alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are...

Alum induces innate immune responses through macrophageand mast cell sensors, but these are not required for alum to actas an adjuvant for specific immunity.1,2

Amy S. McKee*,†, Michael W. Munks*,†, Megan K. L. MacLeod*,†, Courtney J. Fleenor¶,Nico Van Rooijen‡, John W. Kappler*,†,§, and Philippa Marrack*,†,¶

*Howard Hughes Medical Institute, National Jewish Health, 1400 Jackson St., Denver CO 80206†Integrated Department of Immunology, National Jewish Health, 1400 Jackson St., Denver CO80206 ‡Department of Molecular Cell Biology, Vrije Universiteit, VUMC, 1081 BT Amsterdam,The Netherlands §Program in Biomolecular Structure, University of Colorado Health ScienceCenter Aurora, CO 80045 ¶Department of Biochemistry and Molecular Genetics, University ofColorado Health Science Center Aurora, CO 80045

AbstractTo understand more about how the body recognizes alum we characterized the early innate andadaptive responses in mice injected with the adjuvant. Within hours of exposure, alum induces atype 2 innate response characterized by an influx of eosinophils, monocytes, neutrophils, DCs, NKcells and NKT cells. In addition, at least thirteen cytokines and chemokines are produced within 4hours of injection including IL-1β and IL-5. Optimal production of some of these, including IL-1β,depends upon both macrophages and mast cells, while production of others, such as IL-5, dependson mast cells only, suggesting that both of these cell types can detect alum. Alum induceseosinophil accumulation partly through the production of mast cell derived IL-5 and histamine.Alum greatly enhances priming of endogenous CD4 and CD8 T cells independently of mast cells,macrophages and of eosinophils. In addition, antibody levels and Th2 bias was similar in theabsence of these cells. We found that the inflammation induced by alum was unchanged incaspase-1 deficient mice, which cannot produce IL-1β. Furthermore, endogenous CD4 and CD8 Tcell responses, antibody responses and the Th2 bias were also not impacted by the absence ofcaspase-1 or NLRP3. These data suggest that activation of the inflammasome and the type 2 innateresponse orchestrated by macrophages and mast cells in vivo are not required for alum's adjuvanteffects on endogenous T and B cell responses.

Keywordsinnate immune responses; vaccine adjuvant; eosinophils; TLR; NLR

1This work was supported by USPHS grants AI-18785, AI-52225 and AI 22295.2Abbreviations: TLR, toll-like receptor; BSS, balanced salt solution; Tg, transgenic; CTM, complete tumor medium; IL, LTB4Leukotriene B4; PAF, platelet activating factor; MIP, macrophage inflammatory protein; MIG, Monokine induced by gammainterferon; IP10, 10kDa IFN induced protein; KC, keratinocytes-derived chemokine; MCP, monocytes chemotactic protein; PB,pacific blue3 Address correspondence to Philippa Marrack, Howard Hughes Medical Institute, Dept. of Immunology, 1400 Jackson St., Denver,CO 80206. Phone: (303) 398-1324; Fax: (303) 398-1396; [email protected].

NIH Public AccessAuthor ManuscriptJ Immunol. Author manuscript; available in PMC 2010 July 30.

Published in final edited form as:J Immunol. 2009 October 1; 183(7): 4403–4414. doi:10.4049/jimmunol.0900164.

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IntroductionAluminum salts, here referred to collectively as alum, have been used for almost a century toenhance antibody responses in animals and humans with very little understanding about howthey mediate their effects on the immune system (1). In human vaccines, aluminumhydroxide, aluminum phosphate and aluminum sulfate are used and the choice of theformulation depends on how well it adsorbs the protein components of the vaccine.Traditionally, all of the adjuvant effects of alum have been attributed to its ability to prolongantigen exposure to the immune system. It is now clear, however, that alum can berecognized by the innate immune system leading to multiple downstream effects including,perhaps, its ability to act as an immunological adjuvant.

Alum elicits a type 2 inflammatory response, characterized by the accumulation ofeosinophils at the site of injection in vivo, and this may contribute to the effects of alum onspecific immune responses (2). The eosinophils increase MHCII levels and signaling in Bcells via IL-4 and promote early IgM responses. Alum also induces the conversion ofmonocytes into antigen presenting dendritic cells (DCs) (3).

A number of studies have investigated how alum achieves these inflammatory effects. It isknown that engagement of toll-like receptors (TLRs) by their ligands, such as LPS,stimulates innate immunity and has powerful adjuvant effects on specific immuneresponses(4). However, several studies have shown that alum's ability to act as an adjuvantdoes not depend on either MyD88 or TRIF, adaptor molecules in the signaling pathways thatact downstream of TLR ligation(5,6).

Other pattern recognition receptors such as the NOD-like receptors (NLRs) can alsopromote innate responses that, in turn, stimulate specific immunity (4) (7-10). Uponactivation, members of the NLR family, such as NLRP3, form complexes with ASC andpro-caspase-1. The complex formed by these molecules is referred to as the inflammasome.The NLRP3 inflammasome is activated by a number of materials, including alum (11-15).Its activation may be caused directly by the material in question, or indirectly via theproducts of cell damage induced by the material. Such products include uric acid crystalsand/or enzymes released by lysosomes in damaged cells (14,16). Whatever the cause ofinflammasome activation, the consequences include production of active caspase-1 thusconversion of inactive precursor cytokines of the IL-1 family, including IL-1β, IL-18 andIL-33, to their active forms (17). Since these cytokines are potent stimulators of adaptiveresponses in some contexts (7-10), the NLRP3 inflammasome is an attractive candidate asand intermediary of alum's adjuvant effects.

Several reports have in fact come to that conclusion, showing that the ability of alum toinduce migration of antigen loaded DCs to the LN (3) or to increase antibody production(12,13), or even to induce cellular infiltrates (3,12) is greatly reduced in animals that lackNLRP3 or other components of the inflammasome, ASC or caspase-1. Moreover, somestudies have shown that uricase inhibits the effect of alum on antigen presentation and T cellpriming (3), suggesting that uric acid is involved in the activation of the NLRP3inflammasome by alum. However, although it is universally agreed upon that alum's abilityto induce IL-1β production depends on the NLRP3 inflammasome (3,12-15), some studieshave found no role for NLRP3 in the ability of alum to enhance IgG antibody responses(3,15). Such results are supported by the early findings, mentioned above, that MyD88, arequired intermediary in the signaling pathways of IL-1-related cytokines, is not needed foralum to improve antibody responses(5,6).

