Properties and protective value of the secondary versus primary T helper type 1 response to airborne Mycobacterium tuberculosis infection in mice.

Mice immunized against Mycobacterium tuberculosis (Mtb) infection by curing them of a primary lung infection were compared with naive mice in terms of the ability to generate a Th1 cell immune response and to control growth of an airborne Mtb challenge infection. Immunized mice generated and expressed Th1 cell immunity several days sooner than naive mice, as demonstrated by an earlier increase in the synthesis in the lungs of mRNA for Th1 cytokines and for inducible nitric oxide synthase, an indicator of macrophage activation. This Th1 cytokine/mRNA synthesis was accompanied by an earlier accumulation of Mtb-specific Th1 cells in the lungs and the presence of CD4 T cells in lesions. An earlier generation of immunity was associated with an earlier inhibition of Mtb growth when infection was at a 1-log lower level. However, inhibition of Mtb growth in immunized, as well as in naive, mice was not followed by resolution of the infection, but by stabilization of the infection at a stationary level. The results indicate that there is no reason to believe that the secondary response to an Mtb infection is quantitatively or qualitatively superior to the primary response.

Carbon flux rerouting during Mycobacterium tuberculosis growth arrest.

A hallmark of the Mycobacterium tuberculosis life cycle is the pathogen's ability to switch between replicative and non-replicative states in response to host immunity. Transcriptional profiling by qPCR of ∼ 50 M. tuberculosis genes involved in central and lipid metabolism revealed a re-routing of carbon flow associated with bacterial growth arrest during mouse lung infection. Carbon rerouting was marked by a switch from metabolic pathways generating energy and biosynthetic precursors in growing bacilli to pathways for storage compound synthesis during growth arrest. Results of flux balance analysis using an in silico metabolic network were consistent with the transcript abundance data obtained in vivo. Similar transcriptional changes were seen in vitro when M. tuberculosis cultures were treated with bacteriostatic stressors under different nutritional conditions. Thus, altered expression of key metabolic genes reflects growth rate changes rather than changes in substrate availability. A model describing carbon flux rerouting was formulated that (i) provides a coherent interpretation of the adaptation of M. tuberculosis metabolism to immunity-induced stress and (ii) identifies features common to mycobacterial dormancy and stress responses of other organisms.

Disseminated and rapidly fatal tuberculosis in mice bearing a defective allele at IFN regulatory factor 8.

The interferon regulatory factor (IRF) family member IRF-8 participates in IFN-gamma-dependent transcriptional activation of genes containing in their promoter regions IFN-stimulated response element or IFN-gamma activation site elements. To test the role of IRF-8 in host defenses against tuberculosis, BXH-2 mice, which bear a defective IRF-8(R294C) allele, were challenged with low doses of virulent Mycobacterium tuberculosis via the i.v. and aerosol routes. BXH-2 mice were found to be extremely susceptible to M. tuberculosis, as demonstrated by rapid and uncontrolled microbial replication in spleen, liver, and lungs leading to very early death. The BXH-2 defect was expressed very early (10 days postinfection) as uncontrolled intracellular pathogen replication in NOS2-expressing lung macrophages, impaired granuloma formation, rapid dissemination of the infection to distant sites, and rapid necrosis of infected tissues. There was complete absence of IL-12p40 induction, severely reduced IFN-gamma production, and impaired T cell priming in the lungs of infected BXH-2, highlighting the critical role of IRF-8 in this process. Collectively, these results identify IRF-8 as a critical regulator of host defenses against tuberculosis.

Genetic and functional characterization of the mouse Trl3 locus in defense against tuberculosis.

