key molecules

Geneticcontrol of innateresistanceto mycobacterialinfections Erwin Schurr, Danielle Malo, Danuta Radzioch, Ellen Buschman, Kenneth Morgan, Philippe Gros and Emil Skamene The Mendelian segregation of resistance to infection in different strains of mice infected with mycobacteria, Salmonella and Leishmania spp, all ofwhich live in macrophages, is currently under close scrutiny. Here, Erwin Schurr and colleagues review the nature and function of the Beg gene in controlling innate resistance to mycobacterial infection in mice and speculate on the occurrence of a possible human equivalent. According to estimates by the World Health Organization, about 100 million people are infected with Mycobacterium tuberculosis, 10 million of which develop clinical forms of tuberculosis resulting in three million deaths annually’-3. Ten to 15 million people suffer from leprosy, a chronic debilitating mycobacterial (M. leprae) disease that can result in extensive physical deformation’. Effective chemotherapeutic treatment is available for both diseases; however, it is not routinely available in less developed areas, is dependent on long-term compliance to the drug regimen and is complicated by the emergence of drug-resistant mycobacteria. Consequently, the control of tuberculosis and leprosy relies heavily on vaccination program$. By far the most common vaccine for this purpose is an attenuated strain of Mycobacterium bovis Bacille Calmette Guerin (BCG), which has been used for almost 70 year@. One of the unsolved problems in vaccine immunology is the variable efficacy of BCG vaccination against tuberculosis and leprosy that has been observed in several large studie@. Genetic differences among trial populations is one of several possible factors that may account for these surprising variations. In mice, resistance to early growth of M. bovis/EXG is controlled by a single, dominant autosomal gene named Beg’. The same locus also controls innate resistance and susceptibility to M. lepraemuriu&, M. intracellulare9 and M. avium’O and is presently believed to be identical to the Lsh and 1~ loci, which control innate resistance to infection with L. donovani and S. typhimurium, respectively”. This article focuses on the results obtained using mycobacteria as infectious agents but it must be stressed that strategies similar to those described here have been used for the study of the 1~ and Lsh genes. The conclusions and hypotheses about how, when and where the host resistance locus might be expressed have been suggested by workers from several laboratoriesi2. It is now well established that the Beg gene is expressed by mature tissue macrophages. Experiments in viuo have shown that resistance to mycobacterial infection is inde0 1991,ElwvicrScicncc

pendent of T, B and natural killer (NK) cells, is susceptible to silica treatment but not to radiation and is a property of bone-marrow-derived precursor cells13J4. Similarly, cultivated niacrophages and retrovirusimmortalized macrophage lines displayed differential anti-mycobacterial activity as predicted by their allelic composition at the Beg IOCUS’~. From phenotypes to genes In the absence of any known or suspected gene product for Beg, we decided to use a genome approach for cloning this genei6. The first aim was to identify genetic markers that are tightly linked to Beg. Such markers are useful anchor points for the construction of a physical map. The molecular framework provided by a physical map is indispensable for the identification of short chromosomal segments harboring the Beg gene which can be used for cloning candidate genes. The Beg locus was unambiguously assigned to the proximal part of mouse chromosome 1 between ldh-1 and Pep-3 by linkage analyses in recombinant inbred strains and backcross progeny derived from inbred strains carrying either the Beg’ (resistant) or the Beg’ (susceptible) alleles Ii. This portion of mouse chromosome 1 was, therefore, targeted for saturation mapping experiments. Restriction fragment length polymorphisms (RFLP) for numerous probes were tested for linkage to the resistant or susceptible phenotype segregating in Beg congenic mice, recombinant inbred strains and backcross animals. So far, ten cloned loci have been located in the proximity of Beg. Genetic mapping studies have indicated the order of these genes as: centromere, Col3a1, Len2, Fn, (VillBcg), Des, InhA, Akp3, Acrg, Sag and Col6a3 (Fig. 1) r7,18. Three marker loci are at a genetic distance of less than 1 centiMorgan (CM) from Beg: Vi1 is the closest gene to Beg, followed by Des and InhA. However, distances between markers on a genetic map can be affected by a number of parameters such as interference or suppression of crossing-over, and usually do not provide an accurate estimate of the number of

