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

Abstract

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.

Figure 1

Mice were infected with approximately 10² colony-forming units (CFU) of Mycobacterium tuberculosis (Mtb) via the airborne route, 10⁵ CFU via the intravenous (i.v.) route, or 10² via the airborne plus 10⁵ via the i.v. route. Lung infection in mice infected concurrently via the i.v. and airborne routes was controlled earlier than in mice infected via the respiratory route alone, but was controlled earlier still in mice infected via the i.v. route alone. Spleen infection was initiated immediately and controlled after approximately 10 days in mice infected via the i.v. route, or via both routes. It was not initiated until after about a 12-day delay in mice infected via the respiratory route alone, and was not controlled until after day 20. The draining lymph nodes (LNs) became infected on day 10 in mice infected via the respiratory route alone, and probably earlier in mice infected via the i.v. route, or via both routes. Control of LN infection occurred on day 15 in mice infected via the i.v. route or via both routes, and on day 20 in mice infected via the respiratory route alone. Means ± SD of four mice per group per time-point.

Figure 2

Lung infection initiated via the intravenous (i.v.) route progressed more slowly than when initiated via the airborne route. Mice were infected with approximately 10² colony-forming units (CFU) of wild-type (WT) H37Rv via the airborne route, 10⁵ CFU of SR H37Rv via the i.v. route, or 10² WT H37Rv via the airborne plus 10⁵ SR H37RV via the i.v. route. (a) CFU as determined by plating lung homogenates on standard nutrient agar show that lung infection progressed more slowly in mice infected via the i.v. route. (b) CFU as determined by plating lung homogenates of streptomycin-containing agar show that infection caused by SR H37Rv that reached the lungs after i.v. infection progressed more slowly and was controlled earlier at a lower level in the presence or absence of WT H37Rv delivered via the airborne route. Means ± SD of four mice per group.

Figure 3

Kinetics of the splenic T helper type 1 (Th1) response to infection initiated via the airborne, intravenous (i.v.), or both routes as determined by flow cytometry. (a) An increase in the percentage of interferon-γ (IFN-γ)-producing CD4 cells in the spleen was evident as early as day 6 in mice infected via the i.v. route or via both routes, and increased to peak on day 15 before undergoing a decline. In mice infected via the airborne route alone an increase in the percentage of these cells was not evident until day 15–20, and had increased by only a relatively small amount by day 30. (b) An increase in the total number of IFN-γ-producing CD4 cells per spleen was not evident until day15 in mice infected via the airborne route alone, but was evident as early as day 6 in mice infected via the i.v. route or both routes. In mice infected via the i.v. route or both routes, the number increased rapidly to peak on day 15 before declining, whereas it increased relatively slowly after day 10 in mice infected via the airborne route. Means ± SD of four mice per group per time point.

Figure 4

Kinetics of accumulation of interferon-γ (IFN-γ)-producing CD4 cells in the lungs of mice infected via the airborne route, intravenous (i.v.) route, or both routes. (a) Mice infected via the i.v. route alone showed a substantial increase in the percentage of CD4 T cells in the lungs between days 6 and 15 of infection. Mice infected via both routes showed a large increase between days 6 and 20. By contrast, mice infected via the airborne route alone did not show a progressive increase until after day 15. (b) As with the increase in percentage, a progressive increase in the total number of IFN-γ-producing CD4 cells per lung was evident between days 6 and 15 in the case of mice infected via the i.v. route alone, between days 6 and 20 in mice infected via both routes, but not until after day 15 in mice infected via the airborne route. Means ± SD of four mice per group per time point.

Figure 5

Changes in the total number of CD4 cells capable of making interferon-γ (IFN-γ) in response to ESAT-6 (1-20) peptide, Ag85B (240-54) peptide, or a Mycobacterium tuberculosis (Mtb) sonicate, according to the IFN-γ enzyme-linked immunosorbent spot-forming cell assay. In the lungs of mice infected via the respiratory route alone Mtb-specific CD4 began to increase after about day 20 of infection to peak on day 30 before undergoing a decline. By contrast, in the lungs of mice infected via the i.v. route or via both routes Mtb-specific cells began to increase in number much earlier to peak on day 15 and day 20, respectively. In the spleens of mice infected via the i.v. route or via both routes the production of antigen-specific IFN-γ-producing cells began after about day 5 to peak on day 15, whereas in mice infected via the airborne route alone production did not begin until after day 20 and did not to peak until day 30. In the draining lymph nodes (LNs) of mice infected via the airborne route day 30 was again the time of peak production of IFN-γ-producing cells. The number in the LNs of mice infected via the i.v. route or both routes was too small to make comparisons. Means ± SD of results obtained with cells of four mice per group per time-point.