Factors associated with severe granulomatous pneumonia in Mycobacterium tuberculosis-infected mice vaccinated therapeutically with hsp65 DNA

Abstract

Resistant C57BL/6 mice infected in the lungs with Mycobacterium tuberculosis and then therapeutically vaccinated with Mycobacterium leprae-derived hsp65 DNA develop severe granulomatous pneumonia and tissue damage. Analysis of cells accumulating in the lungs of these animals revealed substantial increases in T cells secreting tumor necrosis factor alpha and CD8 cells staining positive for granzyme B. Stimulation of lung cells ex vivo revealed very high levels of interleukin-10, some of which was produced by B-1 B cells. This was probably an anti-inflammatory response, since lung pathology was dramatically worsened in B-cell gene-disrupted mice.

FIG. 1.

Hsp65 DNA vaccination induced an increase of (A) TNF-α and (B) granzyme B in the lungs, but not (C) IFN-γ. Lung cells from hsp65 DNA-vaccinated mice previously infected by aerosol M. tuberculosis were incubated with fluorescently labeled antibodies for surface markers CD4, CD8, and intracellular TNF-α, IFN-γ, and granzyme B. Data are expressed as the mean percentage of cells from five mice per group. Open squares, vector control-treated mice; solid squares, hsp65-treated mice. Error bars represent ± standard error of the mean. *, P < 0.05; **, P < 0.001 compared to the vector control group using Student's t test.

FIG. 2.

Hsp65 DNA vaccine induced an increase in IL-10 production in the lungs. Lung cells isolated from hsp65 DNA-vaccinated mice previously infected with M. tuberculosis were incubated with culture filtrate protein at 37°C and frozen after 72 h. Supernatants from five mice per group were pooled, and a cytometric bead array was run to detect secreted IL-10 protein. Open squares, vector control-treated mice; solid squares, hsp65-treated mice.

FIG. 3.

Representative photomicrographs of lungs from M. tuberculosis-infected B-cell-knockout mice 68 days after the last of four vaccinations with hsp65 DNA. Panel A, mouse did not receive a vaccine. Panels B and C, mice received four intramuscular vaccinations with hsp65 DNA. Panel A shows a moderate, multifocal granulomatous pneumonia. Panel B shows a severe, diffuse, fibrinous, necrotizing granulomatous pneumonia. Panel C shows that in some cases, the lumens of large airways are plugged with fibrin and necrotic cellular debris (*). Multiple foci of neutrophils and eosinophils (arrows) were visibly associated with areas of necrosis. The middle right lung lobe was inflated with 10% formalin, processed and sectioned routinely, and stained with hematoxylin and eosin. A and B: 10× total magnification. C: 40× total magnification.

FIG. 4.

Representative photomicrographs showing immunohistochemical staining for CD8⁺ (left panels) and B220-positive (right panels) T cells within saline controls (A and B) and mice vaccinated four times with hsp65 (C and D). Well-organized lung lesions from C56BL/6 mice consisting of both macrophages and lymphocytes developed through 90 days postinfection. Arrow bars depict centralized aggregates of CD8⁺ T cells (A). Lung lesions from hsp65-vaccinated mice were larger and showed tissue necrosis depicted by parenchymal thickening and cholesterol deposition (photomicrograph not shown). Mice vaccinated with hsp65 showed disorganized lesions with scattered foci of CD8⁺ T cells (C). Peribronchial and perivascular cuffing of lymphocytes from saline controls showed moderate numbers of lymphocytes with aggregates of B220-positive cells depicted by arrow bars (B), while hsp65-vaccinated mice showed increased numbers of lymphocyte aggregates consisting of more B220-positive cells (D). A to D: 10× total magnification.