Publishers Ltd.UK.0167-4919/91/502.00

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key ridecules base pairs separating different loci. The physical distance between genetic markers can be obtained by pulse field gel electrophoresis (PFGE) 19.Since Vil, Desand InhA are located in close proximity to Beg, these DNA markers were used to define the physical map around the Beg gene. Pulse field mapping data, using repeat-free sequenceslocated within the marker genesand anonymous probesobtained by chromosomewalking in recombinant cosmid libraries provided the physical map of two genomit domains overlapping 1000 kb in the vicinity of the Beg gene.These resultsclearly show that the Beg geneis now within reach of long-range cloning techniques. The DNA segmentsoverlapping Vi1 and Des are particularly rich in recognition sitesfor the enzymesBssHII, Mlul, Not1 and Sacll, which define the dinucleotide ‘CpG islands’ often associated with transcriptionally active geneslo.The identification of CpG-rich islandsand the detection of cross-hybridizing sequencesin other species (zoo blot) are two criteria that can be used to identify candidate gene sequenceson short DNA segments.Sequencesthat are candidates for the Beg locus will be tested for the presenceof corresponding cellular RNA transcripts in the tissuesthat are expected to expressthe resistancephenotype. Cloned candidate genesmay then be testedby transfection into macrophagecell lines from Beg’ and Beg’ mice and thesecells may be monitored for the differential expressionof functional and phenotypic parameters characteristic of the resistancephenotype. However, the ultimate test for any cloned candidate gene is the creation of a transgenic mouse with an altered resistance phenotype due to the introduction of the transgene. From mice to humans Most individuals infected with M. leprae or M. tuberczrlosis develop effective immunity without clinically evident forms of the disease,whereas those people who develop tuberculosisor leprosy presentwith a spectrum of clinical manifestations”,“. There is substantial evidence that genetic factors are crucial elementsin determining both susceptibility and the clinical spectrum of the disease23-25 and numerousinvestigationshave associated HLA-DR haplotypes with the subtypesof leprosy or tuberculosis’6-19.Conversely, none of the HLA studies has provided significant evidence that susceptibility to diseaseper se is controlled by HLA polymorphisms. Segregationand linkage analysessuggesta more complex genetic model and are compatible with the hypothesis that susceptibility to the establishment of clinically significant infection is mediated by a non-HLA-linked recessive or co-dominant locus, possibly a human homologue of the mouseBeg gene30a3’. Our experimentsare presently focusing on the localization of a locus for susceptibility to leprosy and tuberculosisin the human genome.Although it isnow possibleto screenmore than 90% of the total human genomefor the position of a locus, this is still a laborious task and we have chosena homology mappingstrategy. We speculate that, owing to the phenomenologicalsimilarity between innate susceptibility to mycobacterial infections in mice and in humans, a human homologue of the mouseBeg locus is indeed the prime candidate for a diseasesusceptibility gene. Experiments in our laboratory have ident-

Mouse chromosome

Map

Human chromosome

localization

a NEB Break-w

2q31-32.2 ELN

2q31-32.3

CRYG14

2q33-35

FN VIL

2q34-36 2q35-36 CHRNG

Break-

1

2q31 -q ler

COLBAl

COMA3

2q32-q 2q37

GCG

2q36-37

lel

I

Fig. 1. Schematic representation of proximal mouse chromosome 1 and the telomeric end of human chromosome 2~7. The proposed breakpoirrts of a conserved synteny between mouse chromosome 1 and human chromosome 2q are indicated by arrows. The distance of the mcxlse loci is given in centiMorgans on the left of the chromosome. The map location of the human loci on chromosome 2q as derived by in situ hybridization is given on the right-hand side of the diagram.

ified a 35 CM chromosomalsegmenton proximal mouse chromosome 1 that includes Beg and that is precisely conserved on the telomeric end of human chromosome 2q (Fig. l)lx. A number of polymorphic DNA probesthat localize to this segmentof human chromosome2q have been generated and usedfor familial linkage analysisof tuberculosis and leprosy. In linkage analysis,the significance of the evidence in favor of linkage at a specified recombination fraction is measuredby the LOD score (logarithm to the base 10 of the odds ratio)31. A LOD scoreof + 3 betweentwo randomly chosenloci indicates odds in favor of linkage of 1000: 1 at the particular recombination fraction. Recent advancesin the statistical evaluation of complex traits have provided tools to improve the detection of linkage between genetic markers and complex traits33.34.These methods allow analysis of complicated genetic models including the interaction of genesand incomplete penetrance of trait expression. Preliminary analysisof our data has provided evidence for a linkage between chromosome 2q polymorphisms and susceptibility to tuberculosis, although theseresultsare not yet statistically significant. However, since LOD scoresare additive between different families, the investigation of additional pedigreeswill confirm whether or not the diseaseresistancelocusis linked with the markers located on human chromosome2q. Pleiotropic effects of the Beg gene Based on the observation that innate resistance is expressedby macrophages,we and others developed the hypothesisthat the mechanismof action of the Beg gene might be better understood by exploring the parameters of macrophageactivation in mice congenic for this gene. There is wide agreementthat the magnitude of both the hexose monophosphateshunt and the respiratory burst