The genetic control of susceptibility to tuberculosis in DBA/2J and C57BL/6J mice is complex and influenced by at least four tuberculosis resistance loci (Trl1-Trl4). To further study the Trl3 and Trl4 loci, we have created congenic mouse lines D2.B6-Chr7 and D2.B6-Chr19, in which resistant B6-derived portions of chromosome 7 (Chr.7) and chromosome 19 (Chr.19) overlapping Trl3 and Trl4, respectively, were independently introgressed onto susceptible D2 background. Transfer of B6-derived Trl3 chromosome 7 segment significantly increased resistance of D2 mice, as measured by reduced pulmonary microbial replication at day 70, and increased host survival following aerosol infection. However, transfer of B6-derived chromosome 19 (Trl4) onto D2 mice did not increase resistance by itself and does not improve on the protective effect of chromosome 7. Further study of the protective effect of Trl3 in D2.B6-Chr7 mice indicates that it does not involve modulation of timing or magnitude of Th1 response in the lung, as investigated by measuring the number of Ag-specific, IFN-gamma-producing CD4(+) and CD8(+) T cells. Rather, Trl3 appears to affect the intrinsic ability of activated macrophages to restrict intracellular mycobacterial replication in an NO synthase 2-independent fashion. Microarray experiments involving parental and congenic mouse lines identified a number of genes in the Trl3 interval on chromosome 7 the level of expression of which before infection or in response to Mycobacterium tuberculosis infection is differentially regulated in a parental haplotype-dependent fashion. This gene list represents a valuable entry point for the identification and prioritization of positional candidate genes for the Trl3 effect on chromosome 7.

Virulent but not avirulent Mycobacterium tuberculosis can evade the growth inhibitory action of a T helper 1-dependent, nitric oxide Synthase 2-independent defense in mice.

Control of infection with virulent Mycobacterium tuberculosis (Mtb) in mice is dependent on the generation of T helper (Th)1-mediated immunity that serves, via secretion of interferon (IFN)-gamma and other cytokines, to upregulate the antimycobacterial function of macrophages of which the synthesis of inducible nitric oxide synthase (NOS)2 is an essential event. As a means to understanding the basis of Mtb virulence, the ability of gene-deleted mice incapable of making NOS2 (NOS2(-/-)), gp91(Phox) subunit of the respiratory burst NADPH-oxidase complex (Phox(-/-)), or either enzyme (NOS2/Phox(-/-)), to control airborne infection with the avirulent R1Rv and H37Ra strains of Mtb was compared with their ability control infection with the virulent H37Rv strain. NOS2(-/-), Phox(-/-), and NOS2/Phox(-/-) mice showed no deficiency in ability to control infection with either strain of avirulent Mtb. By contrast, NOS2(-/-) mice, but not Phox(-/-) mice, were incapable of controlling H37Rv infection and died early from neutrophil-dominated lung pathology. Control of infection with avirulent, as well as virulent Mtb, depended on the synthesis of IFN-gamma, and was associated with a substantial increase in the synthesis in the lungs of mRNA for IFN-gamma and NOS2, and with production of NOS2 by macrophages at sites of infection. The results indicate that virulent, but not avirulent, Mtb can overcome the growth inhibitory action of a Th1-dependent, NOS2-independent mechanism of defense.

Fibrotic response as a distinguishing feature of resistance and susceptibility to pulmonary infection with Mycobacterium tuberculosis in mice.

The differential susceptibility of inbred mouse strains DBA/2J (susceptible) and C57BL/6J (resistant) to pulmonary tuberculosis following aerosol infection is under complex genetic control. In this report, transcriptional profiling with RNAs from Mycobacterium tuberculosis-infected lungs was used to investigate the physiological response, cell type, and biochemical pathways underlying differential susceptibility to infection. Statistical analysis of cDNA-based microarrays revealed that 1,097 transcripts showed statistically significant changes in abundance (changes of > or = 1.5-fold) in at least one of four experimental group comparisons (C57BL/6J [day 0] versus DBA/2J [day 0] mice, C57BL/6J [day 90] versus DBA/2J [day 90] mice, C57BL/6J [day 90] versus C57BL/6J [day 0] mice, or DBA/2J [day 90] versus DBA/2J [day 0] mice). A group of genes showing very high degrees of significance (changes of > or = 2.0-fold) displayed enrichment for transcripts associated with tissue remodeling and the fibrotic response. The differential expression of fibrotic response genes (Sparc, Col1a1, Col1a2, Col4a1, and Col4a2) in the infected lungs of the two mouse strains was validated by another microarray platform (Affymetrix oligonucleotide chips) and by reverse transcription-PCR. Furthermore, the differential expression of additional genes known to be associated with fibrosis (Mmp2, Timp1, and Arg1) was also validated by these approaches. Overall, these results identify the differential fibrotic response as a pathological basis for the high susceptibility of DBA/2J mice to pulmonary tuberculosis.