Here, we decided to study the effects of alum on innate and specific immunity in moredepth, to find out which cells respond most immediately to the introduction of alum into the

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body and which of the effects of alum depend on the NLRP3 inflammasome. Our resultsshow that many proinflammatory cytokines and chemokines are rapidly produced in vivoafter exposure to alum. Eosinophils, neutrophils, monocytes, NK cells, NKT cells and DCsare rapidly recruited to the site of injection. Simultaneously, mast cell, macrophage and Bcell numbers at the site of injection fall. The recruitment of eosinophils was promoted byboth macrophages and mast cells, the latter cells via their alum-induced production of IL-5and histamine, but did not require the inflammasome component, caspase 1.

Alum not only induced enhanced endogenous CD4 T cell responses and antibody productionbut also enhanced CD8 T cell priming. None of these effects of alum on the specific immuneresponse required macrophages, mast cells, or eosinophils, or, most significantly, theinflammasome components, NLRP3 and caspase 1. Only IL-1β production was affected bythe absence of inflammasome activity. Thus, although mast cells and macrophages are earlysensors of alum, and in spite of the fact that these cells make many stimulatory substances inresponse to alum, they are not required for alum's adjuvant effects. Furthermore, althoughthe NLRP3 inflammasome is required for optimal IL-1β production following exposure toalum in vivo, alum promotes its effects on CD4 and CD8 T cell priming and enhancingantibody responses by mechanisms that are independent of the inflammasome.

Materials and MethodsMice

Wild-type (WT) C57BL/6 (B6), WT BALB/c, CCR3-/-, 5-Lipoxygenase-/-, 15-Lipoxygenase-/-, GATA1Δ and W/+ and Wv/+ breeder mice were purchased from TheJackson Laboratory (Bar Harbor, ME). B6 IL-4 reporter (4Get) mice were obtained from Dr.Richard Locksley (University of California at San Francisco, CA). Phil Tg mice wereprovided by Dr. Jamie Lee (Mayo Clinic, Scottsdale, Arizona). BLT1-/- mice developed byDr. Bodduluri Haribabu (James Graham Brown Cancer Center, Louisville, Kentucky) andIL5-/- mice were obtained from Dr. Erwin Gelfand at National Jewish Medical Center(NJMC), Denver CO. Caspase-1 +/- mice generated by Dr. Richard Flavell were providedwith permission by Dr. Kenneth Rock (University of Massachusetts Medical Center, Boston,MA) and were bred in house to generate caspase-1 +/-, +/+ and -/- mice for experiments.NLRP3-/- mice, backcrossed for over 9 generations onto a B6 background, were obtainedfrom Dr. Richard Flavell (Yale University, New Haven, CT). All animals were housed andmaintained at the Biological Resource Center at NJMC in accordance with the researchguidelines of the Institutional Animal Care & Use Committee.

Antibodies and ReagentsAlum was precipitated in the laboratory as previously described (18) and endotoxin levels inthe material were determined to be <1ng/ml using the Limulus amebocyte lysate assay(LAL) (Endpoint Chromogenic LAL, Lonza). Alhydrogel was purchased from AccurateChemical and Imject from Pierce and endotoxin levels were determined to be <1ng/injectionusing the LAL. The following monoclonal antibodies were purchased from BD Biosciences:PE αCD19 (1D3), PE αNK1.1 (PK136), FITC αCD11c (HL3), PE αSiglec F (E50-2440),PE-Cy7 αCD117 (2B8), AF647 αCCR3 (83103), PerCP αGr1 (RB6-8C5), APC αTCRβ(H57-597), APC-Cy7 αCD4 (GK1.5), PerCP αCD8 (536.7), and FITC αCD62L (MEL-14).FITC αFcERI (MAR-1), PB αF480 (BM8), PB αB220 (RA3-6B2), and PE-Cy7 αCD44(IM7) were purchased from eBioscience. PE labeled αGalCer mouse CD1d tetramers wereproduced as previously described in the laboratory of Dr. Laurent Gapin (19). PE labeledIAb/3K tetramer and APC labeled Kb/SIINFEKL tetramers were produced as described inour laboratory (20,21). 3K-Ova was generated using the Imject Maleimide Activated Ova kitfrom Pierce Biotechnology and a cysteine linked 3K peptide (FEAQKAKANKAVDGGGC)

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purchased from Genemed. Endotoxin free ovalbumin protein was isolated as previouslydescribed (22) by and provided generously by Ross Kedl at NJRC. The Proteome ProfilerMouse Cytokine Array Panel A kit was purchased from R&D Systems. Cobra venom factor(CVF) from Naja naja kaouthia was purchased from Calbiochem. Clodronate and PBScontaining liposomes were synthesized using Cl2MDP (clodronate) a gift of RocheDiagnostics, Phosphatidylcholine, obtained from Lipoid GmbH, (Ludwigshafen,Germany)and cholesterol purchased from Sigma, as previously described (23). J113863 andUCB35625 were purchased from Tocris Bioscience. Neutralizing IL-5 monoclonal antibodywas purified from TRFK5 hybridoma supernatants (24). WEB2086 and CV-3968, werepurchased from Biomol. Pyrilamine and famotidine, and G-200 Sephadex beads werepurchased from Sigma. Diphtheria toxoid and toxin were both purchased from ListBiological Laboratories, Inc.

Injections2-5 mg of Alhydrogel or, in some experiments, alum precipitated in our laboratory wereinjected i.p. or i.m.. We did not notice significant differences between the innate responseinduced by these two different formulations of alum at 18 or 24 hours (data not shown). 50μlof the 7mg/ml liposomal clodronate suspension was injected i.p. 24 h prior to alum or PBSinjection. In some experiments mice were immunized i.p or i.m (into the hind calf muscle)with 10μg 3K-Ova or diphtheria toxoid precipitated in 2mg Alhydrogel or Imject alum asnoted in the figure legends. For treatment of mice with J113863 or UCB35625 inhibitors,mice were injected i.p with 10mg/kg of each inhibitor 2 h prior to injection with either PBSor alum. For in vivo neutralization of IL-5, we injected 500μg of αIL5 antibody i.v. 24 hprior to injection with PBS or alum. Complement depletion was induced by three i.p.injections of 4 U of CVF at 12 h intervals with the last injection 12 h before injection ofalum. This treatment resulted in ∼90% depletion of complement. Famotidine (200μg/mouse)and pyrilamine (100μg/mouse) were injected into mice i.p. 1 h prior to injection of PBS oralum. For inhibition of PAF, mice were treated 1 h prior to PBS or alum treatment withWEB2086 (100μg/mouse) or CV-3968 (100μg/mouse) i.p.

