Extensive Mycobacterium bovis BCG infection of liver parenchymal cells in immunocompromised mice.

A histologic study was performed on the livers of wild-type (WT), severe combined immunodeficient (SCID), hydrocortisone acetate (HC)-treated WT, and HC-treated SCID mice infected intravenously with 10(5) CFU of Mycobacterium bovis BCG. It was found that infection progressed faster in SCID mice than in WT mice and that HC treatment caused exacerbation of infection in both types of mice. In all cases infection in the liver was confined to granulomas that were populated predominantly by macrophages. Higher levels of infection in HC-treated SCID mice, but not HC-treated WT mice, were associated with extensive infection and destruction of parenchymal cells at the margins of granulomas. The results indicate that in the absence of T-cell-mediated immunity and of HC-sensitive T-cell-independent defense mechanisms, macrophages are incapable of restricting BCG growth and of confining infection to their cytoplasm. Consequently, BCG bacilli are released into the extracellular environment, where they are ingested by neighboring parenchymal cells.

The relative importance of T cell subsets in immunity and immunopathology of airborne Mycobacterium tuberculosis infection in mice.

Wild-type (WT) and targeted-mutant mice incapable of making alphabeta T cells, gammadelta T cells, class I major histocompatibility complex (MHC), class II MHC, interferon (IFN)-gamma, or inducible nitric oxide synthase (NOS2), were infected with Mycobacterium tuberculosis (Mtb) by aerosol, and monitored over time for their ability to (a) control infection, (b) develop histopathology at sites of infection, and (c) survive. WT mice acquired the ability to control and to hold infection at a stationary level from day 20 on. This was associated with the development of a macrophage-dominated alveolitis at sites of infection, with increased synthesis of IFN-gamma and NOS2 mRNA, and with an median survival time (MST) of 258.5 d. In the absence of alphabeta T cells, Mtb grew progressively and rapidly to induce a necrotic, neutrophil-dominated lung pathology that killed mice with an MST of 48 d. In the absence of CD4-mediated immunity (class II(-/-) mice), progressive bacterial growth continued in the lungs and in other organs beyond day 20, resulting in an MST of 77 d. By contrast, in the absence of CD8 T cell-mediated immunity, lung infection was controlled at a 1 log higher stationary level that induced a similar histopathologic response to that of WT mice, and resulted in an MST of 232 d.

Genetic control of susceptibility to infection with Mycobacterium tuberculosis in mice.

Genetic factors play a key role in host response, disease severity, and ultimate outcome of infection with Mycobacterium tuberculosis in humans. In the mouse, the DBA/2J strain is very susceptible to M. tuberculosis H37Rv infection, while the C57Bl/6J strain is resistant. In DBA/2J, a heavier bacterial burden causes a unique phenotype, that includes very severe and rapidly fatal pulmonary disease with extensive exudation of neutrophils and tissue necrosis, as opposed to slower progressive pulmonary disease characterized by the accumulation of epithelioid macrophages with protective immune and inflammatory responses in C57Bl/6J. To identify the genes responsible for differences in host response to M. tuberculosis in these two strains, 95 animals of an informative (C57Bl/6J x DBA/2J) F2 cross were infected intravenously with M. tuberculosis (1 x 10(5) CFU) and duration of survival was used as a quantitative phenotypic measure of susceptibility in a whole genome scan. Quantitative trait locus analysis (QTL) showed that the genetically controlled susceptibility was multigenic. QTL analysis identified two significant linkages on the distal portion of chromosome 1 (Trl-1, LOD, 4.80) and on the proximal portion of chromosome 7 (Trl-3, LOD, 4.66) that each account for approximately 21% of the phenotypic variance. A third suggestive linkage was identified on the proximal portion of chromosome 3 (Trl-2, LOD, 3.93; additional 18% of the variance). At each locus, homozygosity for the parental C57Bl/6J alleles was associated with increased resistance to infection. These novel mouse loci provide the basis for evaluating a possible association of the corresponding syntenic chromosomal regions in humans with susceptibility to tuberculosis.