'Immunization' against airborne tuberculosis by an earlier primary response to a concurrent intravenous infection.

Tuberculosis in mice is a lung disease. Airborne infection of this host species with Mycobacterium tuberculosis (Mtb) resulted in 20 days of Mtb growth in the lungs before further growth was inhibited and the level of infection stabilized. Inhibition of Mtb growth was associated with the production of interferon-gamma (IFN-gamma)-producing T cells in the lymph nodes and spleen and with the progressive accumulation of these cells in the lungs. Production of IFN-gamma-producing T cells was not discernable until about day 15 of infection, presumably because Mtb did not disseminate from the lungs to the draining lymph nodes and spleen until after an approximate 10-day delay. By contrast, in mice infected via the intravenous (i.v.) route, the spleen became infected almost immediately, resulting in much earlier production of IFN-gamma-producing T cells and earlier control of spleen and lung infection. In mice infected concurrently via both routes, earlier generation of immunity to the i.v. infection resulted in earlier accumulation of IFN-gamma-producing T cells in the lungs and earlier control of lung infection that was initiated via the airborne route. This protection against airborne infection afforded by an earlier primary immune response is equivalent to that expressed by mice vaccinated with bacillus Calmette-Guérin or certain other vaccines.

Transcriptional characterization of the antioxidant response of Mycobacterium tuberculosis in vivo and during adaptation to hypoxia in vitro.

Transcriptional profiling of antioxidant genes of Mycobacterium tuberculosis was performed by real-time RT-PCR during mouse lung infection and during adaptation to gradual oxygen depletion in vitro. M. tuberculosis genes involved in major detoxification pathways of oxidative stress were not up-regulated during chronic mouse lung infection, which is established in response to expression of host adaptive immunity. This result suggests that a major function of bacterial antioxidant enzymes is to protect from oxidants generated during the early, acute phase of infection. In vivo transcription profiles of bacterial antioxidant enzymes differed from those seen under adaptation to low oxygen in vitro, indicating differences between growth arrest in vivo and that induced by hypoxia in vitro.

Superior virulence of Mycobacterium bovis over Mycobacterium tuberculosis (Mtb) for Mtb-resistant and Mtb-susceptible mice is manifest as an ability to cause extrapulmonary disease.

Mice of a Mycobacterium tuberculosis-resistant (BALB/c) and of a M. tuberculosis-susceptible (DBA/2) strain proved considerably more susceptible, and equally so, to infection with Mycobacterium bovis than with M. tuberculosis when infection was initiated via the iv route. Infection with M. tuberculosis was eventually controlled at an approximately stationary level in the lungs, livers, spleens and kidneys of BALB/c mice, and in all of these organs except the lungs in DBA/2 mice. M. tuberculosis-infected DBA/2 mice died with a much shorter median survival time (MST) than M. tuberculosis-infected BALB/c mice. By contrast, infection with M. bovis killed mice of both strains with the same and much shorter MST. Unexpectedly, M. bovis caused progressive infection and pathology in the livers of BALB/c mice, but not in this organ in DBA/2 mice. More importantly, this pathogen caused progressive infection and infection-induced pathology in the kidneys and adrenal glands of both strains of mice. It is proposed that disease of the adrenal glands might serve to explain why M. bovis caused mice of both strains to die with the same much shorter MST.

Differences in the ability to generate type 1 T helper cells need not determine differences in the ability to resist Mycobacterium tuberculosis infection among mouse strains.

BACKGROUND: C57BL/6 (B6) and BALB/c mice are considerably more resistant to infection with Mycobacterium tuberculosis than DBA/2 mice. METHODS: To determine whether the difference in resistance is because DBA/2 mice generate a type 1 T helper (Th1) immune response of lower magnitude, the Th1 response to airborne infection of mice of all 3 strains was measured in terms of the number of interferon (IFN)-gamma-producing CD4 and CD8 T cells generated. RESULTS: Despite the superior resistance of BALB/c mice compared with DBA/2 mice, both strains generated a similarly low number of Th1 cells. On the other hand, B6 mice, despite being approximately equal in resistance compared with BALB/c mice, generated a much larger number of Th1 cells. In DBA/2 mice, a higher level of lung infection was associated with larger numbers of M. tuberculosis bacilli in individual macrophages at sites of infection, indicating lower levels of macrophage mycobacteriostatic function. Despite this, infected macrophages from DBA/2 mice stained positive for nitric oxide synthase type 2 (NOS2) by immunocytochemistry as intensely as did infected macrophages from B6 and BALB/c mice, indicating the acquisition of NOS2-dependent mycobacteriostatic function in all cases. CONCLUSION: The ability to generate a large number of Th1 cells need not determine the ability to resist M. tuberculosis infection.

