Special Modelling of Cerebral Tuberculosis: Hope for ... · PDF filechildren. It is thought...
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www.mjms.usm.my © Penerbit Universiti Sains Malaysia, 2011 For permission, please email:[email protected] Abstract Cerebral tuberculosis is a severe type of extrapulmonary disease that is highly predominant in children. It is thought that meningeal tuberculosis, the most common form of cerebral tuberculosis, begins with respiratory infection followed by early haematogenous dissemination to extrapulmonary sites involving the brain. Host genetic susceptibility factors and specific mycobacteria substrains could be involved in the development of this serious form of tuberculosis. In this editorial the different animal models of cerebral tuberculosis are commented, highlighting a recently described murine model in which BALB/c mice were infected by the intratracheal route with clinical isolates, which exhibited rapid dissemination and brain infection. These strains were isolated from the cerebrospinal fluid of patients with meningeal tuberculosis; they showed specific genotype and induced a peculiar immune response in the infected brain. This model could be a useful tool to study host and bacilli factors involved in the pathogenesis of the most severe form of tuberculosis. Keywords: experimental models, infectious diseases, meningeal tuberculosis, mice, Mycobacterium, virulence Tuberculosis and the Central Nervous System Tuberculosis involvement of the central nervous system (CNS) is a significant and serious type of extrapulmonary disease. It constitutes approximately 5%–15% of the extrapulmonary cases, and in developing countries, it has high predominance in children (1). There are different clinical/pathological manifestations of cerebral tuberculosis; the most common is tuberculous meningitis, followed by tuberculoma, tuberculous abscess, cerebral miliary tuberculosis, tuberculous encephalopathy, tuberculous encephalitis, and tuberculous arteritis (2). Cerebral tuberculosis is often fatal and mainly caused by Mycobacterium tuberculosis; other non-tuberculous mycobacteria such as M. avium-intracellulare can also produce CNS tuberculosis, mainly in human immunodeficiency virus (HIV)-infected persons (2). It is believed that cerebral tuberculosis, like any other forms of tuberculosis, begins with respiratory infection followed by early haematogenous dissemination to extrapulmonary sites, including the CNS. On the basis of their clinical and experimental observations, Rich and McCordock (3) suggested that cerebral tuberculosis develops in two stages. Initially, small tuberculous lesions (Rich’s foci) develop in the brain during the stage of bacteraemia of the primary tuberculosis infection or shortly afterwards. These early tuberculous lesions can be located in the meninges, the subpial or subependymal surface of the brain, and may remain dormant for long time. Later, rupture or growth of one or more of the small lesions produces development of various types of CNS tuberculosis. Rupture into the subarachnoideal space or into the ventricular system produce meningitis, the most common form of cerebral tuberculosis. Modelling of Cerebral Tuberculosis Experimental animal models of cerebral tuberculosis have been established in rabbits (4,5), mouse (6,7), and pigs (8). Although they reproduce in some extend the human lesions, Special Communication Modelling of Cerebral Tuberculosis: Hope for Continuous Research in Solving the Enigma of the Bottom Billion’s Disease Rogelio Hernández Pando Experimental Pathology Section Department of Pathology National Institute of Medical Sciences and Nutrition “Salvador Zubirán” Calle Vasco de Quiroga 15 Tlalpan, CP 14000, México DF, México Submitted: 23 Oct 2010 Accepted: 25 Oct 2010 12 Malaysian J Med Sci. Jan-Mar 2011; 18(1): 12-15
Transcript of Special Modelling of Cerebral Tuberculosis: Hope for ... · PDF filechildren. It is thought...
www.mjms.usm.my © Penerbit Universiti Sains Malaysia, 2011 For permission, please email:[email protected]
Abstract Cerebraltuberculosisisaseveretypeofextrapulmonarydiseasethatishighlypredominantinchildren.Itisthoughtthatmeningealtuberculosis,themostcommonformofcerebraltuberculosis,beginswithrespiratoryinfectionfollowedbyearlyhaematogenousdisseminationtoextrapulmonarysites involving thebrain.Hostgenetic susceptibility factorsandspecificmycobacteria substrainscould be involved in the development of this serious form of tuberculosis. In this editorial thedifferentanimalmodelsofcerebraltuberculosisarecommented,highlightingarecentlydescribedmurinemodelinwhichBALB/cmicewereinfectedbytheintratrachealroutewithclinicalisolates,which exhibited rapid dissemination and brain infection. These strains were isolated from thecerebrospinal fluid of patients with meningeal tuberculosis; they showed specific genotype andinducedapeculiarimmuneresponseintheinfectedbrain.Thismodelcouldbeausefultooltostudyhostandbacillifactorsinvolvedinthepathogenesisofthemostsevereformoftuberculosis.