Cell preparationPeritoneal cells and in some experiments, spleens, were harvested into BSS. In someexperiments blood was harvested by cardiac puncture and sera stored at −20° C untilanalysis. For analysis of CD4 and CD8 cells, spleens were harvested 9 d after immunizationand processed into single cell suspensions using nylon mesh. Red cells were lysed usingammonium chloride and nucleated cells enumerated using a Coulter Counter.

Flow cytometryPeritoneal cells were incubated with 2.4G2 hybridoma supernatant (αFcγRI/II) and stainedusing antibodies against the cell surface markers indicated in the figure legends. Splenocyteswere stained as described previously (18) with PE labeled IAb/3K and APC labeled Kb/SIINFEKL tetramers, APC-Cy7 αCD4, PB αB220, PB αF480, and PE-Cy7 αCD44 andwells were washed and analyzed on a Cyan Flow Cytometer using Summit Software(DakoCytomation). After data acquisition, data was analyzed using FlowJo software (Tree-Star Inc).

Analysis of cytokines and chemokinesMice were injected and peritonea washed with 1ml BSS. Cells were spun down and fluidwas passed through a 0.2μm syringe filter. Fluid was analyzed using the Proteome Profilerkit (R&D systems) or the histamine enzyme immunoassy (Immuno Biological Laboratories,Inc.) according to the manufacturer's instructions.

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ELISAs96-well Immulon plates (Thermo) were coated with ovalbumin or diphtheria toxin at 10μg/ml in PBS. Plates were washed using ELx405 autoplate washer (BioTek Instruments) andthen blocked with 10%FCS/PBS for 2hr RT. Plates were washed and antibody serumsamples diluted in 10%FCS/PBS were added to the plates. To determine relative units weused a positive control pooled serum sample from B6 and Balb/c mice that contained ova ordiphtheria toxin specific Th2 and Th1 antibody isotypes. The samples were incubatedovernight at 4°C. Plates were washed and alkaline phosphatase conjugated anti-IgG1 (X56),anti-IgG2a/c (R19-15), biotinylated anti-IgE (R35-119) (Pharmingen) or anti-kappa(Southern Biotech) detection antibodies were added for 2hr RT. For IgE alkalinephosphatase conjugated streptavadin was added for 1hr RT. Substrate (p-nitrophenylphosphate) diluted in glycine buffer was added to each well and 405nm absorbance valueswere collected on Elx808 microplate reader.

StatisticsStatistical significance between selected groups was determined using Student's two tailed Ttest and in some experiments, a one-way ANOVA was performed with Bonferonni post hoctest. All statistical analysis was done using GraphPad Prism software version 4.

ResultsAlum induces rapid accumulation of innate IL-4 expressing cells that are primarilycomposed of eosinophils

Alum usually generates Th2-related specific immune responses. In part this bias is causedby alum's ability to induce production of IL-4. IL-4 made in response to alum is not requiredto drive Th2 responses but rather, acts by suppressing Th1-related phenomena, leading to amore polarized immune response (18,25). Because of this we tracked the appearance of type2 innate cells after alum injection. To follow inflammation following exposure to alum, weused a peritoneal model in order to be able to follow cell infiltrates and production ofsoluble mediators in the peritoneal cavity (18). Alum was into mice that express GFP froman internal ribosomal entry site immediately downstream of the IL-4 stop site (4Get mice)(26,27). Previous experiments using these mice, showed that Gr1intIL-4 expressing cells arerecruited to the peritoneal cavity in response to i.p. injection of alum by 24 hours (18).

To characterize the nature of innate IL-4 expressing cells that respond to alum, 4Get micewere injected i.p. with alum and IL-4+ (GFP+) cells at the site of injection examined 24hours later for markers of eosinophils (Siglec F), mast cells (cKit) and basophils (FcERI)(Figure 1A). In PBS injected mice, most of the IL-4 expressing cells in the peritoneal cavitywere mast cells (Siglec Fnegckit+) and only a few were eosinophils (Siglec F+ckitneg) (Fig.1A, left column, Fig. 1C). The mast cells dropped in numbers after alum injection (Fig. 1A,right hand column and Fig. 1B). In alum injected mice, almost all of the IL-4 expressingcells were eosinophils (Fig. 1A) and these cells co-expressed low levels of Gr1 (data notshown). Consistent with previous findings (2, 3), the numbers of eosinophils rose after alumadministration, beginning their increase a few hours after the disappearance of the mast cells(Fig. 1B). Basophils also express IL-4 and have been described to play a role in Th2responses (28-32). We did not find any basophils (FcER1+ckitneg) in the peritoneum prior toor following injection of alum (Fig. 1C). Thus, almost all of the innate IL-4+ cells in alum-induced inflammation had staining characteristics of eosinophils (Siglec F+ckitneg) (Fig. 1A,right column).

Further examination of the inflammation induced by alum confirmed that, as describedpreviously (3), the numbers of peritoneal macrophages (F4/80hi) fell following alum

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injection (Fig. 1A, B), and with the same kinetics as the mast cells (Fig. 1B). As predicted(3), the numbers of neutrophils (Gr1hiSSChi), Gr1 intermediate (Gr1int) Ly6ChiSSClo

monocytes (Supplemental F. 1), and DCs (Supplemental Fig. 2B) all increased in responseto alum. In addition, NK cells and NKT cell also increased in number (Supplemental Fig.2C,D), while T cell numbers were unchanged and B cell numbers dropped modestly(Supplemental Fig. 2A and C).

