3 allows the recruitment and autocatalytic activation of

the cysteine protease caspase-1 in a large cytosolic protein com-plex named the “inflammasome” (8). Once activated, theinflammasome mediates caspase-1 cleavage of the inactive pre-cursor of the proinflammatory cytokine IL-1β resulting in releaseof mature IL-1β. The adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)bridges the interaction between NLRP3 and caspase-1, making itessential for activation of the inflammasome (8). Whether anyof the NLRP3 inflammasome components (NLRP3, ASC, andcaspase-1) play a role in MF59-mediated immune stimulation hasnot been addressed.The goal of this study was to understand the roles of the dif-

ferent NLRP3 inflammasome components (NLRP3, ASC, andcaspase-1) in MF59-mediated immune stimulation when com-bined with H5N1 subvirion inactivated vaccines. We found that inASC−/− mice, unlike NLRP3−/− and caspase-1−/− mice, the pro-duction of H5-specific IgG antibodies was significantly reducedafter immunization with MF59-adjuvanted H5N1 vaccines incomparison with WT mice. This effect was adjuvant specific, be-cause ASC−/− mice elicited an H5-specific IgG antibody responsecomparable with that of WT mice when immunized with unad-juvanted H5N1 vaccines. In addition, we found that the de-velopment of germinal center (GC) B cells in ASC−/− draininglymph node cells was reduced. These defects extended to memory,because antigen-specific recall antibody responses were impaired

Author contributions: A.H.E., T.-D.K., and R.J.W. designed research; A.H.E., C.L., H.E.G.,and J.D. performed research; A.H.E., T.-D.K., and R.J.W. analyzed data; and A.H.E., T.-D.K.,and R.J.W. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1T.-D.K. and R.J.W. contributed equally to this work.2To whom correspondence may be addressed. E-mail: Thirumala-Devi.Kanneganti@StJude.org or richard.webby@stjude.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1012455108/-/DCSupplemental.

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significantly in ASC−/− mice. Finally, inflammatory cytokine pro-duction by ASC−/− bone marrow-derived dendritic cells (BMDCs)was lower than that of WT cells. Therefore, ASC plays a caspase-1(and thus inflammasome)-independent role in the inductionof antigen-specific humoral immunity in response to MF59-adjuvanted vaccines.

ResultsMF59 Is Superior to Alum in Priming for Robust H5-Specific AntibodyResponses. HA-specific antibodies are considered the most im-portant correlate of protection elicited by inactivated influenzavaccines. It was shown previously that in mice MF59 is superior toalum and several other adjuvants in priming for a robust antigen-specific humoral immune response when combined with seasonalinfluenza vaccines (13). To confirm this observation, we primedfour groups of WT C57BL/6 mice with H5N1 subvirion vaccines(MF59- or alum-adjuvanted or unadjuvanted) or with sublethalH5N1 influenza virus (homologous to the vaccine strain) in-fection. After boosting all groups with the unadjuvanted H5N1subvirion vaccine in PBS, we first determined the serum levels ofH5-specific IgG antibodies induced. We found that mice primedwith the MF59-adjuvanted vaccine elicited significantly higherantibody titers than the mice primed with alum-adjuvanted orunadjuvanted vaccine (Fig. 1A). The H5-specific IgG titers in theMF59-adjuvanted group were comparable to those elicited in theinfection-primed mice (Fig. 1A). We next determined the fre-quency of H5-specific antibody-forming cells (AFCs) in the bonemarrow and spleen after the boosting immunization. For themice primed with the unadjuvanted vaccine, the number of AFCswas below the assay detection limit (Fig. 1 B and C). Consistentwith the serum IgG results, however, the number of H5-specificIgG-forming cells was comparably elevated in the MF59- andinfection-primed mice and was significantly higher than that ob-served in alum-primed mice (Fig. 1 B and C).