Expression levels of Mycobacterium tuberculosis antigen-encoding genes versus production levels of antigen-specific T cells during stationary level lung infection in mice.

Mycobacterium tuberculosis lung infection in mice was controlled at an approximately stationary level after 20 days of log linear growth. Onset of stationary level infection was associated with the generation by the host of T helper type 1 (Th1) immunity, as evidenced by the accumulation of CD4 Th1 cells specific for the early secretory antigen (ESAT-6) of M. tuberculosis encoded by esat6, and for a mycolyl transferase (Ag85B) encoded by fbpB. CD4 T cells specific for these antigens were maintained at relatively high numbers throughout the course of infection. The number of CD4 T cells generated against ESAT-6 was larger than the number generated against Ag85B, and this was associated with a higher transcription level of esat6. The total number of transcripts of esat6 increased during the first 15 days of infection, after which it decreased and then approximately stabilized at 10(6.5) per lung. The total number of fbpB transcripts increased for 20 days of infection before decreasing and then approximately stabilizing at 10(4.8) per lung. The number of transcripts of esat6 per colony-forming unit of M. tuberculosis fell from 8.6 to 0.8 after day 15, and of fbpB from 0.3 to less than 0.02 after day 10, suggesting that at any given time during stationary level infection the latter gene was expressed by a very small percentage of bacilli. Expressed at an even lower level was an M. tuberculosis replication gene involved in septum formation (ftsZ), indicating that there was no significant turnover of the M. tuberculosis population during stationary level infection.

Changes in energy metabolism of Mycobacterium tuberculosis in mouse lung and under in vitro conditions affecting aerobic respiration.

Transcription profiling of genes encoding components of the respiratory chain and the ATP synthesizing apparatus of Mycobacterium tuberculosis was conducted in vivo in the infected mouse lung, and in vitro in bacterial cultures subjected to gradual oxygen depletion and to nitric oxide treatment. Transcript levels changed dramatically as infection progressed from bacterial exponential multiplication (acute infection) to cessation of bacterial growth (chronic infection) in response to host immunity. The proton-pumping type-I NADH dehydrogenase and the aa3-type cytochrome c oxidase were strongly down-regulated. Concurrently, the less energy-efficient cytochrome bd oxidase was transiently up-regulated. The nitrate transporter NarK2 was also up-regulated, indicative of increased nitrate respiration. The reduced efficiency of the respiratory chain was accompanied by decreased expression of ATP synthesis genes. Thus, adaptation of M. tuberculosis to host immunity involves three successive respiratory states leading to decreased energy production. Decreased bacterial counts in mice infected with a cydC mutant (defective in the cytochrome bd oxidase-associated transporter) at the transition to chronic infection provided initial evidence that the bd oxidase pathway is required for M. tuberculosis adaptation to host immunity. In vitro, NO treatment and hypoxia caused a switch from transcription of type I to type II NADH dehydrogenase. Moreover, cytochrome bd oxidase expression increased, but cytochrome c oxidase expression decreased slightly (nitric oxide) or not at all (hypoxia). These specific differences in respiratory metabolism during M. tuberculosis growth arrest in vitro and in vivo will guide manipulation of in vitro conditions to model bacterial adaptation to host immunity.

Immunity to tuberculosis.

Only 5 to 10% of immunocompetent humans are susceptible to tuberculosis, and over 85% of them develop the disease exclusively in the lungs. Human immunodeficiency virus (HIV)-infected humans, in contrast, can develop systemic disease that is more quickly lethal. This is in keeping with other evidence showing that susceptible humans generate some level of Th1 immunity to Mycobacterium tuberculosis (Mtb) infection. Tuberculosis in mice is also exclusively a lung disease that is progressive and lethal, in spite of the generation of Th1-mediated immunity. Thus mouse tuberculosis is a model of tuberculosis in susceptible humans, as is tuberculosis in guinea pigs and rabbits. Inability to resolve infection and prevent disease may not be a consequence of the generation of an inadequate number of Th1 cells but of an intrinsic deficiency in macrophage function that prevents these cells from expressing immunity. If this proves to be true, vaccinating susceptible humans against tuberculosis will be a difficult task.