Induction of multiple chemokines and cytokines occurs within hours of exposure to alumTo characterize the soluble factors released during the inflammatory response induced byalum, we used a multiplex membrane bound ELISA assay to test the peritoneal fluid frommice injected previously with PBS or alum. A time course showed that the amounts ofinduced factors peaked 4 hours after injection (data not shown). In alum injected mice, wedetected increased levels of 14 soluble factors including the Th2-associated cytokine IL-5(Fig. 2A). No Th1- or Th17-associated factors were detected over background levels (Fig.2A). In addition to IL-1β (Fig. 2A and (11)), several other inflammation associatedcytokines were elevated after alum injection, including IL-1ra and IL-6, while levels ofIL-1α, TNFα, and IL-10 remained unchanged (Fig. 2A). In addition to MCP-1, KC andeotaxin, which have been shown to increase after alum injection (Fig. 2B and (11)), aluminduced increased levels of additional cell-attractive proteins, including C5a, MIG, IP10,and MIP2 (Fig. 2B).

Macrophages and mast cells promote type 2 inflammationThe rapid disappearance of macrophages and mast cells after alum injection may result fromactivation, adherence to the peritoneal cell wall, mast cell degranulation, or cell death as hasbeen observed in macrophages exposed to alum in vitro (14), and suggests that these cellsmay respond rapidly to alum and participate in the production of cytokines followingexposure to alum. To test the idea, macrophage or mast cell deficient mice were injectedwith alum and the appearance of cytokines and chemokines in the peritonea analyzed 4hours later. To deplete macrophages, WT B6 mice were injected with the macrophagedepleting agent, clodronate liposomes (33). As expected, liposomal clodronate was highlyefficient at depleting macrophages, but not mast cells, DC, neutrophils, or eosinophils(Supplemental Fig. S3). To determine the role of mast cells, we used mast cell deficient W/Wv mice.

Of the cytokines and chemokines that were elevated significantly above controls by aluminjection (Fig. 2), the amounts of IL-1β, IL-1Ra, IL-6, and the chemokine, eotaxin, were allsignificantly decreased in clodronate liposome treated mice (Fig. 3, left column). However,to our surprise, all of these factors were also decreased in alum injected mast cell deficientmice. This result suggests that these factors are potentially made by both macrophages andmast cells in response to alum or that interactions between these cell types promote optimalcytokine production. In contrast, levels of IL-5, IL-16, G-CSF, KC, and MIP2 were alldecreased in mast cell deficient but not macrophage depleted mice (Fig. 3, middle column).Finally the levels of MIG, IP10, KC and MCP-1 were not significantly affected by absenceof either macrophages or mast cells (Fig. 3, right column) suggesting that they may be madeby other cell types including endothelial cells, fibroblasts, neutrophils and/or DC (34). Theseresults suggest a complex inflammatory response to alum mediated by multiple cell typesthat culminates in the production of a wide range of soluble mediators.

Alum promotes mast cell mediated recruitment of eosinophils via IL-5 and histamineIn mast cell deficient mice, we found a partial reduction in the number of eosinophilsrecruited in response to alum, compared to the numbers in WT mice (Fig. 4A). IL-5, whichis decreased in the mast cell deficient mice (Fig. 3), is known to play a role in the

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recruitment of eosinophils in response to other Th2 driving substances such as helminth eggs(35,36). Eosinophil recruitment did not occur in IL-5-/- mice given alum (data not shown).However, IL-5 is needed for eosinophil development as well as optimal eosinophilmovement (35,37) and therefore lack of IL-5 could have reduced eosinophil accumulationbecause of reduced numbers of the cells in IL-5-/- mice. To test this, we transiently depletedIL-5 in vivo. Mice were injected with neutralizing anti-IL-5 antibody 1 day before theinjection of PBS or alum, and the effect of these treatments on eosinophil accumulation wasmeasured 24 hours later. We found that blocking IL-5 resulted in a reduction in theaccumulation of eosinophils in response to alum (Fig. 4B). In contrast, anti-IL-5 did notfurther reduce the small numbers of eosinophils that appear in response to alum in W/Wvmice (Fig. 4D). These data suggest that, like helminth eggs (36), alum attracts eosinophilsby promoting mast cell dependent IL-5 production.

Mast cell degranulation and the release of histamines have been shown to be involved in therecruitment of inflammatory cells in response to implanted biomaterial particles (38) andhave been implicated in eosinophil recruitment as well (39). Accordingly, we were able todetect histamine release in the peritoneal fluid of alum injected WT mice within 10 minutesof alum injection but not in similarly treated mast cell deficient W/Wv mice (Fig. 4C). Toevaluate whether histamine production had any impact on eosinophil recruitment, we used acombined treatment of famotidine and pyrilamine to block signaling through the H1 and H2receptors. Treatment of mice with these antihistamines 2 hours prior to exposure to alumresulted in a partial reduction in the total number of eosinophils recruited in response toalum particles in WT but not mast cell deficient W/Wv mice (Fig. 4D). Treatment of micewith αIL-5 antibody together with antihistamines did not have an additive effect (Fig. 4D).Thus mast cells, IL-5 and histamine all promote eosinophil recruitment in response to alumand appear to be acting together.

Despite the reduced influx of eosinophils in the absence of mast cells, IL-5 or histaminereceptor signaling, there still were increased numbers compared to those in PBS injectedcontrol mice, therefore an alternative pathway must promote eosinophil responses to alum.We observed a marked reduction of eosinophils in mice depleted of macrophages (Fig. 4E),suggesting that they also respond to alum by promoting eosinophil recruitment. Surprisingly,several factors expected to play a role, including eotaxin, platelet activating factor,complement, and leukotriene B4, were not required for the alum induced eosinophilresponse (Supplemental Figs. 4, 5). Therefore, besides the mast cell/IL-5/histamine pathway,there are other pathways involved in the eosinophil response to alum. Macrophages maypromote eosinophil recruitment through as yet undefined factors, or perhaps several solublefactors tested here, may act redundantly.

