DTIC ADA519544: Cutting Edge: Resistance to Bacillus anthracis Infection Mediated by a Lethal Toxin Sensitive Allele of Nalp1b/Nlrp1b

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Cutting Edge: Resistance to Bacillus
anthracis Infection Mediated by a
Lethal Toxin Sensitive Allele of
This information is current as
Nalp1b/Nlrp1b

of May 10, 2010

Jill K. Terra, Christopher K. Cote, Bryan France, Amy L.
Jenkins, Joel A. Bozue, Susan L. Welkos, Steven M.
LeVine and Kenneth A. Bradley

J. Immunol. 2010;184;17-20; originally published online
Nov 30, 2009;
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doi:10.4049/jimmunol.0903114 o
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http://www.jimmunol.org/cgi/content/full/184/1/17 n
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1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED
10 MAY 2010 N/A -
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Cutting edge: resistance to Bacillus anthracis infection mediated by a
5b. GRANT NUMBER
lethal toxin-sensitive allele of Nalp1b/N1rp1b
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Terra, JK Cote,CK France, B Jenkins, AL Bozue, JA Welkos, SL LeVine,
5e. TASK NUMBER
SM Bradley, KA
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
United States Army Medical Research Insitute of Infectious Diseases, REPORT NUMBER
TR-09-118
Fort Detrick, MD
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Approved for public release, distribution unlimited
13. SUPPLEMENTARY NOTES
The original document contains color images.
14. ABSTRACT
Pathogenesis of Bacillus anthracis is associated with the production of lethal toxin (LT), which activates the
murine Nalp1b/Nlrp1b inflammasome and induces caspase- 1dependent pyroptotic death in macrophages
and dendritic cells. In this study, we investigated the effect of allelic variation of Nlrp1b on the outcome of
LT challenge and infection by B. anthracis spores. Nlrp1b allelic variation did not alter the kinetics or
pathology of end-stage disease induced by purified LT, suggesting that, in contrast to previous reports,
macrophage lysis does not contribute directly to LT-mediated pathology. However, animals expressing a
LT-sensitive allele of Nlrp1b showed an early inflammatory response to LT and increased resistance to
infection by B. anthracis. Data presented here support a model whereby LT-mediated activation of Nlrp1b
and subsequent lysis of macrophages is not a mechanism used by B. anthracis to promote virulence, but
rather a protective host-mediated innate immune response.
15. SUBJECT TERMS
Bacillus anthracis, anthrax, lethal toxin, allelic variation, macrophages, dendritic cells
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON

a. REPORT b. ABSTRACT c. THIS PAGE SAR 4
unclassified unclassified unclassified
Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std Z39-18
Bacillus anthracis
Cutting Edge: Resistance to Infection
Nalp1b/
Mediated by a Lethal Toxin Sensitive Allele of
Nlrp1b
† † †
Jill K. Terra,* Christopher K. Cote, Bryan France,* Amy L. Jenkins, Joel A. Bozue,
Susan L. Welkos,† Steven M. LeVine,‡ and Kenneth A. Bradley*,x
PathogenesisofBacillusanthracisisassociatedwiththe As a result, LT cripples the host innate immune system by
production of lethal toxin (LT), which activates the blocking cytokine production from numerous cell types,
murineNalp1b/Nlrp1binflammasomeandinducescas- inhibitingchemotaxisofneutrophils,andinducingapoptosis
pase-1–dependent pyroptotic death in macrophages in activated macrophages (3). At high concentrations, sim-
and dendritic cells. In this study, we investigated the ilar to those found late in infection, LT induces cytokine-
effect of allelic variation of Nlrp1b on the outcome of independent shock and death in animals that is associated
withvascularcollapse(1,2,4).
LT challenge and infection by B. anthracis spores.