Humoral Immune Response to MF59-Adjuvanted Vaccines RequiresASC but Not NLRP3 or Caspase-1. The NLRP3 inflammasome hasbeen implicated in the adjuvant effect of alum. Therefore, weasked whether the NLRP3 inflammasome plays any role in me-diating the adjuvant activity of MF59 (7, 12). We first determinedthe HA inhibition (HI) titers of WT, NLRP3−/−, ASC−/−, andcaspase-1−/− mice after vaccination with MF59-adjuvanted H5N1subvirion vaccine. We found that ASC−/− mice, but not NLRP3−/−

or caspase-1−/− mice, elicited significantly lower HI titers thanWT

mice (Fig. 2A). We next determined the serum levels of the dif-ferent H5-specific antibody isotypes in immunized mice 3 wk aftera booster immunization. Consistent with the HI results, circulatinglevels of H5-specific IgM, IgG, IgG1, IgG2b, and IgG2c antibodieswere comparably elevated in the serum of WT, NLRP3−/−, andcaspase-1−/−mice (Fig. 2B–F). ImmunizedASC−/−mice, however,elicited significantly less IgG, IgG1, IgG2b, and IgG2c antibodiesthan WT mice (Fig. 2 C–F). Interestingly, circulating levels of H5-specific IgM antibodies in ASC−/− mice were comparable to thoseof WT mice (Fig. 2B). As a control, we examined the H5-specificantibody response to the unadjuvanted vaccine in WT, NLRP3−/−,ASC−/−, and caspase-1−/− mice (Fig. S1). ASC−/− mice respondedrobustly to the unadjuvanted vaccine, indicating that the defectiveresponse observed with the MF59-adjuvanted vaccine was notcaused by a general defect in these mice in response to this par-ticular inactivated influenza vaccine. We also tested the antibodyresponse to H5N1 vaccine adjuvanted with incomplete Freund’sadjuvant (IFA) in WT and ASC−/− mice. Again, there were nosignificant differences in H5-specific IgG1 or IgG2c antibody titers(Fig. S2). In addition, infection with a sublethal dose of an atten-uated H5N1 virus resulted in comparable anti-H5 IgG titers inWTand ASC−/− mice when measured 3 wk after infection (Fig. S3).These results suggested that ASC is specifically required for theoptimal generation of antigen-specific antibodies in response toMF59-adjuvanted H5 vaccines. In addition, class switching anti-H5antibodies from IgM to IgG, rather than antibody production itselfwas impaired in ASC−/− mice.

ASC Is Required for GC B-Cell Formation After Immunization withMF59-Adjuvanted Vaccines. Antibody class switching after vaccina-tion or infection takes place mainly within the GC reaction in thedraining lymph nodes (14). The observed decrease in IgG pro-duction in ASC−/−mice afterMF59-adjuvantedH5N1 vaccinationsuggested a defect in the development of the GC reaction in thesemice. To investigate this possibility, we immunizedWT,NLRP3−/−,ASC−/−, and caspase-1−/− mice with the MF59-adjuvanted H5N1vaccine and harvested the draining (inguinal) lymph nodes as wellas sera 7 d later. H5-specific IgM and IgG antibodies were un-detectable in the sera of ASC−/−mice, whereas both isotypes weredetectable in the other three groups (Fig. 3 A and B), indicatinga delay in the induction of the antibody response in the ASC−/−

mice. In agreement with this observation, WT, NLRP3−/−, andcaspase-1−/− mice all had significantly larger lymph nodes thanASC−/− mice, as evidenced by the difference in cellularity (Fig.

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Fig. 1. Superior priming with MF59-adjuvanted H5N1 vaccines in comparison with the alum-adjuvant or the unadjuvanted formulations. Four groups ofC57BL/6 WT mice (n = 5) were primed i.m. with 1 μg of H5N1 subvirion vaccine (MF59- or alum-adjuvanted or unadjuvanted) or with sublethal A/vietnam/1203/2004 (H5N1) influenza virus infection. All groups were boosted with 3 μg of H5N1 subvirion vaccine in PBS, and sera and other organs were harvested 5 d afterthe boost. (A) Mean circulating levels of H5-specific IgG antibodies. (B and C) Mean number of H5-specific IgG-forming cells in the bone marrow (B) and spleen(C). The results are representative of at least two separate experiments. Statistically significant differences between WT and mutant groups are indicated byasterisks; *P < 0.05. ns, not significant. nd, not detected. Error bars show SE.