Mast cells, macrophages and eosinophils are not required for enhanced priming of T cells,Th2 bias or antibody responses to alum

Our data suggest that mast cells and macrophages sense the presence of alum and respondby producing a number of inflammatory factors, chemokines and cytokines. Because solublefactors, such as IL-1β, have been implicated in alum's adjuvant effects on B and T cells(11-13), it was possible that this is an important early step in alum's adjuvant activity invivo. To test this idea, we first tracked CD4 and CD8 T cell responses in untreated orclodronate liposome injected WT (Kit+/+) or W/Wv mice. To avoid the artifacts that mightoccur if transferred T cells expressing transgenic TCRs are used (40,41), we followedendogenous antigen specific CD4 and CD8 T cell responses using peptide/MHC tetramers.In order to do this we immunized mice with the 3K peptide (21,42) conjugated to ovalbumin(ova) protein (3K-ova), and measured endogenous CD4 T cell responses with 3K/IAb

tetramers and CD8 T cell responses to an ova peptide/Kb with SIINFEKL/Kb tetramers(21,42). We found that injection of WT mice with 3K-ova adsorbed to alum promoted

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endogenous CD4 T cell responses (18). Moreover, this antigen/adjuvant combination also,to our surprise, greatly enhanced CD8 T cell priming (Fig. 5B,C). We found no difference inthe percentage (Fig. 5A,B) or total number (Fig. 5C) of antigen-specific CD4 or CD8 T cellsprimed by alum in mice lacking macrophages, mast cells, or both cell types. Likewise,absence of mast cells had no effect on the ability of alum to induce total Ig and IgG1primary or secondary responses to ova, or IgG2c secondary responses to the same antigen(Fig. 5E).

Similar experiments were performed in B6 or B6 4Get mice in which macrophages weredepleted with clodronate liposomes. Lack of macrophages had no effect on the size of theCD4 and CD8 T cell response (Fig. 5C), the Th2 nature of the response (Fig. 5D) or the sizeand nature of the antibody response to 3K-ova plus alum (Fig. 5E).

Previous results suggest that IL-4 is not required to initiate Th2 responses to alum, but maysuppress Th1 responses (25). Treatment of mice with a Gr1 specific antibody had a similarbut partial effect on the bias of the response (18). Although Gr1 is expressed at low levels oneosinophils, it is a non-specific marker and may bind to both Ly6G and Ly6C expressingcell types. Thus, we wanted to examine whether adaptive responses were affected ineosinophil deficient mice immunized with alum. We first examined T cell priming andantibody responses in IL-5 deficient mice and found no significant effect on the totalnumber of antigen specific CD4 or CD8 T cells primed compared to WT control animals(Fig. 6A). In addition, we found no effect on the levels of total anti-ova antibody in IL-5deficient mice or in the levels of IgG1 or IgG2c (Fig. 6B), suggesting that eosinophilsneither impact alum's ability to act as an adjuvant nor the bias of the response induced. Wealso saw no decrease in the levels of ova specific Ig, IgG1 or IgG2c antibody responses ineosinophil deficient Phil mice (Fig. 6C). These mice express diphtheria toxin under thecontrol of the eosinophil peroxidase promoter and are congenitally deficient in eosinophils(43). Finally, we saw no decrease in ova specific Ig, IgG1, IgG2a, and IgE levels ineosinophil deficient GATA1Δ mice that contain a mutation in GATA1 that preventseosinophil differentiation (44) compared to WT controls (Fig. 6D). The only effect observedwas an overall increase in IgE levels in the GATAΔ mice (Fig. 6D).

Thus, macrophage and mast cell sensors appear to play a role only in the inflammatoryresponse to alum and do not participate in the ability of alum to act as an adjuvant for thespecific immune response in vivo. While the induction of eosinophils by alum has beenshown to promote early IgM responses to alum and alter MHC II mediated intracellularsignaling (45), they are unlikely to plan an important role in antibody responses relevant toimmunization.

The inflammatory response to alum does not require caspase-1Recent work by others (11,12,15,46,47), and our detection of IL-1β in peritoneal exudates(Fig. 3), suggests that alum can activate caspase-1 rapidly in response to alum. Other workhas indicated that products of this activation, such as IL-1β family members mightcontribute to the downstream consequences of alum injection perhaps via effects on DCs(11,12), while others have found no role for the inflammasome in the adjuvant activity ofalum (15). To test whether the inflammasome promotes recruitment of inflammatory cellsincluding those likely to be required for alum's adjuvant effects, we injected caspase-1-/-mice with PBS or alum and examined the numbers of resident and inflammatory cells intheir peritoneal cavities 18 hours later. Absence of caspase-1 had no impact on the ability ofalum to reduce the numbers of macrophages and mast cells in the peritoneal lavage, or onthe alum-induced increases in the numbers of eosinophils, neutrophils, monocytes or DCs inthe peritoneal cavity (Fig. 7A). This was observed even though IL-1β production in responseto alum was drastically reduced in these same animals (Fig. 7B). This result was surprising,

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considering the previous report that accumulation of granulocytes, monocytes and DC aredependent upon NLRP3, an adaptor which is thought to mediate its effects by promotingactivation of caspase-1 (11).

Together, these data strongly suggest that the inflammatory response does not requirepathways dependent on caspase-1. The discrepancies between these results and ours are notclearly understood, although they may suggest that another pathway downstream of NLRP3could be playing a role in early inflammatory responses to alum (11).

The adjuvant effects of alum on T and B cell responses are not dependent on NLRP3 orcaspase-1

To examine, in our own hands, the issues surrounding the controversy about the role of theNLRP3 inflammasome in alum induced responses (11-13,15), we immunized NLRP3-/- andcontrol WT mice and caspase-1+/+ and +/- littermate control mice i.p. with 3K-ovaadsorbed to alum and followed their endogenous antigen-specific CD4 and CD8 T cellresponses in the spleen and mediastinal LN (the LN that drains the peritoneal cavity (3)),using MHC/tetramers (Fig. 8A-F). Alum improved responses to its accompanying antigensince, as expected, T cell responses were much smaller in animals given antigen withoutalum (Fig. 8A,B). The absence of NLRP3 or caspase-1 had no effect on the magnitude ofCD4 T cell and CD8 T cell responses to antigen plus alum, either in spleen (Fig. 8A,B,E,F)or the mediastinal LN (Fig 8C,D and data not shown). In addition, we assayed antigenspecific antibodies in the sera of these mice and found that caspase-1-/- mice had levels ofanti-ova IgG1 antibody that were similar to those of heterozygous or WT mice (Fig. 8G).Levels of antigen specific IgG2c were undetectable at this time in all the mice (data notshown). Because these experiments were done very early in the immune response in order tolook at T cell priming, the possibility remained that although early IgG1 responses wereunaffected, long term antibody responses may be impacted in mice unable to activatecaspase-1. However, when we followed antigen specific antibody responses to ova in ova/alum injected caspase-1 +/+, +/- and -/- mice at later time points and during the secondaryresponse, we were unable to see any significant difference in the antibody response (Fig.8H).