Consequence of Nramp1 deletion to Mycobacterium tuberculosis infection in mice.

129sv mice functionally deleted of the antimicrobial resistance gene, Nramp1, were found to be as resistant as wild-type mice to infection with the virulent H37Rv strain of Mycobacterium tuberculosis, as determined by monitoring bacterial growth in major organs and recording host survival times. Death of infected mice of both types was associated with extensive infection-induced pathology in the lungs but not in other major organs. These findings are in keeping with the view that Nramp1 is of limited importance in resistance to tuberculosis in mice.

Growth rate of mycobacteria in mice as an unreliable indicator of mycobacterial virulence.

The CDC1551 strain of Mycobacterium tuberculosis was compared with the H37Rv strain of M. tuberculosis and the Ravenel strain of Mycobacterium bovis for virulence in mice. Although all three strains gave rise to the same level of stationary infection in major organs, mice infected with the Ravenel strain died much earlier from lung disease.

Genetically susceptible mice remain proportionally more susceptible to tuberculosis after vaccination.

DBA/2 mice are much more susceptible to infection with Mycobacterium tuberculosis than major histocompatibility complex-compatible BALB/c mice. It is shown here that, although vaccination provided mice of both strains with a capacity to reduce the level of infection in their lungs, vaccinated DBA/2 mice remained much more susceptible in this organ than vaccinated BALB/c mice. Consequently, the former mice developed more lung pathology and died much earlier than the latter. On the other hand, colony-forming unit counts and histology suggest that vaccination provided mice of both strains with an increased and equal ability to express immunity in the liver and spleen, thereby indicating that they possessed equal systemic levels of vaccine-induced immunity at the time of M. tuberculosis challenge. The results indicate that inefficient expression of immunity in the lungs is likely to prove an obstacle to successful vaccination against tuberculosis in resistant and susceptible mouse strains, but more so in the latter strains.

Vaccinated mice remain more susceptible to Mycobacterium tuberculosis infection initiated via the respiratory route than via the intravenous route.

Mice given Mycobacterium tuberculosis bacilli via the respiratory route succumbed much sooner to infection than mice given 1,000 times more bacilli via the intravenous route. Vaccination provided increased protection to an M. tuberculosis challenge infection; however, mice infected via the respiratory route remained much more susceptible.

How important is Nramp1 in tuberculosis?

The assumption that the antimicrobial resistance gene Nramp1 is a major determinant of resistance of mice to infection with virulent Mycobacterium tuberculosis can now be challenged on the basis of published evidence to the contrary. It is likely that, with tuberculosis, Nramp1-determined defenses are subordinate to other defenses.

Mice incapable of making IL-4 or IL-10 display normal resistance to infection with Mycobacterium tuberculosis.

With a view to determining whether production of Th2 cytokines, IL-4 or IL-10, is responsible for the inability of mice to resolve infection with Mycobacterium tuberculosis, mice with targeted disruption of their IL-4 or IL-10 gene were compared with wild-type mice in terms of their ability to defend against an M. tuberculosis infection initiated via the respiratory route. The results show that mice that are unable to make either IL-4 or IL-10 are no more capable than wild-type mice at defending against tuberculosis (TB). Therefore, the results are inconsistent with the proposition that the inadequacy of Th1-mediated anti-tuberculosis immunity is due to its down-regulation by either of these Th2 cytokines.

Resistance ranking of some common inbred mouse strains to Mycobacterium tuberculosis and relationship to major histocompatibility complex haplotype and Nramp1 genotype.