Keywords:experimental models, infectious diseases, meningeal tuberculosis, mice, Mycobacterium, virulence
Tuberculosis and the Central Nervous System Tuberculosis involvement of the centralnervoussystem(CNS)isasignificantandserioustype of extrapulmonary disease. It constitutesapproximately 5%–15% of the extrapulmonarycases, and in developing countries, it has highpredominanceinchildren(1).Therearedifferentclinical/pathological manifestations of cerebraltuberculosis; the most common is tuberculousmeningitis,followedbytuberculoma,tuberculousabscess,cerebralmiliarytuberculosis,tuberculousencephalopathy, tuberculous encephalitis, andtuberculousarteritis(2).CerebraltuberculosisisoftenfatalandmainlycausedbyMycobacterium tuberculosis;othernon-tuberculousmycobacteriasuch as M. avium-intracellulare can alsoproduce CNS tuberculosis, mainly in humanimmunodeficiency virus (HIV)-infected persons(2). It is believed that cerebral tuberculosis,like any other forms of tuberculosis, beginswith respiratory infection followed by earlyhaematogenousdisseminationtoextrapulmonary
sites, including the CNS. On the basis of theirclinical and experimental observations, Richand McCordock (3) suggested that cerebraltuberculosis develops in two stages. Initially,small tuberculous lesions (Rich’s foci) developin the brain during the stage of bacteraemia ofthe primary tuberculosis infection or shortlyafterwards. These early tuberculous lesionscan be located in the meninges, the subpial orsubependymal surface of the brain, and mayremain dormant for long time. Later, ruptureor growth of one or more of the small lesionsproduces development of various types of CNStuberculosis. Rupture into the subarachnoidealspace or into the ventricular system producemeningitis, the most common form of cerebraltuberculosis.
Modelling of Cerebral Tuberculosis
Experimental animal models of cerebraltuberculosis have been established in rabbits(4,5), mouse (6,7), and pigs (8). Although theyreproduce in some extend the human lesions,
Special Communication
Modelling of Cerebral Tuberculosis: Hope for Continuous Research in Solving the Enigma of the Bottom Billion’s DiseaseRogelio Hernández Pando
Experimental Pathology SectionDepartment of PathologyNational Institute of Medical Sciencesand Nutrition “Salvador Zubirán” Calle Vasco de Quiroga 15 Tlalpan, CP 14000, México DF, México
Submitted: 23Oct2010Accepted: 25Oct2010
12Malaysian J Med Sci. Jan-Mar 2011; 18(1): 12-15
Special Communication |Modellingofcerebraltuberculosis
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thesemodels are artificial because they use thedirect intracerebral or intravenous route ofinfection,insteadofthenaturalrespiratoryroute.Thus,itisimportanttoestablishanexperimentalmodel which reproduces more closely thehuman disease, including the initial respiratorynatural routeof infection.However, suchmodelis difficult to achieve because of the highlyefficientCNSprotectionconferredby theblood-brain-barrier (BBB).BBB is composedof tightlyassociated brain microvascular endothelial cellscoveredbypericytesandoutgrowthsofastrocytes(cytoplasmic end feet). This structure efficientlypreventsCNSinfectionbymanymicroorganisms,including mycobacteria. Thus, to produce CNSinfection, some microorganisms have evolvedspecific virulence factors that permit, first,endothelial attachment and internalization,followed by brain parenchyma invasion (9).This is thecaseofbacterialproteinsIbeA,IbeB,AslA,YijP,andOMPAexpressedbyneurotropicEscherichia coli, or meningococcal surfaceproteinsOpa,Opc,andPiICamongothers. Recent in vitro studies have shown thatM. tuberculosis can adhere, invade, andtraverse endothelial cells (10), and clinical–epidemiological studies have