It has been suggested that different laboratories may find different requirements for theNLRP3 inflammasome because they use different alum formulations. To check this, wefollowed antibody responses to antigen adsorbed to Alhydrogel or Imject alum, and stillfound no requirement for caspase-1 (Supplemental Fig. 6A). Likewise, the site ofsensitization did not govern whether or not caspase-1 were required, since responses toantigen plus alum were unaffected in mice immunized ip followed by intranasal challengewith antigen (Supplemental Fig. 6A,B) or after intramuscular injection of antigen plus alum(Fig. 9A).

Although, consistent with published reports (5,6), we found no effect on alum's ability toincrease either T cell priming or antibody production in MyD88 deficient mice (data notshown), it was possible that low levels of endotoxin present in our antigen preparation(<1.3ng/injection) could influence our results, and could provide an explanation for thediscrepant results regarding the role of the caspase-1 and NLRP3 in the adjuvant activity ofalum. To test this, we obtained ova that had no detectible levels of endotoxin by the LALassay (endotoxin <0.008ng/injection) for immunization. Levels of IgG1 antibodies inducedagainst ova were similar in WT and caspase-1 deficient mice injected with endotoxin freeova plus alum (Fig. 9A,B).

To check that our findings reflected the response to an antigen used in man, we comparedprimary and secondary antibody responses to diphtheria toxin in WT or NLRP3-/- mice

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immunized with diphtheria toxoid adsorbed to alum. To control for any potential adjuvantproperties of the toxoid alone, we also compared antibody responses induced in the absenceof alum. Alum adsorbed diphtheria toxoid greatly enhanced antibody production againstdiphtheria toxin compared to injection of diphtheria toxoid alone. We confirmed thatequivalent amounts of antibody that recognized diphtheria toxin were circulating in WT andNLRP3 deficient mice that had been immunized with diphtheria toxoid adsorbed to alum(Fig. 9E).

Thus overall, we could not find any compulsory role for the inflammasome in the ability ofalum to improve T and B cell responses to alum.

DiscussionAlum is a mysterious adjuvant and, in spite of a significant amount of work, many questionsabout the effects of this material remain. Others have previously shown that macrophagesand DCs can respond directly to the presence of alum in vitro through activation ofcaspase-1 (11,12,47). The work described here confirms the finding that macrophages aretargets in vivo and extends them to include responses of mast cells, suggesting that boththese cell types have the machinery needed to detect alum particles. In addition, our resultsindicate that other resident cells, perhaps DCs or stromal cells, may also have this capacity,since removal of both macrophages and mast cells from the site of alum injection did notcompletely ablate responses to the material.

How macrophages and mast cells detect alum is an intriguing question and in fact, alum maybe detected in several, non-mutually exclusive ways. A number of in vitro experiments haveshown that alum activates the NLRP3 inflammasome in macrophages which, in turn,activates caspase-1 and consequent production of IL-1β. In vitro, but not in vivo, alum'sability to induce IL-1β production requires the additional stimulus of LPS (11,12). Theactivation of the NLRP3 inflammasome in macrophages by alum requires intact phagocyticmachinery (12) and may involve K+ efflux (12) and may be initiated by so-called frustratedphagocytosis (48), or alternatively, by phagocytosis followed by fracture of lysosomes,release of lysosomal enzymes into the cytosol (14). Some data implicate uric acid inactivating the inflammasome and production of IL-1β in vivo (3,47).

The experiments described in this manuscript show that eosinophil, neutrophil, DC andmonocytes cellular infiltrates induced in response to aluminum hydroxide are unaffected byabsence of the enzyme, caspase-1, needed to generate IL-1β. The accumulation of one celltype, the eosinophil is promoted in part by mast cell derived IL-5 and histamine (37).Surprisingly, eosinophil accumulation did not require any of the many chemokines,including eotaxin, that are secreted in response to alum administration. Histamine isproduced by activated mast cells and basophils, and, since we found so few basophils inperitoneal cavities, this suggested an additional role for mast cells. Eosinophils responddirectly to histamine via H4 histamine receptors, while H1 and H2 receptors are expressedby the endothelium (49). The experiments described here showed that eosinophilrecruitment was inhibited by antagonists of histamine H1 and H2 receptors. Thus, in thiscase the histamine probably acted by promoting vascular leakiness, allowing eosinophilmigration into the peritoneal cavity, rather than by affecting eosinophils directly.

The processes by which mast cells detect alum are not clear. It is known that alum activatesseveral sets of serum enzymes, including those of the complement cascade (50) and thesemay activate cells such as macrophages and mast cells. However, although mast cells areactivated to release histamine and other factors by complement fragments (51,52), we hereshow that depletion of complement does not affect the appearance of eosinophil exudates in

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response to alum, suggesting that this is not the means whereby mast cells, at least, detectalum.

Despite the ability of mast cells and macrophages to respond to alum particles, theirpresence has no impact on the downstream adaptive responses initiated by alum. In addition,eosinophils are not required for T cell priming, and do not enhance the magnitude or changethe overall nature of the antibody response to alum. Thus, although eosinophils express IL-4,they play no role in the suppression of Th1 associated isotypes that has been observed inIL-4 deficient or Gr1 depleted mice (18,25). Perhaps T cells themselves, or basophils(28-32), are the important source of IL-4 that mediates this effect in vivo.

We, like some (15), but not all (3,12,13) have completely failed to find a connectionbetween inflammasome activation and the adjuvant effects of alum. Our data suggest thatthis negative result is not due to contamination of our preparations with LPS, or with thetype of alum we use, or with the timing or route of alum administration. Thus it seems thatresponses to antigen plus alum have a variable requirement (in our case, no requirement) forthe NLRP3 inflammasome.

There are three, not mutually exclusive, explanations for the discrepant results. One is thatsubtle experimental differences, such as the precise status of the mice, allow the presence orabsence of factors that can substitute for the products of the NLRP3 inflammasome.Assuming that are IL-1-related cytokines are responsible for mediating adjuvant effects,what could these compensating factors be? They are unlikely to be products of alum-activated macrophages, such as IL-6, since macrophages, the major source of alum-inducedIL-1β and IL-6 (Fig. 5) are not involved in the adjuvant activity of alum.

