The
Brazilian
Journal
of
INFECTIOUS
DISEASES
w w w .e l s e v i e r . c o m / l o c a t e / b j i d
Special
article
Revisiting
the
Rich’s
formula:
an
update
about
granulomas
in
human
tuberculosis
João
Carlos
Coelho
Filho
a,∗,
Iukary
Takenami
b,
Sérgio
Arruda
baLaboratóriodePatologia,Fundac¸ãoJoséSilveira,Salvador,BA,Brazil
bLaboratórioAvanc¸adodeSaúdePública,CentrodePesquisasGonc¸aloMoniz,FIOCRUZ,Salvador,BA,Brazil
a
r
t
i
c
l
e
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n
f
o
Articlehistory:
Availableonline5March2013
Keywords: Rich’sformula Tuberculosis Immunopathogenesis Granuloma Hypersensitivity
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t
TheformulaproposedbyRichin1951explainedtheformationinatuberculouslesionin aperiodthatwasunknowncellularfunctions,cytokinesandotherimmunologicalaspects involvedingranulomaformationbytuberculosis;itscomponentsareassembled concep-tuallyto explainthe pathogenic mechanisms involvedin thegranulomatouslesion in tuberculosis.Inthismanuscript,wereportanupdateofRich’sformulabasedonthenew andoldconceptsaboutpathogenicmechanismsinvolvedinthegranulomatouslesionin tuberculosis.Currentknowledgeallowsustoconcludethatthebalancebetweenthe charac-teristicsofthebacillusandhostprotectiveresponseisnecessarytoindicatetheoutcomeof pathogenesis,infectionoractivediseaseandthenecrosisdegreeofthetuberculosislesion. ©2013 ElsevierEditoraLtda.Allrightsreserved.
Introduction
Tuberculosis (TB) is the most frequent cause of granulo-masand isthemostprevalent infectiousdisease inBrazil, remainingonethemostimportantproblemsofpublichealth intheworld.In2010,71,000caseswerereported,witha mor-talityof4800/year.1Brazilisthe19thinrankamongcountries withthemajorityofTBcasesintheworld.Bahia,the north-eastStateofBrazil,isranked3rdinnumberofcases,afterSão PauloandRiodeJaneiro.TBincidenceinBahiais39.5/100,000 habitants.1–3
TB is a granulomatous disease characterized by a cell-mediatedhypersensitivity reactioninresponseto com-ponents of the wall structure of Mycobacterium tuberculosis
especiallycordfactor and lipoarabinomannan.4 Granuloma
∗ Correspondingauthorat:LaboratóriodePatologia,Fundac¸ãoJoséSilveira,JoãoCarlosCoelhoFilho,Fundac¸ãoJoséSilveira,Ladeirado
CampoSanto,Federac¸ão,s/n,Salvador,BA40210-320,Brazil. E-mailaddress:[email protected](J.C.CoelhoFilho).
was first described in 1679 by Sylvius, and etymologically comesfromtheoriginalwordtubercle.5Nowadays, granulo-masaremuchbetterunderstoodbasedontheknowledgeof cellularimmunology(cellmediatedhypersensitivityreaction) anditsinteractionwithcytokinesandchemokines.
In1951,Richproposedthatthepathogenesisoftuberculous lesionwasdirectlydependentofinoculum,bacillusnumber (N),bacillivirulence(V)andhosthypersensitivity(H),by coun-terbalancinganddecreasingthelesionsize,thenaturaland acquired resistanceofthe host(Rn+a).Thisformulacanbe representedby6:
L=N+RV+H
(n+a)
Rich’sformulashowscomponentsthatareassembled con-ceptuallytoexplainthepathogenicmechanismsinvolvedin
1413-8670/$–seefrontmatter©2013 ElsevierEditoraLtda.Allrightsreserved. http://dx.doi.org/10.1016/j.bjid.2013.01.006
thegranulomatouslesioninTB.ConsideringtheTB patho-genesisintwodifferentclinicalforms,infectionanddisease, whereininfection,thebalancedependsonthehostnatural resistance.Incontrast,indisease,thebalancedependsonthe bacillipropertiessuchasvirulenceandhypersensitivity phe-nomenon.Inbothclinicalforms,eitherinfectionordisease, granulomasaredetectedinthetissue.7–9Bothstagesare tak-ingplaceinsideofthegranuloma,wherethecomponentsof Rich’sformulaoccur.Theancientconceptwasthata granu-lomawasacellularagglomeratewallingofthebacilli;thusin Rich’sformulathisshouldbeconsideredinthedenominator. Thiscanbeobservedinsomehost,inwhichthegranuloma formationis impairedand therefore the bacillus growth is faster. In fact, inthese almost 60 years since the proposi-tionofRich’sformula,thesciencehasadvancedsignificantly intheknowledgeofthepropertiesofM.tuberculosis,aswell asinunderstandingofhostdefensemechanisms.Thesenew conceptshavebeenincorporatedintotheaboveformula.