Six common inbred strains of mice and their F1 hybrids were examined for resistance to infection with the H37Rv strain of Mycobacterium tuberculosis. According to survival times after inoculation of 10(5) CFU intravenously (i.v.), the mice could be classified as being either highly susceptible (CBA, DBA/2, C3H, 129/SvJ) or highly resistant (BALB/c and C57BL/6). F1 hybrids of susceptible and resistant strains were resistant. Although an examination of a limited number of H-2 congenic strains showed that the H-2k haplotype could confer susceptibility on a resistant strain, it was evident that non-major histocompatibility complex (MHC) genes were much more important. Resistant strains all possessed the susceptibility allele of the anti-microbial resistance gene, Nramp1. Results obtained with selected strains infected with 10(2) CFU of M. tuberculosis by aerosol agreed with the results obtained with mice infected i.v. The size of the bacterial inoculum was important in distinguishing between resistant and susceptible strains, in that a 10(7) inoculum overcame the resistance advantage of one strain over another.

Murine listeriosis as a model of antimicrobial defense.

Murine listeriosis was introduced 35 years ago as a model with which to analyze mechanisms of antibacterial defense that are independent of antibodies. Listeria monocytogenes was shown to be an intramacrophage pathogen with capacity to induce the generation of a state of specific immunity in the form of DTH and a macrophage system with enhanced non-specific bactericidal activity. The demonstration that anti-Listeria immunity and DTH can be passively transferred with T cells was taken to indicate that the T cells responsible for DTH function upregulate the listericidal function of macrophages. This interpretation is contradicted by recent research showing that CD8 T cells, rather than CD4 T cells, are responsible for mediating adoptive immunity. However, T-cell depletion studies show that primary infection can eventually be resolved in the absence of either CD8 or CD4 T cells. On the other hand, infection becomes lethal in the absence of neutrophils or NK cells. It is apparent, therefore, that the most important defense against primary listeriosis resides with the functions of neutrophils and NK cells that are mobilized early in infection. Antigen-specific T cells function at a later time to resolve infection more efficiently. It is apparent that T cells are much more important in defense against secondary infection.

Identification of nitric oxide synthase as a protective locus against tuberculosis.

Mutagenesis of the host immune system has helped identify response pathways necessary to combat tuberculosis. Several such pathways may function as activators of a common protective gene: inducible nitric oxide synthase (NOS2). Here we provide direct evidence for this gene controlling primary Mycobacterium tuberculosis infection using mice homozygous for a disrupted NOS2 allele. NOS2(-/-) mice proved highly susceptible, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids, and allowed Mycobacterium tuberculosis to replicate faster in the lungs than reported for other gene-deficient hosts. Susceptibility appeared to be independent of the only known naturally inherited antimicrobial locus, NRAMP1. Progression of chronic tuberculosis in wild-type mice was accelerated by specifically inhibiting NOS2 via administration of N6-(1-iminoethyl)-L-lysine. Together these findings identify NOS2 as a critical host gene for tuberculostasis.

Persistent infection with virulent but not avirulent Mycobacterium tuberculosis in the lungs of mice causes progressive pathology.

A strain of Mycobacterium tuberculosis (H37Rv) considered virulent for mice and a strain (R1Rv) considered relatively avirulent were compared for their ability to survive host immunity in the lungs and to induce lung pathology. Although both strains of M. tuberculosis were capable of causing a slowly progressive infection in the lungs of immunocompetent mice, only the H37Rv strain was capable of inducing progressive destructive pathology and of causing loss of lung function over a 300-day period. Therefore, the ability to survive host immunity in the lungs and the ability to cause lung pathology are separate manifestations of mycobacterial virulence.

The Nramp1 antimicrobial resistance gene segregates independently of resistance to virulent Mycobacterium tuberculosis.

Nramp1 is a recently cloned gene that is involved in resistance of mice to infection with certain microbial pathogens, including the attenuated bacillus Calmette-Guérin (BCG) strain of Mycobacterium bovis, as well as certain other mycobacteria. With a view to determining whether Nramp1 influences resistance of mice to infection with virulent M. tuberculosis, BALB/c mice homozygous for the susceptibility allele of Nramp1, and DBA/2 mice homozygous for the resistance allele, as well as their F1 and F2 progeny, were typed according to their possession of these alleles using a 'hot start' polymerase chain reaction (PCR) procedure. As assessed by the ability of the mice to survive infection, the results show that Nramp1 plays no discernible role in resistance to tuberculosis.