Interestingly,LTinducesrapidcelllysisinmacrophagesand
Nlrp1ballelicvariationdidnotalterthekinetics orpa-
dendritic cells derived from a subset of inbred mouse and rat
thology of end-stage disease induced by purified LT, D
suggesting that, incontrastto previousreports,macro- strains (3, 5). This finding led to the model that the cytokine ow
phagelysisdoesnotcontributedirectlytoLT-mediated burstresultingfromLT-inducedmacrophagelysiscontributesto nlo
pathologyassociatedwiththistoxin(6,7).Suchamodelisat- ad
pathology. However,animalsexpressing a LT-sensitive e
tractive,asrapidreleaseofproinflammatorycytokinesconcom- d
allele of Nlrp1b showed an early inflammatory re- itant with macrophage lysis could, in theory, exacerbate the fro
sponse to LT and increased resistance to infection by m
vascular damage associatedwith anthrax and LT-mediated pa- w
B. anthracis. Data presented here support a model thology(3).Furthermore,macrophagesplayanimportantrole w
w
whereby LT-mediated activation of Nlrp1b and sub-
sequent lysis of macrophages is not a mechanism used in limiting B. anthracis infection (8–10), and their rapid de- .jim
structionbyLTwouldbepredictedtoresultinincreasedbacterial m
by B. anthracis to promote virulence, but rather a pro- fitness.However,thismodelisatoddswiththeobservationsthat un
tective host-mediated innate immune response. The animals resistant to purified LT are sensitive to challenge by ol.o
Journal of Immunology, 2010, 184: 17–20. B. anthracis spores and vice versa (11). A similar inverse re- rg
B lationship exists ininbredmousestrainswhereby manystrains on
acillus anthracis is the pathogenic bacterium re- whose macrophages lyse in response to LT display increased M
a
y
sponsible for the acute disease anthrax. Virulence resistancetoinfectionbyB.anthracis(12).Therefore,contrary 1
of B. anthracis is mediated in large part via the toonemodel,LT-mediatedlysisofmacrophagesappearstobe 0, 2
production of a protein exotoxin called lethal toxin (LT). associatedwithprotectionagainstinfectionbyB.anthracis. 01
0
Indeed, purified LT induces many symptoms associated Asinglegene,Nlrp1b,controlsmacrophageanddendriticcell
with fulminant anthrax including vascular collapse and sensitivity to LT (3, 13), and when heterologously expressed
death (1–3). LT is a bipartite toxin in which the binding with caspase-1 in human fibroblasts, confers susceptibility to
subunit, protective Ag (PA), attaches to anthrax toxin re- LTinthesecells(14). Nlrp1b isamemberofthenucleotide-
ceptorsandsubsequentlydeliversthecatalyticmoiety,lethal bindingdomain–leucinerichrepeatfamilyofproteinsfoundin
factor(LF),intothehostcellcytosol.Onceintracellular,LF plants,calledRproteins,andanimals,termedNLRproteins(6,
functions as a zinc-dependent metalloproteinase, cleaving 13).PlantRproteinsfunctioninhostimmunitybyrecognizing
theNterminiofMAPKkinasesandtherebydisruptingcell pathogensand/ordangersignalsandinitiatingahypersensitive
signalingthroughtheERK1/2,JNK,andp38pathways(3). response that can function locally through induction of cell
*Department of Microbiology, Immunology, and Molecular Genetics, University of Opinions,interpretations,conclusions,andrecommendationsarethoseoftheauthors
California,LosAngeles,LosAngeles,CA90095;†BacteriologyDivision,U.S.Army andarenotnecessarilyendorsedbytheU.S.Army.Researchwasconductedincompli-
MedicalResearchInstituteofInfectiousDiseases,FortDetrick,Frederick,MD21702; ancewiththeAnimalWelfareActandotherfederalstatutesandregulationsrelatingto
‡DepartmentofMolecularandIntegrative Physiology, University ofKansasMedical animalsandexperimentsinvolvinganimalsandadherestotheprinciplesstatedinthe
Center,KansasCity,KS66160;andxCaliforniaNanoSystemsInstitute,LosAngeles, GuidefortheCareandUseofLaboratoryAnimals,NationalResearchCouncil,1996.
CA90095 ThefacilitywherethisresearchwasconductedisfullyaccreditedbytheAssociationfor
AssessmentandAccreditationofLaboratoryAnimal.