Initiation
of
infection
and
granuloma
formation
Aftermycobacterialarrivalinthetissuesoftherespiratory tract,an unspecifiedreaction occurs in which neutrophils trytophagocytizethebacillus.Thisinitialphaseisnotwell studiedinhumans.10 Threeweekslater,thereisa develop-mentwithinthesetissuesofamorespecificreaction,when mononuclearcells reach the focus oftheinfectious lesion, modulatingthegranulomaformation.9,11,12Mononuclearcells including monocytes/macrophages and lymphocytes, cells withtheir predefined morphologiesand functions, partici-pateintherecognitionand presentationofcomponents ofM.tuberculosistotheimmunesystem.11–13 Duringand after
mononuclearcellsnaïvestartscellsdifferentiationinto spe-cificcelltypessuchasmacrophages,dendriticcells,CD4and CD8Thelper.14
Intheprocessofgranulomaformation,manymacrophages with large cytoplasm and organelles withhigh phagocytic andmicrobicidalfunctionswereobserved.15–17Thesecellscan fuseintomultinucleatedgiantcells;however,thesechanges arenotcompletelyunderstood.12,17Maturemacrophagescan undergo a transformation into epithelioid cells (epithelioid histiocytes),whichinterdigitatedtheirmembranes aggregat-ingintoother cellsasthe epithelialbarrier.16CD4andCD8 Thelper(Th)lymphocytesaswellasepithelioidmonocytes andgiantmultinucleatedcellsparticipateinthisgranuloma formationthatisorchestratedbyINF-␥ producedbyCD4and TNF-␣secreted bymacrophages.8,11,12,15,18 Other cells,such asneutrophils,jointhisgranulomaviaadynamicprocess.9,19 Allthesefactshavedynamicmorphologythatisinfluenced bythebalancebetweenhostresistanceandtheM.tuberculosis
virulencegeneexpression.9,11
Cellularapoptosisofcaseousnecrosiscanalsobedetected withinthe centersofgranulomas.Progress hasbeenmade indeterminingthesemechanisms;however, muchremains notcompletelyunderstood.9 Oneinterestingpointthathas beenfoundregardsthefunctionofthegranuloma.Although themainconsensusisthatthegranulomathatsurroundsthe
M.tuberculosis isdoing soinan attemptto restrictit from
spreading.20Otherexperimentsinthezebrafishhaveshown
thatgranulomaalsocontributetobacillispreading.Duringthe granuloma’sformation thetissuesareinjured andnecrosis develops.Thismechanismoflesiondevelopmentdependson theacquiredresistanceversusthehypersensitivitydeveloped bythepatienttothebacillusduringtheinfection.9
Granulomasseenintuberculouspleurisyallowto distin-guish thecellularcomponents comparingthe cells present inpleurafluid.21 Fayyazietal.showedapossibleapoptosis ofepithelioidcellsand thepresenceofCD4Tlymphocytes intheproximityofthefocusofthenecrosis.Suchfindings could suggestadirectrelationbetweenthecells,their sec-retionsandthecentralnecrosisofgranulomas,inadditionto theepithelioidcell’sapoptosis.22Theinteractionofallthese aspectsandalsotheelementsoftheextracellularmatrix9are partofthefinalproduct,thegranulomawithcentralcaseous necrosis.23,24
Bacilli
virulence
ThegenomesequenceofM.tuberculosisallowedtoconduct studiesabouttheroleofunknowngenesthroughfunctional genomics.Theidentificationofgenespresentinhypervirulent strainsofM.tuberculosis,25 butabsentinpathogenicstrains
suchasMycobacteriumbovisBCG,allowedabetter
understand-ingofthepathogenesisofTB.