Mice that carry the resistance allele of the Bcg gene (Bcgr) develop a superior capacity to stabilize bacille Calmette-Guerin (BCG) infection in their lungs and spleen over a protracted period in the absence of specific immunity.

In mice, natural resistance to infection with BCG is under the influence of an autosomal gene designated Bcg. It is shown here in agreement with others that mice that possess the dominant resistant allele of the gene (Bcgr) are more capable than mice that possess the susceptible recessive allele (Bcgs) at restricting the growth of BCG in their lungs, as well as in their spleens, during the first 20 days of infection. It is shown, in addition, that in the absence of specific immunity the resistance difference between Bcgr and Bcgs mice became much more pronounced as infection progressed beyond day 20. Whereas T cell-depleted Bcgr mice developed a capacity after day 20 to cause infection in their lungs and spleens to stabilize and plateau for at least 40 days, T cell-depleted Bcgs mice were unable to prevent infection from progressing in these organs. On the other hand, both types of T cell-depleted mice were capable of causing infection to plateau in their livers and kidneys. Moreover, this T cell-independent mechanism of resistance was essentially abolished in all organs in which it was expressed by treating the mice with hydrocortisone. In the lungs of immunocompetent Bcgs mice, failure to stabilize infection was associated with heavily infected macrophages and failure to contain BCG at original sites of infection.

Evidence inconsistent with a role for the Bcg gene (Nramp1) in resistance of mice to infection with virulent Mycobacterium tuberculosis.

The superior resistance of some strains of mice over others to infection with certain intracellular pathogens, including the vaccine strain of Mycobacterium bovis, bacillus Calmette Guerin (BCG), is determined by a gene associated with a small segment of chromosome 1 designated by Ity/Lsh/Bcg locus, referred to here as the Bcg locus. DBA/2 mice containing the dominant resistant allele of the Bcg gene (Bcgr), major histocompatibility complex-compatible BALB/c mice containing the recessive susceptible allele (Bcgs), and congenic C.D2-N20 Bcgr, which are genetically the same as BALB/c mice except for possessing a small piece of DBA/2 chromosome 1 containing the Bcg locus, were used to determine whether the Bcg gene determines resistance to infection with the virulent H37Rv strain of Mycobacterium tuberculosis (Mtb). According to the survival times of Bcgr and Bcgs mice infected via either the intravenous or respiratory route, Bcgr mice proved much less, rather than more, resistant to Mtb infection than Bcgs mice. Shorter survival times of Bcgr mice were associated with an inferior capacity to control Mtb growth in their lungs and to retard the development of Mtb-induced pathology in this organ. Resistance to Mtb infection was a dominant trait in the F1 progeny of Bcgr and Bcgs mice. The results show that resistance to Mtb is not determined by the resistance allele of the Bcg gene nor by the recently isolated candidate Bcg gene Nramp1, located in the Bcg locus.

Mycobacterium tuberculosis is strikingly more virulent for mice when given via the respiratory than via the intravenous route.

Fewer Mycobacterium tuberculosis colony-forming units given by aerosol were substantially more virulent for mice than much larger numbers inoculated intravenously (iv), as shown by a faster rate of bacillary growth in the lungs and much shorter survival of the host. Earlier death of mice infected by aerosol was associated with faster development of lung pathology, even though the number of M. tuberculosis given iv resulted in the same number of bacilli initially implanting in the lungs as the number given by aerosol. Mice depleted of CD4 T cells died of infection much sooner than immunocompetent mice but at about the same time after being infected via either route. The results indicate that a small number of M. tuberculosis colony-forming units given by aerosol is less immunogenic than a larger number given iv.