Another possibility is that alum enhances immune responses via redundant effects on DCsand is enhanced but not required by the activity of the NLRP3 inflammasome (3). Thus, inone study, alum enhanced trafficking of antigen-bearing monocytes and antigen presentationto a fairly large number of transferred T cell receptor transgenic CD4 T cells in draininglymph nodes, a process that was abrogated by neutralization of IL-1β (3,11). Despite theseeffects on antigen presenting cells, however, lack of NLRP3 had no negative effect onantigen specific IgG levels in this study (11), a result that is consistent with our data.Perhaps the dependence of specific immune responses on the NLRP3 inflammasomedepends on whether or not APC activity is limiting. In animals containing large numbers ofnaïve antigen specific T cells, APCs may be limiting and the large immune response maythen be dependent on NLRP3 activity for an optimal response. In animals containing smallnumbers of endogenous antigen specific T cells, NLRP3-mediated stimulation of APCs maynot be needed and alum may act through additional pathways to support T cell priming andT dependent antibody responses.

Finally, assuming IL-1β or other related cytokines are the required product of the NLRP3inflammsome, perhaps they can be produced via other redundant pathways. IL-1β cytokinecan be produced by additional enzymes, which include proteases such as proteinase-3,elastase and granzyme A (53). This latter possibility seems unlikely given that IL-1β levelsare drastically reduced in alum-injected macrophage deficient or caspase-1 deficient mice(Figs. 3, 7), yet alum's adjuvant activity in these animals is unabated.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

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AcknowledgmentsWe would like to thank the NIH core facility for supplying the biotinylated recombinant CD1d protein and Dr.Laurent Gapin for the assembled αGalCer CD1d tetramers. We would also like to thank Drs. Peter Henson andLeonard Dragone, and the members of the Kappler Marrack laboratory for their intellectual contributions to thisproject, and especially to Janice White, Frances Crawford, Tibor Vass, and Alexandria David for technical support.We would like to thank Dr. Richard Locksley for the IL-4 reporter mice, Drs. Richard Flavell and Ken Rock for theCaspase-1-/- and NLRP3-/- mice, and Drs. Bodduluri Haribabu and Erwin Gelfand for the BLT1-/- mice.

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Figure 1. Characterization of the innate response 24 hours after injection with alumA. 4Get mice were injected with either PBS or alum i.p. and peritoneal cells harvested andstained 24 h later. IL-4 expressing (GFP+) cells from PBS or alum injected mice wereanalyzed for expression of the eosinophil marker, Siglec-F, and mast cell marker, ckit andtotal cells were analyzed for expression of F4/80. B. The kinetics of appearance ofeosinophils, mast cells and macrophages following injection of PBS (open circles) or alum(solid squares) are shown. Total numbers of eosinophils were determined from the percentof total cells that were Siglec-F+/IL-4+, total numbers of mast cells were determined usingthe percent of total cells expressing ckit and IL-4 and total numbers of macrophages weredetermine using the percent of total cells expressing high levels of F4/80 as in A. C. Gated

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IL-4+ cells from PBS or alum injected mice were analyzed for expression of ckit and FcERIto determine if ckitnegFcERI+ basophils were present. Scatter plots in A are representativeof 3 individual mice. Numbers on scatter plots indicate the percent of cells that fall in theindicated gates for the gated population in the sample shown. Points on line graphs indicatethe mean total number of each indicated cell type for 3 individual mice and error barsindicate SEM. Asterisks indicate a significant difference was detected between PBS andalum injected mice (p<0.05) using a Student t-test. The experiment was performed morethan 6 times with similar results.

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Figure 2. Cytokines and chemokines induced 4 hours after exposure to alumMice were injected with either PBS (open bars) or alum (solid black bars) and peritonealfluid harvested 4 hours later by lavage and assayed for the presence of cytokines (A) andchemokines (B) as described in methods. Bars indicate mean relative units of each factordetected for 3 individual mice per group. Error bars indicate SEM. The data arerepresentative of two individual experiments. Asterisks indicate a significant difference wasdetected between alum and PBS treated groups as determined by one-way ANOVA andBonferonni post-hoc test. Single asterisks indicate p<0.05 and double asterisks indicatep<0.01.

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Figure 3. Macrophages and mast cells are sensors of alum in vivoCytokines shown to be induced by alum (Fig. 2) were analyzed in kit+/+ untreated mice(WT), kit+/+ mice pretreated with clodronate liposomes (WT+Clod LS) or mast celldeficient mice (W/Wv) injected with either PBS (open bars) or alum (solid bars). The leftcolumn of graphs shows the cytokines whose levels in response to alum were significantlyimpaired in both mast cell deficient and macrophage depleted mice. The center column ofgraphs shows those alum-induced cytokines that were significantly impaired in mast celldeficient mice only. The right hand column shows that the alum-induced cytokines that wereunaffected by absence of either cell type. Bars on graphs show mean relative units of eachfactor for 3 individual mice and error bars indicate SEMs. Data are representative of 2

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individual experiments. Asterisks indicate a significant difference was detected betweensimilarly treated WT control mice by one-way ANOVA and Bonferoni post hoc test(p<0.05).

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Figure 4. Mast cells and macrophages are needed for accumulation of eosinophils in response toalumA. W/Wv mice (open bars) and Kit+/+ (WT) littermate control mice (solid black bars) wereinjected with either PBS or alum and the total number of eosinophils recruited wasdetermined as described in Fig. 1. B. B6 mice were treated neutralizing αIL-5 antibody(open bars) or rat IgG control antibody (solid black bars) and then injected with PBS oralum 24 hours later. C. Kit+/+ (WT) or W/Wv mice were injected with alum and peritonealfluid was harvested 10 min later and tested for the presence of histamine as described inmethods. Graph shows mean concentration of histamine for three mice per group in oneexperiment of 2. D. Kit+/+ (WT) or W/Wv mice were treated with either αIL-5 or anti-

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histamines, as described in the Methods section, and injected with either PBS or alum. Thetotal number of eosinophils recruited was determined as described in figure 1. E. B6 micewere treated i.p. with either PBS (-), PBS containing liposomes (PBS LS) or clodronatecontaining liposomes (Clod LS). 24 h later the mice were injected with either PBS (openbars) or alum (black bars). Data in A and B are from one representative experiment of three.Data in D is from one representative experiment of 2. Bars on graphs (B, D, E) equal meannumber of each indicated cell type × 10-3 for 3 individual mice per group. Error barsindicate SEM and asterisks indicate a significant difference was determined from similarlytreated control mice using a t-test (p<0.05).