2928 | www.pnas.org/cgi/doi/10.1073/pnas.1012455108 Ellebedy et al.

3C).We next looked at the frequency of GCB cells in the drainingipsilateral lymph nodes. We found that in WT, NLRP3−/−, andcaspase-1−/− mice, GC B cells were comparably developed, ac-counting for 30–50%of the B220+ B-cell population (Fig. 3D andE). On the other hand, GC B cell frequencies in the ASC−/− micewere significantly lower (Fig. 3 D and E). The generation of a GCB-cell reaction requires the help of CD4+ T cells, so we askedwhether they exhibited similar defects. Indeed, we found thatabsolute numbers of CD4+ T cells in the ASC−/− mice were sig-nificantly lower than in WT mice (Fig. 3F). These results showthat ASC, independent of the caspase-1 inflammasome, is re-quired for the rapid induction of the antibody response to MF59-adjuvanted H5N1 vaccines. Moreover, the development of a GCreaction in the draining lymph nodes of ASC−/− mice is impaired,explaining the defect observed in antibody isotype switching inthesemice. This defect was not B-cell specific, because CD4T-cellfrequencies also were significantly lower in ASC−/− mice, poten-tially contributing to the GC phenotype.

ASC Is Required for Robust Antigen-Specific Recall B-Cell Responses.The ultimate outcome of a GC B-cell reaction is the generation ofhigh-affinity, antigen-specific memory B cells and long-livedplasma cells (14). Upon antigen reexposure, memory B cells ex-pand rapidly and differentiate into AFCs. We therefore in-vestigated whether the defect in GCB-cell development in ASC−/−

mice after priming would, in turn, result in a defective H5-specificmemory B-cell compartment. To test that notion, we primed WT,NLRP3−/−, ASC−/−, and caspase-1−/−mice with one dose ofMF59-adjuvanted H5N1 vaccine, and all mice were given a booster withthe same vaccine in PBS 3 wk later. Similar to our observationswith the primary response, 5 d after antigen reexposure, WT,NLRP3−/−, and caspase-1−/− mice had significantly larger draininglymph nodes than ASC−/−mice, and that response was mirrored bya greater number of live cells (Fig. 4A). Also, GC B-cell numbers

were significantly lower in the draining lymph nodes of ASC−/−

mice than in the other groups (Fig. 4B). Moreover, the number ofCD138+ plasma cells was significantly lower in ASC−/− mice thanin WT, NLRP3−/−, and caspase-1−/− mice (Fig. 4C). In agreementwith the decreased number of CD138+ cells in ASC−/− mice afterthe boost, the number ofH5-specific AFCs in the bonemarrowwassimilarly lower (Fig. 4D). As expected, when the H5-specific IgGantibody titers in mice sera before and 5 d after the boost werecompared, ASC−/− mice showed a slight (twofold) increase in an-tibody titer (Fig. 4E). In contrast,WT, NLRP3−/−, and caspase-1−/−

mice showed a significantly larger (five- to sixfold) increase in H5-specific IgG titers after the boost, signifying a robust memoryresponse in these mice (Fig. 4E). These results demonstrate thatASC is required for the optimal priming of antigen-specific B-cellmemory responses with MF59-adjuvanted vaccines.

ASC Expression in DCs Is Required for Adequate Production ofInflammatory Chemokines. We have shown that ASC deficiency re-sults in poor B-cell and CD4 T-cell responses to MF59-adjuvantedH5N1 subvirion vaccines. To elucidate further the mechanism bywhich ASC acts, we started from the knowledge that the adjuvanteffect of MF59 and alum depends at least partially on their abilityto induce the secretion of chemokines responsible for recruitinginflammatory cells to the site of injection (6). DCs are the mostpotent antigen-presenting cells capable of stimulating naïveT cells for the subsequent induction of antigen-specific T-cell andB-cell responses. It has been shown that DCs internalize most ofthe adjuvant within 48 h of MF59 i.m. injection, (15). We there-fore investigated whether the defective antigen-specific antibodyresponse in immunized ASC−/− mice was caused by a defectiveinflammatory response by BMDCs. To examine this possibility,BMDCs from WT, NLRP3−/−, ASC−/−, and caspase-1−/− micewere incubated with MF59, and the secretion of proinflammatorychemokines in the culture supernatant was determined. WT