Currently, the virulence of M. tuberculosis is defined by region of difference (RD1)genes, which are absent in BCG strainsandarepresentinallM.tuberculosisvirulentstrains. SeveralstudieshavedemonstratedthatdeletionofRD1from
M. tuberculosis results in attenuationof virulence,
suggest-ingadominantroleinimmuneresponses.26,27Thus,deletion mutantsofvirulent M.tuberculosisstrainsforRD1resemble BCGintheirinfectivityandattenuation.27
Furthermore, another groupof geneshave been associ-ated with virulence of M. tuberculosis in animal model. It is suggested that the mammalian cell entry genes (mce), whichencodesMceprotein,wereoriginallyidentifiedinM.
tuberculosis28andhavebeenassociatedwithsurvivalwithin
macrophagesandanincreaseinvirulence.29,30Disruptionof themcegeneofBCGresultsinamutantthatexhibitsreduced invasivenessepithelialcells,impairingtheirabilitytosurvive andmultiplyinsidephagocyticcells.31
InhibitionofmacrophageapoptosisbyM.tuberculosisstrain H37Rvhasbeenproposedasanothervirulencemechanism.32 This hypothesis is supported by studies that show that bacilli can cause necrosis instead of apoptosis in infected macrophagestoavoidinnatehostdefense.Themacrophage celldeathpathwaybynecrosiscouldbeanexitmechanism
forM.tuberculosistofacilitatethespreadofinfectionand
con-tributetotheformationofnecroticlesionsinTB.33,34Indeed, infection ofmacrophageswiththe attenuatedM. tuberculo-siscausesapoptosis,whichlimitsbacterialreplicationsand promotesT-cellcrossprimingbyantigen-presentingcells.35
Host
hypersensitivity
There isastrong relationshipamongimmunityand hyper-sensitivityinTB.Tlymphocytescanproducedistincttypesof
immuneresponsesassociatedwithdifferentcytokineprofiles andhaveopposingeffectsonresistanceandsusceptibility.14 The result of this balance will determine the degree of lesionininfected individualsand TBdisease.Latent infec-tionisassociatedwithprotectivecellularimmuneresponses whereasadvanced diseaseisassociatedwithimmune sup-pressionandexacerbatedhypersensitivityreaction,causing tissuedamage.Thus,Tlymphocytesmayalsomediate harm-ful tissue-destroying hypersensitivity reactions that cause progressionofdisease.36
Natural
and
acquired
resistance
Macrophages, dendritic cells and neutrophils principally mediate the natural hostresistance to M. tuberculosis. The availabledata on the role of neutrophilsare controversial. Neutrophilsarethefirstcellstobemobilizedandarrivewithin hoursatthesiteofinfection.Theyarenotprofessional phago-cytessuchas macrophages,but contributesubstantially to innateresistancetoinfection.Neutrophilscanalsoforman extracellularfibrilmatrixknownasNETs(neutrophil extracel-lulartraps).TheseNETsweredescribedrecently.37Theyare formedbyactivatedneutrophilsandconsistofaDNA back-bonewith embeddedantimicrobial peptides and enzymes. Thus,NETsrepresentadistinct innatedefensemechanism tocontrolandeliminatemicrobialinfections.38Ontheother hand,thepoorabilityofneutrophilstorestrict mycobacte-rialgrowthcomparedtoalveolarmacrophagesindicatesthat theprevalenceofneutrophilsinTBinflammationcontributes tothedevelopmentofanonspecificcellularreaction,rather thanprotectionofthehost,and thatneutrophilsmay play the role of a“Trojan horse” forM. tuberculosis.10,39 Indeed, interferon-inducible neutrophil-drivenbloodtranscriptional signaturehasbeendescribedinpatientswithactiveTB,which correlateswithlungradiographicdiseaseseverityand impli-catesneutrophilsdirectlyinthepathogenesisofactiveTB.40
Molecular studies show that the human genetic are responsiblefornaturalresistancetoM.tuberculosis.Thereis consensusintheliteraturethathumansexposedto
Mycobac-terium millennially were evolutionarily selected, providing