The requirement of tumour necrosis factor-alpha and interferon-gamma for the expression of protective immunity to secondary murine tularaemia depends on the size of the challenge inoculum.

The present study was conducted to determine the extent to which the cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) are required to protect against primary or secondary murine tularaemia caused by the live vaccine strain of the facultative intracellular bacterium Francisella tularensis. It is shown that non-immune mice treated with neutralizing monoclonal antibodies (mAbs) against TNF-alpha and IFN-gamma are rendered defenceless against otherwise sublethal intravenous inocula of the bacterium. Treatment with either of the anti-cytokine mAbs resulted in even a very small inoculum of 500 c.f.u. of the pathogen multiplying unrestrictedly in the livers, spleens and lungs of non-immune mice to rapidly reach lethal numbers. By contrast, Francisella-immune mice treated with either of the mAbs remained capable of resolving secondary infection with 50-fold larger inocula. However, the need for TNF-alpha and IFN-gamma for controlling secondary tularaemia became critical when challenge inocula exceeded 10(6) c.f.u. Overall, the results imply that different defence mechanisms operate to control primary versus secondary murine tularaemia. Additionally, they show that the need for TNF-alpha and IFN-gamma to combat secondary infection depends on the size of the challenge inoculum.

Expression of NADPH oxidase-dependent resistance to listeriosis in mice occurs during the first 6 to 12 hours of liver infection.

Wild-type mice inoculated with Listeria monocytogenes intravenously were capable of reducing the bacterial load in their livers by 90% within 6 h. In contrast, mice with deletions of the gene for NADPH oxidase were incapable of expressing this early oxygen-dependent anti-Listeria defense and consequently showed higher levels of liver infection at later times.

Evidence inconsistent with a negative influence of T helper 2 cells on protection afforded by a dominant T helper 1 response against Mycobacterium tuberculosis lung infection in mice.

Mice incapable of generating an efficient Th2 response because of functional deletion of the genes for signal transducer and activation of transcription 6 (Stat6), interleukin-4 receptor alpha chain (IL-4Ralpha), or IL-4 plus IL-13 (IL-4/IL-13) were no more resistant than wild-type (WT) mice to airborne infection with virulent Mycobacterium tuberculosis. WT mice were able to control infection and hold it at a stationary level following 20 days of log linear M. tuberculosis growth. Likewise, infection was kept under control and was held at the same stationary level in IL-4/IL-13(-/-) mice but progressed to a slightly higher level in Stat6(-/-) and IL-4Ralpha(-/-) mice. The onset of stationary-level infection in WT mice was associated with the expression of Th1-mediated immunity, as evidenced by an approximately 100- to 1,000-fold increase in the lungs in the synthesis of mRNA for IL-12, gamma interferon (IFN-gamma), and inducible nitric oxide synthase (NOS2) that was sustained for at least 100 days. IL-12 is essential for the induction of Th1 immunity, IFN-gamma is a key Th1 cytokine involved in mediation of immunity, and NOS2 is an inducible enzyme of macrophages and is needed by these cells to express immunity. In response to infection, the lungs of Stat6(-/-) mice showed increases in synthesis of mRNA for IL-12, IFN-gamma, and NOS2 similar to that seen in WT mice. In IL-4/IL-13(-/-) mice, however, synthesis of mRNA for IFN-gamma and NOS2 reached higher levels than in WT mice. These results argue against the notion that a Th2 response is partly or wholly responsible for the inability of Th1-mediated immunity to resolve infection with a virulent strain of M. tuberculosis.

Expression of Th1-mediated immunity in mouse lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating persistence.