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activity are reliable parameters for the activational status of macrophages35. Indeed, phagocytosis or ligandinduced respiratory burst activity of macrophages from resistant animals (Bcf) was found to be significantly superior to that displayedby macrophagesfrom susceptible mice (Beg”) following either infection with BCG or treatment with gamma-interferon (IFN-y)36,37. In line with these results was the finding that phenotypic markersof macrophageactivation were alsoexpressedat increasedlevelson Bcgr macrophages.Phenotypic analysis of normal, uninfected splenic macrophage poplations by fluorescence activated cell sorter (FACS) showed that Beg’ congenic mice contained lo-15% more la+ cellscomparedwith their Beg” counterparts38. IFN-y treatment also causedupregulation of classII la moleculesand of the AcM.1 activation marker in macrophagesfrom Bcgr mice‘3+j9.The unifying hypothesisfrom theseexperimentsis that the Beg generegulatesthe level of macrophageactivation and that macrophagesfrom innately resistant mice (Beg’) are genetically programmed to switch readily to the activated mode, whereas macrophagesfrom innately susceptible mice (Beg”) are lesseasily activated. The effect of the Beg geneon macrophageactivation alsoexplains the previous observationsthat during BCG infection Beg’ miceproduce more interleukin 2 than Beg’ mice40.In vitro, assaysshow a clear associationbetween heightenedT-cell proliferation, increasedIa expression and the antigen-presentingability of macrophagesisolated from Beg’ mice after BCG4’ and L. donovani infection42. In contrast, Begsanimals displayed macrophage suppressionof T-cell proliferation43, significant expansion of the B-cell compartment40and higher levels of serum antibodies to BCG compared with their Bcgg’ counterparts. To reconcile these observations, we are pursuing the hypothesis that the different antigenpresentingfunctions in Bcg’and Beg”macrophagesinfluence the type of helper T cell (T,l (T-cell stimulating) versus T,Z (B-cell stimulating)) activated during BCG infection. To facilitate the study of the molecular nature of the Beg gene,bone marrow macrophagesfrom mousestrains congenic at the Beg locus have been immortalized by infection with recombinant J2 retrovirus. Two cloned macrophagecell lines,BlOS(Bcg’) and BlOR(Bcg$ have been produced, both of which exhibit surface markers and morphology typical of mature tissue macrophages and are similar in their phagocytic activity, in their level of c-fms and transforming growth factor p (TGF-P) mRNA expression and in their rumoricidal activity in responseto IFN-y. However, the BlOR(Bcg’) cell line constitutively expressesincreased bactericidal activity and higher levels of both I-AP mRNA and Ia antigen compared with BlOS cells. Likewise, treatment of cells with IFN-y induced maximal expressionof Ia antigen after 48 h in BlOR but only after 72 h in BlOS macrophages.We conclude from these results that essential features of Beg’ and Beg phenotypes, as expressedby splenic tissuemacrophages,have been preservedin the immortalized macrophagelines. Consequently, we expect these cell lines to be valuable tools for further analyses of the molecular basis of the resistance phenotype.

Conclusion The genetically determineddefect in Beg” macrophage activation and mycobacterial activity could possibly OCcur at a number of points in the seriesof biochemical reactions that connect the membrane perturbation induced by the invading mycobacteria with the emergence of anti-mycobacterial effector mechanisms.At least two generalmodelsexplaining the singlegeneeffect of Beg on macrophageactivation may be considered.First, the two allelesof the Beg genemay encodea functional wild type and nonfunctional mutant form of a (structural) molecule directly involved in signal transduction. Dysregulation of diacylglycerol-inositoltriphosphate metabolism, protein kinase activation or production of secondary messengerssuch as CAMP may occur in the mutant. Second,the Bcggenemay encodea DNA-binding protein that controls transcriptional events associated with macrophageactivation. In this context, it is interesting to note that IFN-7 treatment enhances the pleiotropic effects of the Beg gene, which possibly reflects an interaction of the Beg gene product with IFN-responsive sequences. Erwin

Schurr, Danielle Malo, Danuta Radzioch, Ellen Ken Morgan, Philippe Gras and Emil Skamene are at the McGill Centre for the Study of Host Resistance, Montreal General Hospitcl Research Institute, Montreal, Quebec H3G lA4, Canada. Buschnan,

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