Received for publication September 29, 2009. Accepted for publication October 21,
2009. AddresscorrespondenceandreprintrequeststoDr.KennethA.Bradley,Universityof
California,LosAngeles,609CharlesE.YoungDriveEast,LosAngeles,CA90095.E-
Thisresearch wassupported bytheUniversity ofCalifornia, LosAngeles,Microbial
mailaddress:[email protected]
PathogenesisTrainingGrantT32-AI007323(toJ.K.T.),NationalInstitutesofHealth
GrantAI077791(toK.A.B.andS.M.L.),andtheJointScienceandTechnologyOffice Abbreviationsusedinthispaper:ERP,earlyresponsephenotype;IL-1b,bformofpro-
for Chemical and Biological Defense/Defense Threat Reduction Agency project IL-1;LF,lethalfactor;LT,lethaltoxin;LTR,lethaltoxin-resistant;LTS,lethaltoxin-
1.1A0010-07-RDB(toS.L.W.). sensitive;PA,protectiveAg;PMN,polymorphonuclearneutrophil.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903114
18 CUTTING EDGE:NLRP1B MEDIATESRESISTANCETOANTHRAX
death or distally through production and release of antimi- Surprisingly, B6Nlrp1b(129S1) mice displayed a time to a mori-
crobialproductsandsignalingmolecules.Localizedcelldeath bund state similar to nontransgenic littermate controls fol-
inducedbyRproteinsrepresentsamechanismtolimitbacterial lowing LT challenge (Fig. 1A), indicating that the expression
infectionandcanbetriggeredbyanumberofupstreamstimuli ofaLTSalleleofNlrp1bdoesnotcontributetowhole-animal
includingthepresenceofbacterialproteasesinthehostcytosol susceptibilitytoLT.Histopathologicalanalysisalsorevealedno
(6,15).Wereasonedthatasimilarhypersensitiveresponsemay differences at the end stage of disease (data not shown), con-
also occur in B. anthracis-exposed animals and could explain sistent with earlier reports (1). However, a previously un-
why macrophage susceptibility to LT varies inversely with describedrapidandtransitoryresponsewasobservedfollowing
susceptibilitytosporechallengeasdescribedabove.Therefore, LTchallenge,whichwascharacterizedbyataxia(Fig.1B),bloat,
wesoughttodeterminehowNlrp1binfluencesoutcometoLT dilatedvesselsonpinnea,loose/wateryfeces,laboredabdominal
andsporechallenge. breathing,and/ormildhypothermia(Fig.1C).Thisdistinctive
responsewasdesignatedastheearlyresponsephenotype(ERP)
Materials and Methods
assomeanimalspresentedasearlyas30minafterLTadmin-
Mouse maintenanceandbreeding istration,andtheremaininganimalstypicallypresentedby1to
AllmicewerecaredforinaccordancewiththeUniversityofCaliforniaAnimal 2h.Wild-typeB6andlittermatecontrol(notshown)animals
Research Committee and the U.S. Army Medical Research Institute of In- displayednosignificantERPfollowingLTchallenge(Fig.1B,
fectiousDiseasesAnimalCareandUseCommittee.C57BL/6J(B6)micewere 1C).Surprisingly,B6Nlrp1b(129S1)micerecoveredtoseemingly
purchasedfromtheTheJacksonLaboratory(BarHarbor,ME).Transgenic
mice expressing a 129S1/SvImJ(129S1)-derived lethal toxin-sensitive (LTS) normalbehaviorfollowingtheERPbeforesuccumbingtoLTin
allele of Nlrp1b on a LT-resistant (LTR) B6 background (B6Nlrp1b(129S1)), amannersimilartocontrolanimals(Fig.1B).
