Initiation of acquired immunity in the lungs of mice lacking lymph nodes after infection with aerosolized Mycobacterium tuberculosis

Abstract

Recent evidence points to lung draining lymph nodes as the site that initiates the immune response in mice infected with aerosolized Mycobacterium tuberculosis. Here we expanded these studies and showed that infection of mice that lack lymph nodes with aerosolized M. tuberculosis results in a massive mononuclear cell infiltrate in the lungs within 14 days postinfection. This infiltration clearly resembles an expansion of the bronchus-associated lymphoid tissue. As expected, no bronchus-associated lymphoid tissue was observed in M. tuberculosis-infected wild-type control mice. Importantly, acquired specific immune response to M. tuberculosis antigens could be detected in lung lymphocytes harvested from mice lacking lymph nodes as early as 14 days postinfection. In addition, the bacterial burden in these mice was indistinguishable from that observed in wild-type C57BL/6 control mice. These results indicate that in the absence of lymph nodes, priming of the immune response occurs in the lung tissues after infection of mice with aerosolized M. tuberculosis and clearly illustrate the enormous plasticity of the immune system to develop resistance to foreign pathogens.

Figure 1

Bacterial burden and development in LTα-KO and C57BL/6 mice infected with M. tuberculosis. Mice were infected with 100 to 200 aerosolized M. tuberculosis, and the course of infection (CFU) was determined in the lungs and spleens.

Figure 2

Development of adaptive immune response in LTα-KO and C57BL/6 mice infected with M. tuberculosis. Mice (five per group/time point) were infected with 100 to 200 aerosolized M. tuberculosis and at the designated time points lung and spleen mononuclear lymphoid cells were obtained, stimulated for 48 hours with Ag85B (5 μg/ml), and assayed for IFN-γ production by ELISPOT. Note that although the magnitude of the response is higher in wild-type compared with LTα-KO mice (P < 0.05 for lungs at 14 and 21 days and spleen at 21 days postinfection; P > 0.05 for spleen at 14 days postinfection), no differences are observed in the kinetics of IFN-γ production by the mononuclear cells isolated from both groups. SFC, spot-forming cells. Bars represent SE for the means of the results of five mice per group. This is one representative experiment from three experiments with essentially the same results.

Figure 3

Bacterial burden and initiation of adaptive immunity in lungs of splenectomized LTα-KO mice infected with M. tuberculosis. LTα-KO mice were splenectomized and 10 days later infected with 100 to 200 CFU of aerosolized M. tuberculosis (A). In addition, at designated time points, lung mononuclear lymphoid cells were obtained, stimulated for 48 hours with Ag85B (5 μg/ml), and assayed for IFN-γ production by ELISPOT (B). Five mice were assayed at each time point. SFC, spot-forming cell. Error bars represent the SEM.

Figure 4

Histopathology of lungs from controls and M. tuberculosis-infected LTα-KO and C57BL/6 mice. Mice were infected as described in Figure 1 and euthanized at the indicated time points. Lungs were removed and embedded in paraffin, and tissue sections (5 μm) were stained with H&E. Note the absence of cellular infiltration surrounding the bronchioles of both uninfected control mice as well as in wild-type (WT) mice infected with M. tuberculosis. However, patches of perivascular infiltrations are seen surrounding some vessels of uninfected LTα-KO (red arrowhead). In contrast, in infected LTα-KO animals, mononuclear cell infiltrations are seen in the organ parenchyma at 14 days after infection (black arrowhead) as well as contiguously to the bronchial tree (black arrows) at both days 14 and 21 postinfection. Similar pattern is seen in the lungs of splenectomized LTα-KO. However, no inflammation is seen in C57BL/6 mice at day 14 postinfection. As expected, at day 21, granulomas are seen in the lung parenchyma of these mice (white arrowhead), but no inflammation is seen surrounding the bronchial tree. B, bronchiole; A, artery. Original magnification, ×100.

Figure 5

Immunohistochemistry of lungs from LTα-KO and C57BL/6 mice infected with M. tuberculosis. Mice were infected as described in Figure 1 and euthanized at 14 days postinfection. Lungs were removed and embedded in paraffin and tissue sections (5 μm) were processed for immunohistochemical staining for B cells (anti-B220) and T cells (anti-CD3). Note the intense accumulation of both B and T cell aggregates (B220⁺ and CD3⁺ cells, respectively) surrounding the lung airways (black arrows) of LTα-KO mice. In contrast, no B220⁺ or CD3⁺ cells are seen in the lungs of C57BL/6 mice. B, bronchiole. Original magnification, ×100.