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Figure 5. Mast cells and macrophages are not required for alum to enhance adaptive immunityA,B. WT and W/Wv mice were immunized with either nothing (Naïve), 3K-ova or 3K-ova/alum. 3K-specific CD4 T cells and SIINFEKL-specific CD8 T cells in the spleen wereanalyzed by tetramer staining 9 days later. Numbers on scatter plots indicate the percent ofgated CD4 (A) or CD8 (B) T cells that fall within the indicated gates for the sample shown.Plots are representative of 4 individual mice. C. Bars indicate the mean total number of IAb/3K+CD44hi CD4 positive cells or Kb/SIINFEKL+CD44hi CD8+ cells detected in the spleen9 d after injection for WT or W/Wv mice injected with either nothing (open bars), 3K-Ova(grey bars), or 3K-ova/alum (black bars). D. B6 4Get mice were treated with clodronateliposomes i.p. and injected with 3K-ova/alum and 3K/IAb specific CD4 T-cells in the spleen

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were analyzed 9 days after as described above. Graphs show percent and total number of3K/IAb tetramer positive cells that express GFP in untreated (open bars) and clodronateliposome treated (solid, black bars) mice. E. WT (squares) or W/Wv (circles) mice were leftuntreated (solid symbols) or injected with clodronate liposomes (open symbols). 24 hourslater all mice were immunized and boosted on days 0 and 30 with ova+alum (arrowheads).Ova specific antibody isotypes in the serum were detected by ELISA. Values on graphsindicate mean values for 4 individual mice and error bars indicate SEM. Data in A-C arefrom one representative experiment of three and data in D, E are from one representativeexperiment of 2.

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Figure 6. Eosinophils have no effect on the ability of alum to enhance T cell priming andantibody levelsWT or IL5-/- mice were injected with either nothing (Naïve, open bars), 3K-ova (grey bars)or 3K-ova/alum (black bars) and splenocytes were analyzed for tetramer staining as in Fig.5. Bars indicate the mean total number of IAb/3K+CD44hi CD4 positive cells or Kb/SIINFEKL+CD44hi CD8+ cells detected in the spleen 9 d after injection for 3 individualmice. Data is representative of 2 experiments. B. WT (solid squares) or IL5-/- (open circles)mice immunized and boosted on days 0 and 27 with ova+alum (arrowheads). Ova specificantibody isotypes in the serum were detected by ELISA. C. Eosinophil deficient Phil Tgmice (Phil, open circles) or WT littermate controls (WT, solid squares) were injected and

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antibody responses analyzed as in B. D. WT Balb/c (solid squares) or eosinophil deficientGata1Δ (open circles) mice were immunized and boosted with ova/alum on day 0 and 40and antibody responses analyzed by ELISA. Values on graphs indicate mean values for 4individual mice in B,C and for 5 individual mice in D. Error bars indicate SEM.

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Figure 7. Induction of eosinophil recruitment by alum in caspase-1 deficient mice is normalWT (Cas1+/+) and caspase-1 deficient (Cas1-/-) mice were injected with either PBS (openbars) or alum (solid black bars). Total numbers of macrophages, mast cells, eosinophils,neutrophils, monocytes and DC were determined by flow cytometry as in Fig. 1 and Fig.S1,2. B. WT (Cas1+/+, solid bars) or caspase-1 deficient (Cas1-/-, striped bar) mice wereinjected with either PBS (open bars) or alum (filled bars) and IL-1β levels were determinedfrom the peritoneal fluid obtained from the mice 4 hours later by ELISA. Bars on graphs areequal to the mean number of cells detected (×10-3) in the peritoneal cavities of 3 individualmice per group. Error bars indicate SEM and data is representative data from oneexperiment of 2. Asterisks indicate a significant difference (p<0.05) as determined by t-test.

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Figure 8. NLRP3 and Caspase-1 are not required for alum to enhance endogenous CD4, CD8 Tcell priming and antibody responses to adsorbed antigenA-D. Graphs show total numbers of 3K/IAb CD4 (A,C), and SIINFEKL/Kb CD8 (B,D)CD44hi T cells in the spleen and mediastinal LN 7 days after injection of nothing (Naïve),3K-ova, or 3K-ova/alum (solid, black bars) into WT or NLRP3-/- mice. E,F. WT (Cas1+/+),heterozygous (Cas1+/-) and homozygous caspase-1 deficient mice (Cas1-/-) were injectedwith either nothing (open bars), 3K-ova (hatched bars), or 3K-ova adsorbed to alum (solidblack bars). Bars indicate the mean total number of IAb/3Ktet+CD44hi CD4 positive cells orKb/SIINFEKLtet+CD44hi CD8+ cells detected in the spleen 9 d after injection. G. Graphshows the amount of Ova specific IgG1 detected in mice in A and B. H. WT (Cas1+/+),heterozygous (Cas1+/-) and homozygous caspase-1 deficient mice (Cas1-/-) were injectedand boosted with alum on day 0 and 10 with ova/alum. Graphs show the amount of ova-specific IgG1 detected on day 20. Data on bar graphs indicate mean values plus or minusSEM for 4 individual mice per group. Data shown in A-D are from a representativeexperiment of 2. Data in E-G are from a representative experiment of 3.

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Figure 9. Enhanced T cell priming and antibody responses to alum adjuvant are normal inNLRP3 deficient miceA. The graph shows the amount of ova specific IgG1 detected in WT (Cas1+/+) or caspase-1deficient mice (Cas1-/-) mice 14 days after intramuscular injection with endotoxin free ovaadsorbed to alum and, for comparison, in uninjected WT mice. B. The graph shows therelative levels of ova specific IgG1 detected in WT (Cas1+/+), caspase-1 heterozygous(Cas1+/-) and caspase-1 knockout (Cas-/-) littermates injected with ova that contains lowlevels of endotoxin (Endolo ova) or endotoxin free ova (Endoneg ova). C. The graph showslevels of circulating anti-diphtheria toxin antibody in WT or NLRP3-/- mice in miceimmunized and boosted with diphtheria toxoid alone (grey bars) or adsorbed to alum (solidblack bars) on day 10 of the primary response or on day 20 (10 days after a boost injection).Bars on graphs indicate mean values and SEM for 4 mice per group. Data are from arepresentative experiment of 2.

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alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are...

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