shown distinctgenotype in strains isolated from tuberculouspatients’ cerebrospinal fluid (CSF) (11), whichsuggest strain-dependent neurovirulence andneurotropism.We recently informed the resultsfrom an experimental study in which, using amodel of pulmonary tuberculosis in BALB/cmouse infectedby the intratracheal route, threedifferentM. tuberculosisclinicalisolatesobtainedfromCSFofmeningealtuberculouspatientswereabletorapidlydisseminateandinfectthemousebrain (12). These clinical strains were isolatedfrom patients with meningeal tuberculosis inColombia,andtheyshowedadistinctivegenotype.Theyextensivelydisseminatedbyhaematogenousroute after one day of intratracheal infectionand rapidlyproduced tuberculous lesions in themice brain. As mentioned before, it has beenestablished thatmycobacteria reach theCNSbythehaematogenousroutesecondarytopulmonaryinfection(3).Thisexperimentalmodelisthefirstonethatreproducedthissituation,confirmingthatthestraintypeisdirectlyrelatedwiththeabilitytodisseminatebythehaematogenousroute,andaddM. tuberculosis tothelistofmicroorganismfamilies in which some members or substrainshavecertainabilitytoinfecttheCNS. Comprehensive clinical-epidemiologicalstudies have identified several risk factors formeningeal tuberculosis; these include age less
than40years,(HIV)infection,andcertainethnicpopulations.Thelatterfactorsuggestssignificantinterplay between host genetic background andspecific strains of mycobacteria (11). In fact, ithas been recently shown association betweenthe development of tuberculous meningitis andsingle nucleotide polymorphism in the Toll-interleukin-1receptordomaincontainingadaptorprotein (TIRAP) and Toll-like receptor (TLR-2)genes(13). Our strains are from the Euro-Americanlineagewhichwas recently reported to bemorepulmonarythanmeningeal.Ithasbeenproposedthat Euro–American strains are less capableof extra pulmonary dissemination due the lackof pks 15/1 intact gene. Pks gene participatesin the production of phenolic glycolipid (PGL),which inhibits the innate immune responseand may be responsible for dissemination andCNS infection. Our Euro–American isolates areunable to express PGL; however they efficientlydisseminate and produced brain infection,suggesting that other mycobacterial moleculescould participate in this process. We considermycobacterial heparin-binding haemaglutininadhesinasagoodcandidatebecausethismoleculetriggersreceptor-mediatedbacilliadherenceandinvasion to epithelial cells, and extrapulmonarydissemination. Another potential participatingmolecule is histone-like protein (HLP), whichpermitstoM. lepraeinteractswithlamininonthesurfaceofSchwanncells,facilitatingitsinvasion.HLP is also expressed by M. tuberculosis, andcouldparticipate in the infectionof thenervouscellsafterendothelialbarriertraverse.Thisstudyisnowinprogressinourlaboratory. Thefirststeprequiredforcertainneurotropicmicroorganisms to cause CNS infection is topenetratetheBBB.ThebasicandfirstelementofBBBismicrovascularendothelialcellsthatdifferfrom those in other tissues by tight junctionswith high electrical resistance and a relativelylownumberofpinocytoticvesicles.Recently,aninvitromodelusinghumanbrainmicrovascularendothelialcellsshowedthatthereferencestrainM. tuberculosis H37Rv can invade and traversethese cells, using a process that requires activecytoskeleton rearrangements. By microarrayexpressionprofiling,theauthorsfound33genesthatwereoverexpressedduringendothelial cellsinvasion, suggesting that the products of thesegenes might participate in this process (10).Perhapsdifferentgeneprofilecouldbeexpressedby our strains than the laboratory referencestrainH37Rvusedintheinvitrosystemthatourexperimental model showed limited ability toinfectthebrain.