backcrossedtoB6forsevengenerations,wereobtainedfromDrs.E.Boyden Thepathology,timing,andclinicalpresentationsassociated
and W. Dietrich (Harvard Medical School, Boston, MA). Heterozygous
with the ERP are consistent with an inflammatory response,
B6Nlrp1b(129S1) were intercrossed or crossed with B6, and transgene-positive
offspringwereidentifiedbyPCRgenotypingaspreviouslydescribed(13). therateofmacrophagelysisexvivo,andthepreviouslyreported D
o
cytokineresponse in LTSstrains ofmice (1,2). We therefore w
Toxin preparationandchallenge n
testedwhetherexpressionofaLTSalleleofNlrp1bissufficient lo
PAwasexpressedinEscherichiacoliandpurifiedaspreviouslydescribed(16), toinduceaproinflammatorycytokineresponsetoLT.Activa- ad
followedbySephacrylS-200(GEHealthcare,Piscataway,NJ)sizeexclusion ed
chromatography.LFwasobtainedfromDr.J.Mogridge(UniversityofTor- tionofNlrp1bresultsinformationofacaspase-1–containing fro
onto,Toronto,Ontario,Canada).Adoseof5mgPAand2.5mgLF,dilutedin inflammasome and subsequent proteolytic maturation of the m
pharmaceuticalgradesaline,pergbodyweightwasinjectedi.p.Alternatively, b form of pro-IL-1 (IL-1b) (13, 19). As expected, IL-1b in- w
PAandLFwerepurifiedfromB.anthracisstrainBH450(17).LFproduced w
creasedrapidlyafterLTadministration(Fig.1D).Inaddition, w
farodmosestorafin15BmHg4P50Adanisdpla7y.5edm3g-fLoFldpleorwgerboacdtyivwityeig(h1t8)w,aasnudsecdontoseqauchenietvlye several proinflammatory cytokines not directly activated by .jim
asimilarmortalityrate.Endotoxinwasremovedfromalltoxinpreparationsas caspase-1alsoincreased(Fig.1D)(1,2).Incontrasttoprevious m
u
described(16).Walkingataxiawasscoredasfollows:mild:reducedexploratory findings with LTS strains of mice (1, 2), there was a mild in- n
o
behaviororrearingonhindlimbs,aslowerand/orlesssteadygait,butfree creaseinTNF-ainB6Nlrp1b(129S1)mice(Fig.1D).Nochanges l.o
ammobuuselawtiaosnabthlerotuoggheonuertattheeacsalogwe;,munosdteeraadtye:(ep.rge.f,ewrroebdbsleyd)egnaittaruysustaalltye,fobru,tt7hes wereobservedineitherIL-1aorIFN-g.Endotoxincontami- org
beforeresting;andsevere:typicallyinastationarystate,butonstimulationthe nationofPAorLFwasnotresponsibleforcytokineinduction n M
mousecouldgenerateafewunstablesteps(e.g.,severewobbleand/ortremor) as no response was detected following injection of a 23 dose a
before stopping. Body temperatures were measured following LT injection y
ofindividualtoxincomponents(datanotshown).Further,B6 1
usingarectalthermometer.BaselinetemperaturesweredeterminedpriortoLT 0
injection and no differences were observed between animal groups (not animalsshowednoERPorcytokineresponsetoLT(Fig.1D), , 2
0
shown).Forcytokineanalysis,bloodwascollectedviacardiacpunctureand indicatingthattheseresponsesarearesultofNlrp1bdetection 1
allowedtocoagulate.Serawascollectedandstoredat280˚C.Cytokineswere 0
of LF activity rather than LPS contamination. Therefore, ex-
detected using the Millipore Milliplex MAP Mouse Cytokine Kit per the
manufacturer’sinstructions(Millipore,Billerica,MA). pressionofaLTSalleleofNlrp1binLTRB6miceissufficientto
induceaproinflammatorycytokineresponsetoLTinmice.