Fig. 2. Defective humoral immune response to MF59-adjuvanted H5N1 vaccines in ASC-KO mice. C57BL/6 WT (n = 5), Nlrp3−/− (n = 4), ASC−/− (n = 5), andcaspase-1−/− (n = 5) mice were immunized twice, 3 wk apart, with 1 μg of H5N1 subvirion vaccine adjuvanted with MF59. Sera were collected 3 wk after theboosting immunization. (A) Mean serum HI titers. (B–F) Mean circulating levels of H5-specific IgM (B), IgG (C), IgG1 (D) IgG2b (E), and (F) IgG2c antibodies.Results are representative of at least two separate experiments. Statistically significant differences between WT and mutant groups are indicated by asterisks;*P < 0.05. Error bars show SE.

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BMDCs secreted significantly more proinflammatory chemokinessuch as macrophage inflammatory protein (MIP)1b and MIP2(P=0.002 and 0.005, respectively) than did BMDCs derived fromASC−/− mice but not significantly more than BMDCs derivedfrom NLRP3−/− and caspase-1−/− mice (Fig. 5 A and B). MF59was responsible for the chemokine stimulation, because the dif-ferences in MIP1b secretion were seen in cells treated with MF59alone but not in cells treated with vaccine antigen alone (Fig. S4).These results suggest that the inflammasome-independent role ofASC in generating the proper inflammatory environment afterimmunization may be the underlying cause of the defective in-duction of humoral immunity in the ASC-deficient mice.

DiscussionSince their emergence in 1996, highly pathogenic H5N1 influenzaviruses have posed a threat of becoming the causative agent of thenext influenza pandemic. This situation has been the catalyst for anincrease in the amount of resources applied to preparedness ac-tivities for such a pandemic. Vaccine development has been one ofthe foci of these activities, and a major challenge that has emergedhas been the formulation of conventional influenza vaccines so

that they can effectively prime a population immunologically naïveto H5N1 antigens. Unadjuvanted split and subunit H5N1 vaccineshave been shown reproducibly to be poorly immunogenic inhumans (1, 16). The addition of alum adjuvant induced onlymarginal improvements (17). In contrast, oil-in-water–basedadjuvants (e.g., MF59) have been shown to prime for a robust andbroadly cross-reactive antibody response to subvirion H5N1 vac-cines, with greatly reduced antigen requirements (18). MF59 hasshown encouraging results in several influenza formulations and islicensed for use in seasonal influenza vaccines in Europe (5, 19–21). However, the mechanisms underlying the ability of MF59 andother adjuvants to stimulate the immune system remain poorlyunderstood. To develop more defined next-generation adjuvants,it is critical to determine the immunologic processes that arestimulated by MF59 and other efficacious adjuvants. Recentstudies have shown that induction of antigen-specific antibodyresponses by commonly used vaccine adjuvants, such as alum andFreund’s complete and incomplete adjuvants, did not requiresignaling through Toll-like receptors (22). However, the ability ofalum and other particulate adjuvants to enhance IL-1 secretion viaNLRP3 has been described (7, 12, 23). In addition, some studies