The lung is the primary target of infection with Mycobacterium tuberculosis. It is well established that, in mouse lung, expression of adaptive, Th1-mediated host immunity inhibits further multiplication of M. tuberculosis. Here, real-time RT-PCR was used to define the pattern of expression against time of lung infection of key genes involved in Th1-mediated immunity and of selected genes of M. tuberculosis. Inhibition of bacterial multiplication was preceded by increased mRNA synthesis for IFN-gamma and inducible NO synthase (NOS2) and by NOS2 protein synthesis in infected macrophages. Concurrently, the pattern of transcription of bacterial genes underwent dramatic changes. mRNA synthesis increased for alpha-crystallin (acr), rv2626c, and rv2623 and decreased for superoxide dismutase C (sodC), sodA, and fibronectin-binding protein B (fbpB). This pattern of M. tuberculosis transcription is characteristic of the nonreplicating persistence [Wayne, L. G. & Sohaskey, C. D. (2001) Annu. Rev. Microbiol. 55, 139-163] associated with adaptation of tubercle bacilli to hypoxia in vitro. Based on this similarity, we infer that host immunity induces bacterial growth arrest. In IFN-gamma gene-deleted mice, bacterial growth was not controlled; NOS2 protein was not detected in macrophages; sodC, sodA, and fbpB transcription showed no decrease; and acr, rv2626c, and rv2623 transcription increased only at the terminal stages of lung pathology. These findings define the transcription signature of M. tuberculosis as it transitions from growth to persistence in the mouse lung. The bacterial transcription changes measured at onset of Th1-mediated immunity are likely induced, directly or indirectly, by nitric oxide generated by infected macrophages.

Increased interleukin-10 expression is not responsible for failure of T helper 1 immunity to resolve airborne Mycobacterium tuberculosis infection in mice.

With a view to determining whether failure of mice to resolve Mycobacterium tuberculosis (Mtb) infection is a consequence of downregulation of T helper 1 (Th1) immunity by interleukin (IL)-10, mice deleted of the gene for IL-10 were compared with wild-type (WT) mice in terms of their ability to make IL-10 mRNA, generate Th1-mediated immunity [as measured by synthesis of mRNA for interferon-gamma (IFN-gamma)], IL-12p40 and inducible nitric oxide synthase (iNOS), and to control lung infection. It was found that the response of WT mice to infection included a substantial and sustained increase in IL-10 mRNA synthesis in the lungs. A Th1 response in the lungs of WT and IL-10-/- mice was evidenced by a large and sustained increase in the synthesis of mRNA for IFN-gamma, IL-12p40 and iNOS, with somewhat higher levels of these mRNA species being made in the lungs of IL-10-/- mice, particularly at an early stage of infection. However, IL-10-/- mice were no more capable than WT mice at combating infection.

Susceptibility to tuberculosis: a locus on mouse chromosome 19 (Trl-4) regulates Mycobacterium tuberculosis replication in the lungs.

The mouse DBA/2 (D2) strain is very susceptible to infection with virulent Mycobacterium tuberculosis, whereas C57BL/6 (B6) is much more resistant. Infection of D2 and B6 mice with M. tuberculosis H37Rv by the respiratory route is biphasic: during the first 3 weeks, there is rapid bacterial growth in the lung of both strains, whereas beyond this point replication stops in B6 but continues in D2, causing rapidly fatal pulmonary disease. To identify the genes regulating growth of M. tuberculosis in the lungs of these two strains, 98 informative (B6 x D2) F2 mice were infected by the respiratory route with M. tuberculosis H37Rv (2 x 102 colony-forming units), and the extent of bacterial replication in the lungs at 90 days was used as a quantitative measure of susceptibility in a whole-genome scan. Quantitative trait locus mapping identified a major locus on chromosome 19 (Tuberculosis resistance locus-4, Trl-4; logarithm of odds 5.6), which regulated pulmonary replication of M. tuberculosis and accounted for 25% of the phenotypic variance. B6 alleles at Trl-4 were inherited in an incompletely dominant fashion and associated with reduced bacterial replication. An additional effect of a locus (Trl-3), previously shown to affect survival to i.v. infection with M. tuberculosis, was also noted. F2 mice homozygous for B6 alleles at both Trl-3 and Trl-4 were as resistant as B6 parents, whereas mice homozygous for D2 alleles were as susceptible as D2 parents. These results suggest a strong genetic interaction between Trl-3 and Trl-4 in regulating pulmonary replication of M. tuberculosis.