Sporechallenge andcellularanalysis
B6Nlrp1b(129S1)andnontransgeniclittermate/cagematemicewereinjectedi.p. LTSNlrp1balleles provideprotectionagainstB.anthracisinfection
withz2.53107unencapsulated,toxigenicSternestrain(7702)or43102
Amesstrainsporespermouseandmonitoreddailyfor14d.Forcellularanalysis, TotesttheroleofNlrp1binaninfectionmodel,B6Nlrp1b(129S1)
micewereinfectedi.p.withz1.63107Sternesporesandeuthanizedat4,28, mice and transgene-negative littermate control animals were
52, 76, and 135 h postinfection. Uninfected mice were used to determine
challenged with the unencapsulated, toxigenic B. anthracis
baseline cell populations in the peritoneal cavity of each strain. Peritoneal
exudateswereharvestedbyinjecting7mlofsterileHBSSand3mlofairintothe Sterne strain. Within 6 d, eight of nine control animals suc-
peritoneal cavity, followed by extraction. Samples were stained with fluo- cumbedtoinfection,whereasallB6Nlrp1b(129S1)micesurvived
rescentlyconjugatedAbstosurfacemarkersMac1/Cd11b(Invitrogen,Carls-
forthedurationoftheexperiment(Fig.2A).Totesttheroleof
bad,CA)andLy6G(BDPharmingen,SanDiego,CA)andanalyzedbyflow
aLTSNlrp1balleleinresponsetoafullyvirulentB.anthracis
cytometry.Duetothecross-reactivityoftheanti-Mac1Ab,polymorphonuclear
neutrophils(PMNs)weredefinedasLy6G+andLy6G+/Mac1+.Monocytes infection,B6Nlrp1b(129S1)micewerechallengedwithB.anthracis
weredefinedasMac1+/Ly6G2.Theaveragepercentageofeachcelltypeper Ames strain. Although B6Nlrp1b(129S1) mice displayed a trend
mousewasthenconvertedtototalcellnumberbymultiplyingwiththemean
toward protection, the data were not statistically significant
hemocytometercountforeachmousegroup.Similarvalueswereobtainedby
histochemicalandmicroscopicanalyses(datanotshown). (Fig. 2B).The latter finding isnot surprising given thatviru-
lenceassociatedwiththeAmesstrainisgovernedprimarilyby
Results
thepresenceofapoly-D-glutamicacidcapsuleratherthanLT
Nlrp1b-mediatedresponse toLT
inthemousemodel(20).
To determine whether the presence of a LTS allele of Nlrp1b TodeterminethecellularmediatorscontributingtoNlrp1b-
controls whole animal susceptibility to purified LT, we chal- mediated resistance to infection, peritoneal exudates were
lenged B6Nlrp1b(129S1) mice with LT via i.p. injection (13). collected and analyzed at various time points following spore
TheJournal ofImmunology 19
FIGURE1. InfluenceofNlrp1bontheresponseinmice
toLT.A,B6Nlrp1b(129S1)transgenicmice(Nlrp1bTg)(n5
10)expressingaLTSalleleofNlrp1bortransgene-negative
controlanimals(B6)(n510)werechallengedwith5mg
PA+2.5mgLFpergbodyweightviai.p.injection.Animals
werecloselymonitoredforthefirst4–6hfollowingLT
injectionandthenevery3hfor5dandeuthanizedupon
reachingamoribundstate.B,Heatmaprepresentingataxia
severityoftheanimalsshowninA.Eachhorizontalline
represents an individual animal from time of LT
injection (left) until the end of the experiment (right).
Ataxiaseverityisindicatedbycolor.Dataarerepresentative
ofsevenindependentexperiments.C,Bodytemperatureof
B6Nlrp1b(129S1)(n55)ornontransgeniclittermatecontrol
mice(n55)wasmeasuredfollowingi.p.injectionof15mg
PA + 7.5 mg LF per g body weight. Temperature was
monitoredhourlyandlowesttemperatureobservedduring
first5hposttoxininjectionisplotted.D,B6Nlrp1b(129S1)
D
transgenic mice (closed triangles) or transgene-negative o
w
controlanimals(opensquares)werechallengedwithLTas n
inC.Uninjectedanimalsservedast50controls.Animals loa
d
weresacrificedat0.5,1,2,and4hposttoxininjection,and e
d
saevreurmagecyvtaolukeinseosflfievveelasnwimeraelsm6easSuDre.d.Datarepresentthe from
w
w
w
.jim
m
u
n
o
l.o
rg
challenge. Both strains responded with an increase in the throughaMAPKsignalingpathway(24).However,thepres- on
numberofLy6G+PMNs(Fig.2C).However,thelevelsofPMNs ence of LT blocks this response by cleaving and inactivating M
a
werehigherinB6Nlrp1b(129S1)miceatearlytimepointsfollowing MAPKkinaseproteins(24).LTSallelesofNlrp1bcounteract y 1
sporechallengecomparedwithnontransgeniclittermatecontr2ol this immunosuppressive effect by triggering a rapid proin- 0, 2
animals. This influx of PMNs was followed by more Ly6G / flammatoryprogrammedcelldeath.Interestingly,IL-1bisre- 0
1
Mac1+monocytesinbothstrains(Fig.2D)thatweremaintained leased upon LT-mediated macrophage lysis (19). IL-1b is 0
inB6Nlrp1b(129S1)butnotcontrolmice. a proinflammatory cytokine that recruits PMNs and mono-
cytes, cell types that are predicted to resolve infection (9, 10,
Discussion
25). Although Nlrp1b inflammasome activation in response
Based on LT and spore-challenge data from different animal
toLTisdetrimentaltothetoxin-exposedmacrophage,ourdata
species,Lincolnetal.(11)hypothesizedthatanimalsresistantto
demonstratethatNlrp1bactivationisultimatelybeneficialfor
infection by B. anthracis were susceptible to challenge by its
the host by inducing inflammation (e.g., enhanced cytokine
toxin and that the inverse was true for infection-susceptible
productionandPMNinfiltration)atthesiteofLTproduction.