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B220+Fas+34.7

0 102 103 104 105

B220+Fas+1.42

0 102 103 104 105

B220+Fas+44.8

B220

WT Nlrp3-/- ASC-/- Caspase-1-/-

FAS

C

E F

BA

D

**

* *

*

Fig. 3. Analysis of GC B-cell development after immunization withMF59-adjuvanted H5N1 subvirion vaccine. C57BL/6WT (n = 5), Nlrp3−/− (n = 4), ASC−/− (n = 5),and caspase-1−/− (n = 3) mice were immunized with 5 μg of H5N1 subvirion vaccine adjuvanted with MF59. Sera and draining (inguinal) lymph nodes werecollected 7 d after immunization. (A and B) Mean circulating levels of H5-specific IgM (A) and IgG (B) antibodies were determined as in Fig. 1. (C) Mean number oflive cells in the ipsilateral draining lymph node. (D) Representative FACS plots for lymphocytes from thedraining lymph nodes stainedwithmonoclonal antibodiesto B220 and CD95 (Fas) on day 7 after immunization with 5 μg of H5N1 subvirion vaccine adjuvanted with MF59. The gated population (circled in red) from eachplot represents GC B cells, whichwere defined as cells that were negative for surface CD4, CD8, and CD11b,with low expression of surface IgD and high expressionof surface B220 and FAS (CD195). (E) Total number of GC B cells as determined by FACS analysis of the cells with the phenotype defined in D. (F) Total number ofCD4+ lymphocytes. Statistically significant differences between WT and mutant groups are indicated by asterisks; *P < 0.05. Error bars show SE.

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have shown that enhancement of antigen-specific humoral im-munity by alum also was NLRP3 dependent (7, 12), although laterreports suggested that NLRP3 was not required (24, 25). Here, wefound that the antibody response to H5N1 subunit influenzavaccines adjuvanted with MF59 critically depend on the presenceof the adapter molecule ASC. After immunization with MF59-adjuvanted H5N1 subvirion vaccines, the amount of H5-specificantibodies of IgG1, IgG2b, and IgG2c subtypes were all signifi-cantly lower in ASC−/− mice than in WT mice. In contrast tostudies with alum, we found that neither NLRP3 nor caspase-1was required for the antigen-specific antibody responses toMF59-adjuvanted H5N1 influenza subvirion vaccines, indicating that theproposed role of ASC is independent of the caspase-1 inflam-masome. It is interesting, however, that the murine antibody re-

sponse to antigens emulsified in Freund’s complete adjuvant hasbeen shown to be unaffected by the absence of ASC (7). Thisresult suggests that, even among emulsion-based adjuvants, themolecular requirement for the proper induction of an adaptiveimmune response varies. It also is possible that the complex na-ture of Freund’s complete adjuvant affords immune stimulationthrough a number of redundant mechanisms.Complexities of the response to adjuvants aside, our data sup-

port recent findings in the murine model of collagen-induced ar-thritis as well as in the development of experimental autoimmuneencephalomyelitis that show an inflammasome-independent rolefor ASC in the induction of various forms of immunity and im-munopathology (26, 27). There are several molecular mechanismsby which ASC may control the antigen-specific antibody response

Fig. 4. Poor H5-specific antibody recall responses in immunized ASC−/− but not in WT, NLRP3−/−, or caspase-1−/− mice after antigen reexposure. C57BL/6 WT(n = 5), Nlrp3−/− (n = 4), ASC−/− (n = 3), and caspase-1−/− (n = 3) mice were primed with 1 μg of H5N1 subvirion vaccine adjuvanted with MF59 and were boosted3 wk later with 5 μg of H5N1 subvirion vaccine in PBS. Sera were collected before and 5 d after the boost. Ipsilateral draining lymph nodes and bone marrowwere collected 5 d after the boost. (A) Mean number of live cells in the draining ipsilateral draining lymph nodes. (B) Mean number of plasma cells in thedraining lymph nodes as determined by FACS analysis of cells that were negative for surface CD4, CD8, and CD11b, with low expression of surface IgD andhigh surface expression of CD138. (C) Mean number of GC B cells determined as in Fig. 2E. (D) Mean frequencies of H5-specific AFCs in the bone marrow ofWT, Nlrp3−/−, ASC−/−, and caspase-1−/− mice 5 d after antigen reexposure. (E) Fold increase in H5-specific IgG titers 5 d after antigen reexposure. Statisticallysignificant differences between WT and mutant groups are indicated by asterisks; *P < 0.05. Error bars show SE.

Fig. 5. Defective inflammatory response to MF59in ASC−/−

BMDCs. BMDCs were stimulated for 24 h with MF59 incomplete medium (1:100 vol/vol), and the quantities of (A)MIP1β and (B) MIP2 were determined in cell-culture super-natants. Tests were assayed in triplicate or quadruplicate.Statistically significant differences between WT and mutantgroups are indicated by asterisks; *P < 0.05. Error barsshow SE.

