Immunopathologic effects of tumor necrosis factor alpha in murine mycobacterial infection are dose dependent

Abstract

In experimental mycobacterial infection, tumor necrosis factor alpha (TNF-alpha) is required for control of bacillary growth and the protective granulomatous response, but may cause immunopathology. To directly examine the positive and detrimental effects of this cytokine, a murine model was used in which different amounts of TNF-alpha were delivered to the site of infection. Mice with a disruption in the TNF-alpha gene (TNF-KO) or wild-type mice were infected with low or high doses of recombinant Mycobacterium bovis BCG that secreted murine TNF-alpha (BCG-TNF). Infection of TNF-KO mice with BCG containing the vector (BCG-vector) at a low dose led to increased bacillary load in all organs and an extensive granulomatous response in the lungs and spleen. The mice succumbed to the infection by approximately 40 days. However, when TNF-KO mice were infected with low doses of BCG-TNF, bacillary growth was controlled, granulomas were small and well differentiated, the spleen was not enlarged, and the mice survived. Infection with high inocula of BCG-TNF resulted in bacterial clearance, but was accompanied by severe inflammation in the lungs and spleen and earlier death compared to the results from the mice infected with high inocula of BCG-vector. Wild-type mice controlled infection with either recombinant strain, but showed decreased survival following high-dose BCG-TNF infection. The effects of TNF-alpha required signaling through an intact receptor, since the differential effects were not observed when TNF-alpha receptor-deficient mice were infected. The results suggest that the relative amount of TNF-alpha at the site of infection determines whether the cytokine is protective or destructive.

FIG. 1

Effect of TNF-α on the bacillary load in the tissues of infected mice. TNF-KO mice (solid symbols) or wild-type mice (open symbols) were infected intravenously with a low or high dose of BCG-vector (squares) or BCG-TNF (circles). ∗, statistically significant (P < 0.05) differences between BCG-vector- and BCG-TNF-infected TNF-KO mice. Results are means ± SD of two independent experiments for each dose with 4 mice per group per time point.

FIG. 2

Morphology of the lungs of mice infected with low-dose recombinant BCG. TNF-KO mice were infected intravenously with a low dose of BCG-TNF (A to C) or BCG-vector (D to F). The lungs were examined at 28 days (A and D) or 45 days (B, C, E, and F). Sections were stained with hematoxylin and eosin (B, C, E, and F) or for iNOS protein expression (brown-staining cells) (A and D). Magnifications, panels B and E, ×10; panels A, C, D, and F, ×40.

FIG. 3

Morphology of the lungs of mice infected with high-dose recombinant BCG. TNF-KO mice were infected with a high dose of BCG-TNF (A to C) or BCG-vector (D to F). Wild-type mice were infected with a high dose of BCG-vector (G to I). The lungs were evaluated at 28 days. Sections were stained with hematoxylin and eosin (A, B, D, E, G, and H) or with Ziehl-Neelsen (C, F, and I). Fluid in the lungs is dark pink in hematoxylin and eosin sections or light pink in Ziehl-Neelsen sections. Magnifications, panels A, D, and G, ×10; panels B, C, E, F, H, and I, ×40.

FIG. 4

Effect of TNF-α on the weight of spleens of TNF-KO mice (solid symbols) or wild-type mice (open symbols) infected with BCG-vector (squares) or BCG-TNF (circles). ∗, statistically significant (P < 0.05) difference between mice infected with BCG-vector and those infected with BCG-TNF. Results are means ± standard deviations of two independent experiments for each dose with 4 mice per group per time point.

FIG. 5

Effect of TNF-α on survival of mice infected with recombinant BCG. TNF-KO mice (solid symbols) or wild-type mice (open symbols) were infected with BCG-vector (squares) or BCG-TNF (circles). Results expressed as percent survival represent the means of two independent experiments for each dose with 12 mice per group. A significant (P < 0.001) difference in survival of TNF-KO mice infected with low doses of BCG-vector versus those infected with BCG-TNF was noted. (For P values for the high doses of infection, see text.)

FIG. 6

Effect of TNF-α on survival of TNFR-KO mice infected with recombinant BCG. TNFR-KO mice were infected with BCG-vector (open triangles) or BCG-TNF (solid triangles). Results expressed as percent survival are from a single experiment for each dose with 12 mice per group. No significant difference in survival of mice infected with BCG-TNF versus those infected with BCG-vector was noted (P > 0.1).