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IntravenousinfectionofC57BlmicewithM. avium induces brain infection, and the numberofbacteria increaseswith thedurationand levelof bacteraemia, which depend of the inoculumsize(7).Oneofourstrains(code209)washighlyvirulent;itinducedmorerapidanimaldeathandhigh bacilli burdens in the lung, liver, spleen,andparticularlyintheblood.Inordertostudyifhypervirulencecouldberelatedtodisseminationand brain infection, we infected animals withother highly virulent strains isolated from thesputumofpatientswithpulmonarytuberculosis.Interestingly, these pulmonary strains producedminimal brain infection, suggesting thathypervirulence is not always related with theability to CNS infection. Moreover, infection ofmice with the other CSF-isolated strains (codes136and28),whichefficientlyinfectedthebrain,produced higher or similar mice survival andbacilli loads than infection of mice with mild,virulentpulmonarystrains. The most distinctive histological featureswere small or middle size nodules constitutedby lymphocytesandmacrophages located in thecerebralparenchymaneartopiamadreorbelowtheependimalcelllayer(Rich’snodules).Duringlateinfectionthesenoduleswerelargerandconnectedwith the subarachnoideal space producing mildorextensiveinflammatoryinfiltrateinmeninges.Another interesting histological observationwas the presence of extracellular positive acidfast bacilli or intracellular in astrocytes ormicroglia cells without inflammatory response.Immunohistochemicaldetectionofmycobacterialantigens showed strong positivity in activatedmicroglia, astrocytes, ependimal cells, andmeningothelial cells, indicating that all thesecell types are able to phagocytosemycobacteriaor their debries. Indeed, under physiologicalconditions,basalexpressionofsignificantinnateimmunity receptors involved in mycobacteriaphagocytosis (such as TLR-2 and TLR-4) weredetected in the meninges, choroids plexus, andcircumventricularbrainarea,whichlackBBBandare more exposed to pathogens. Microglia andastrocytescanalsoexpressthesereceptors.Thus,inbrainmycobacterialinfectionspecificbacterialantigensand theirhostcell receptors,aswellasinnateimmunityreceptorsareinvolved. In many areas where we found strongmycobacterial-antigen immunostaining in CNScells, there were not inflammatory infiltrate.Thus, an efficient modulation of inflammationis produced in the brain which could avoid ordelay tissue damage and signs of neurologicallesion, even in the presence of high amount of
bacilli.Th-2cellscouldparticipateinthisprocess,dueto itsefficientactivitytosuppressTh-1highactivitythat inducesexcessiveinflammationandtissue damage. Indeed, the Th-2 response alsohasbeneficialactivityforCNS,favouringhealingand supporting neuronal survival. We foundprogressive IL-4expression in thebrainofmiceinfectedwitheitherofthethreedifferentclinicalstrains isolated from the CSF of meningealtuberculous patients, while reference strainH37RvinducedlowexpressionofTh-2cytokine.Another significant anti-inflammatory cytokinelocalized in activated microglia is transforminggrowth factorbeta (TGFβ).We foundhighgeneexpressionofTGFβinthebrainofmiceinfectedwith CSF clinical isolates, and our histologicalstudies showed positive immunostaining inmacrophageslocatedinRich’snodules,aswellasin activated microglia and capillary endothelialcells from distant areas of the inflammatoryresponse.Thus,neuroprotectionandsuppressionby specific cytokines should be a significantfactortoavoidexcessiveinflammationandtissuedestructioninmycobacterialCNSinfection.Thisisinagreementwiththeobservationthatduringlate infection, when high bacilli loads in thebrainweredetermined,noneoftheinfectedmicedeveloped evident clinical signs of neurologicaldamage,suchasseizuresorparalysis.Thesamesituation has been reported in mice infectedwithhighdosesofM. aviumby the intravenousroute (7), or even in mice infected directly inthe brain (6).However, we observed significanthistological damage in the hippocampus area,wheremanyneuronsshowedacidophilicnecrosisand extensive gliosis in completely absence ofinflammation. These histological abnormalitiesshould be relatedwithmemory and cognocitivedisturbances. We consider that this experimental model,whichdemonstratesforthefirsttimetheexistenceof apparentlyneurotropicmycobacterial strains,could be a useful tool to study host and bacillifactors involved in thepathogenesisof themostsevereformoftuberculosis.
Special Communication |Modellingofcerebraltuberculosis
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Correspondence
DrRogelioHernándezPandoMD,MScandPhDImmunology(NationalUniversityofMexico)ExperimentalPathologySectionDepartmentofPathologyNationalInstituteofMedicalSciencesandNutrition“SalvadorZubirán”CalleVascodeQuiroga15,TlalpanCP14000,MéxicoD.F.MéxicoTel:(+52-55)54853491Fax:(+52-55)54853491Email:[email protected],[email protected]
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