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to MF59-adjuvanted vaccines. A key underlying feature in theASC−/− mice was the significantly reduced production of key in-flammatory cytokines by BMDCs compared withWT-derived cellsafter stimulation with MF59. Consistent with a defect in antigen-presenting cells, it has been suggested that antigen-specific activa-tion of T cells by ASC-deficient BMDCs was significantly impaired(26). In linewith this notion, after a single immunizationwithMF59-adjuvanted H5N1 vaccine, we observed a delayed antigen-specificantibody response and significantly lower numbers of CD4+ T cellsin the draining lymph nodes in ASC−/− but not NLRP3−/− orcaspase-1−/− mice in comparison with their WT counterparts. Inhumans, the early development of an antigen-specific CD4+ T-cell response was shown to be a successful predictor of the ro-bustness and persistence of an antibody response followingMF59-adjuvanted H5N1 vaccination (28). We also demonstrated thatafter immunization the development of GC B cells in the draininglymph nodes was significantly impaired in ASC−/− mice in com-parison with that seen in WT, NLRP3−/−, and caspase-1−/− mice.In addition, we showed that these defects resulted in a poormemory B-cell response in ASC−/− mice. Because the aggregateevidence demonstrates a critical role for ASC independent ofinflammasome, an entire body of literature linking chronicmodelsof inflammation and infection to inflammasome activation byexperiments performed in ASC−/− mice should be reassessed.The promising nature of MF59 use in humans and the iden-

tification of ASC as a critical factor for the proper induction ofthe adaptive immune response to MF59-adjuvanted vaccineswould have important implications for the formulation of futureinfluenza vaccines.

Materials and MethodsMice and Immunization. Cryopyrin/Nlrp3-/-, ASC-/-, and caspase-1-/- mice werebackcrossed to a C57BL/6 background for at least 10 generations. Mice werehoused in a pathogen-free facility, and the animal studies were conductedunder protocols approved by the St. Jude Children’s Research Hospital Com-mittee on Use and Care of Animals. Animals were immunized intramuscularlytwice with 1, 3, or 5 μg of monovalent H5N1 influenza (rgA/Vietnam/1203/2004) subvirion vaccine (Aventis Pasteur Inc., Swiftwater, PA) either unad-juvanted (in PBS), mixed with MF59 (1:1) or adsorbed to aluminium hydroxideat 14.1 mg/ml Alum, 3 μg/ml antigen and 5 mM Histidine buffer pH 6.5 at 4 °Covernight. Incomplete Freund’s adjuvant or IFA (Sigma, St. Louis, MO) wasmixed with the vaccine solution in 1:1 (vol/vol) ratio and emulsified just beforeinjection. Each mouse dose contained 500 μg Alum. The intranasal infectionswere performed with 100 egg infectious dose 50 of the rgA/Vietnam/1203/2004 (ΔHA) diluted in PBS to a final volume of 30 μl.

Standard procedures and methods such as hemagglutination inhibitionassay, flow cytometry, antigen-specific ELISA and ELISPOT assays, cytokineanalysis and statistical analyses are described in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Anthony Coyle, John Bertin, Ethan Grant,Gabriel Nunez, Richard Flavell, and Shizuo Akira for generous supply ofmutant mice. We thank the Division of Microbiology and Infectious Diseases,National Institute of Allergy and Infectious Diseases, National Institutes ofHealth for a generous supply of MF59. This work was supported by GrantAR056296 from the National Institutes of Arthritis, Musculoskeletal and SkinDiseases (NIAMS), and a National Institute of Allergy and Infectious Diseases(NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS)grant (to T.D.K.), Contract No. HHSN266200700005C from the NationalInstitute of Allergy and Infectious Diseases (to R.J.W.), and by grants fromthe American Lebanese Syrian Associated Charities (to T.D.K. and R.J.W.) andthe University of Tennessee Health Science Center Clinical and TranslationalScience Institute (T32 Scholar) (to A.H.E.).

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