relativelymoreresistantcohorts.Oneexampleofthisisthe infectionofNativeAmericanswhenEuropeansfirstcameto NorthAmericawheretheApachestribewasrapidlyinfected byacuteanddeadlyTB.41Also,indigenousBraziliansshowa highTBrate,whichcanbeattributabletoanintrinsic suscep-tibilityoftheseindividuals.42
TheresistancemechanisminTBinvolvesagreatnumber ofcellularinteractions(macrophage,Tcell).Severaldifferent T-cellpopulationsare requiredforthesuccessfulcontrolof thepathogen.Thisdynamicinterplayunderlyingprotection isthe reasonforthelong-termpersistence ofM. tuberculo-sis.From the cellular pointof view the Tlymphocytes are differentiated inthe thymus. CD4and CD8 T-cellsactivate macrophages against M. tuberculosis. These cells recognize specific parts of the bacillus and secrete cytokines, which activate macrophages.9,11–15,18 T cells also act, induced by componentsofthebacillus,producingdefensemechanisms. Byreactingwithproteins,astateofhypersensitivityis cre-ated that can be measured by the tuberculin test. In this
context,cytokinesplayanimportantroleinthepathogenesis ofTB.Thecytokinesareweightedmoleculesofabout8–14kDa whoseproductionisafunctionofthecomponentsofM.
tuber-culosis.Thesecytokinesattractmacrophagesthattransform
intoepithelioidcells.8,9,14IFN-␥,TNF-␣andinterleukin-(IL)-12 arethemostfundamentalcytokines,buttherearecurrently about40classifiedcytokinesandotherchemokinesthatactas receptorsinvolvedinthecellulardynamicofgranulomas.43
The events underlying the structural reorganization of immune cells toforma stablegranuloma orprogress toa pathologiclesionaredependentonothercomponents. Itis relatedwithsomecomponentsofthestructureofM.
tuber-culosis, including some partsof its geneticmaterial. These
elementscanmobilize,recruit,andamplifyTcellsreaction throughtheinfluenceofcytokines.44Glycolipids,components
oftheM.tuberculosiswall(sero-lipidicmembrane),alsoinvolve
thebacillusandDNA.Theycontaina6kDaprotein,designed asanESAT-6,thatmobilizestheTcellandactivatesthe pro-ductionofTNF-␣andIFN-␥.Bothoftheseactivationsleadthe macrophagestodestroytheMycobacterium.19,45Followingthe aboveevents,aswellasthearrivalofbacilliinthetissues(for example,pulmonaryalveoli),theformationofagranuloma withcentralcaseousnecrosisiscompleted.
Directed by T lymphocytes, modified monocytes aggre-gate and transforminto epithelioid cells.With this fusion, giantmultinucleatedcells(Langerhanscells)arecreated.The dynamicpreviouslydescribedisalsoinfluencedbyTNF-␣.44 In addition toLangerhanscells, TNF-␣also playsarole in the formationofcaseousnecrosiswithinthecenter ofthe granuloma.9,44Theanaerobicenvironmentandtheactionof phagolysosomes withinthe granulomas inhibit the spread ofbacilli. Thehypersensitivity promotesthe progressionto necrosis,makingthecellsmoresensitivetothecomponentsof thebacillus(forexample,tuberculostearicacid).Thefinal dif-ferentiationofthegranulomawilldependontheinterplayof allthefactorscitedinthispaper.Whenthereishighimmunity andlowhypersensitivitytherewillbealowlevelofnecrosis andthegranulomawillmostlikelyscarundertheactionof miofibroblasts.Ifthehypersensitivityisdominant,the necro-siswillspreadandthelesionwillgrowwithanincreaseinthe proliferationofbacilli.46
Inconclusion,althoughRich’sformulawasproposedinthe 1950s,itstillholdssomerelevancetothedistinctcomponents withinthegranulomatouslesions.Evenafterincorporationof currentknowledgeaboutpathogenesisofM.tuberculosis,the balance betweenthecharacteristics ofthebacillusand the hostprotectiveresponseisnecessarytoindicatetheoutcome ofinfection.
Conflict
of
interest
Theauthorshavenoconflictofinteresttodeclare.
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