species.Usinginbredandrecombinantstrainsofmice,Welkos
Of note, a similar mechanism has been described in plants
andcolleagues(12,21,22)substantiatedthisproposedinverse
where R proteins recognize bacterial virulence factors in the
correlationbetweenthesensitivityofanimalstochallengewith
host cell cytosol and induce localized cell death to limit in-
purified LT and with B. anthracis spores and explored the ge-
fection. Importantly,thefindingthattheNlrp1b-mediatedin-
netic basis for this phenomenon. Specifically, mice whose
flammatoryresponseisprotectiveagainstB.anthracisinfectionis
macrophagesrapidlylyseinresponsetoLTweremoreresistant
consistent with previous data that mice deficient in caspase-1,
to spore challenge than mice whose macrophages were LTR
IL-1b,orIL-1Rdisplayincreasedsensitivitytoanthrax(25,26).
(12, 13, 23). Further, mice resistant to spore challenge had
Therefore,weproposethatNlrp1b-mediatedcelldeathprovides
increased rates of PMN infiltration at early time points and
aselectiveadvantagetothehostratherthanpathogen.
sustained higher monocyte numbers at the site of B. anthracis
infections (22). Here we report that allelic variation at Nlrp1b Acknowledgments
accounts for these previously observed phenomena, thereby
WethankDrs.E.BoydenandW.DietrichforprovidingB6Nlrp1b(129S1)mice,
providing molecular insight into host defense against anthrax.
Dr. G. Lawson for histopathological analyses, AlyssaLeiva, Sylvia Trevino,
B.anthracistriggersactivationofTLRsandNOD2inhuman
andSonelaSchlottmannfortheirtechnicalassistance,andDianaFisherfor
and mouse macrophages, resulting in production of TNF-a statisticalassistance.
20 CUTTING EDGE:NLRP1B MEDIATESRESISTANCETOANTHRAX
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aBn6iNmlrapl1sb((1g2r9aSy1)batrrsa)nwsgeerneicchmalliecnege(bdlai.cpk.wbiatrhs)1.o6r3tr1an0s7gsepnoer-ensegoaftBiv.eanctohnrtarcoisl ROonlee3o:feN31-t3e0r.minalaminoacidsinthepotencyofanthraxlethalfactor.PLoS from
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dmeetaenrmvianluedesa(snd5esc2riabte1d3i5n-hthteimMeapteoriinatlsaannddnM5et3hoadtsaslelcottihoenr.tDimateaproeipnrtess)e6nt 20. WAmeleksosst,rSai.nL.o,fNB.aJc.ilVluisetarni,tahnradciPs.aHre.fGulilbybvs.ir1u9le9n3t.Nfoornm-tiocxe:igreonliecodferpivlaastmiviedsopfXt0h2e .jim
m
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21. Welkos,S.L.,andA.M.Friedlander.1988.Pathogenesisandgeneticcontrolof n
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Disclosures 22. rWeseislktaonsc,eS.toL.t,hRe.SWte.rnTerosttrtaeirn,Dof.MBa.cBilleucskearn,tahnradciGs..MOi.crNobel.soPna.th1o9g8.94.:R5e3s–is6t9an.ceto l.org
Theauthorshavenofinancialconflictsofinterest. theSternestrainofB.anthracis:phagocyticcellresponsesofresistantandsuscep- o
tiblemice.Microb.Pathog.7:15–35. n
23. Friedlander,A.M.1986.Macrophagesaresensitivetoanthraxlethaltoxinthrough M
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