Resistance of virulent Mycobacterium avium to gamma interferon-mediated antimicrobial activity suggests additional signals for induction of mycobacteriostasis

Abstract

The cytokine gamma interferon (IFN-gamma) plays a major role in the control of Mycobacterium avium infections. We assessed whether the progressive growth of virulent strains of M. avium was associated with alterations in the production of this cytokine as evaluated by reverse transcription-PCR and detection of immunoreactive cytokine in the serum and in spleen homogenates. We found that IFN-gamma was induced during infection by a virulent strain of M. avium to similar or even higher extents than the levels found during infections by a less virulent strain whose growth was controlled. IFN-gamma produced during infection by both mycobacterial strains was partly derived from T cells and led to activation of macrophages, namely, those that were infected. Concomitant with the development of the infection with the virulent strain of M. avium there was an extensive depletion of lymphocytes in the spleen. Thymectomy alone promoted the proliferation of the virulent, but not of the less virulent, strain of M. avium. Our data indicate that virulent strains of M. avium resist the antimicrobial mechanisms of IFN-gamma-activated macrophages and raise the possibility that a second, T-cell-dependent signal is required for the effective control of mycobacterial replication inside macrophages.

FIG. 1

Proliferation of two M. avium strains in the organs of C57Bl/6 mice (A) or IFN-γ^−/− mice and their heterozygous controls (B and C). (A) C57Bl/6 mice were infected with similar inoculum doses of strain 25291 (circles) or of strain 2447 (squares), and viable counts were done at the indicated time points in the spleens, livers, and lungs. (B and C) IFN-γ^−/− (solid symbols) and (IFN-γ^−/− × BALB/c)F1 (open symbols) mice were infected with strains 2447 (B) or 25291 (C), and viable counts were done in the same organs as for panel A at the indicated time points. Statistically different values are indicated by ∗ for P < 0.01 and ∗∗ for P < 0.05. Each time point represents the geometric mean of CFU values from four mice ± 1 standard deviation.

FIG. 2

Amounts of immunoreactive IFN-γ in the sera (A) and spleen homogenates (B) of C57Bl/6 mice infected at the indicated time points with M. avium strains 2447 (open squares) or 25291 (solid circles). Each point represents the determination of IFN-γ from one animal. (C) Analysis by RT-PCR of IFN-γ gene expression in the spleens, livers, and lungs of C57Bl/6 mice infected with M. avium strains 2447 or 25291 at days 15 and 30 of infection and of uninfected control mice. Southern blotting of the PCR products for HPRT and IFN-γ was performed with ³²P-labeled probes, and the photographic plates were scanned and analyzed. The data are presented as the means ± 1 standard deviation of the corrected results (standardized for the HPRT message) in terms of the intensity of the reading in pixels. The open bars represent the signal for uninfected mice, the hatched bars represent those of strain 2447-infected mice, and the solid bars represent those of strain 25291-infected mice. Independent analysis of the results for days 15 and 30 was done, and therefore, exposure of the photographic plates differed between those time points, leading to different readings of the uninfected material. ND, not detected. Note that linear increases in intensity (in pixels) correspond to exponential increases in PCR product under the conditions used here. Statistically significant differences according to Student’s t test are labeled ∗ (P < 0.05) or ∗∗ (P < 0.01).

FIG. 3

Kinetic analysis of macrophage activation. Peritoneal cells from infected animals were collected at different time points of infection, cultured, and used to assess their ability to secrete nitrite in response to LPS (A) or superoxide in response to PMA (B). The open symbols represent the results from mice infected with strain 2447, and the closed symbols represent those from mice infected with strain 25291. Each value represents the mean ± 1 standard deviation of triplicates from a pool of cells from four mice. ∗, P < 0.05; ∗∗, P < 0.01.

FIG. 4

Immunohistological evidence of macrophage activation in hepatic granulomas. (A) Liver sections were stained simultaneously with a polyclonal antibody for the expression of iNOS, with reactivity revealed through a peroxidase-conjugated secondary antibody, and for the presence of acid-fast bacilli. Sections from mice with the iNOS gene disrupted that were infected for 2 months with M. avium 25291 were used as a negative control for specificity (magnification, ×600) (B and C) Extensively labeled granulomas were observed in BALB/c mice infected for 1 month with strain 2447 (B) (magnification, ×1,200) or strain 25291 (C) (magnification, ×1,200). (D) Note the presence of acid-fast bacilli in the granuloma induced by strain 25291 (magnification, ×2,400) despite the marked reaction for iNOS. (E and F) IFN-γ gene-deficient mice on a BALB/c background infected with strain 2447 (E) (magnification, ×1,700) or strain 25291 (F) (magnification, ×1,700) showed reduced granuloma formation as well as reduced reactivity for iNOS. Note the extensive proliferation of strain 25291 inside macrophages of IFN-γ^−/− mice in panel F.

FIG. 5

Kinetics of the effect of infection on numbers of CD4⁺ and CD8⁺ cells in the spleens of mice infected with strains 2447 (open symbols) or 25291 (solid symbols).

FIG. 6

Involvement of the thymus in systemic infection by M. avium 2447 or 25291. (A) Cellularity of the thymus in terms of numbers of CD4⁺ CD8⁻ (open bars), CD4⁻ CD8⁺ (solid bars), CD4⁺ CD8⁺ (hatched bars), or total cells (shaded bars) of the thymi of mice infected for 1 or 60 days with strains 2447 (left) or 25291 (right). Results are means ± 1 standard deviation (SD). (B) Effects of thymectomy alone (shaded bars) or thymectomy followed by CD4⁺-T-cell depletion (solid bars) on the proliferation of M. avium 2447 (left) or 25291 (right) compared to that in untreated mice (open bars) at 40 days of infection. The results are shown as means ± 1 SD (n = 5). (C) Infection of SCID mice with M. avium 25291. The growth of M. avium was evaluated in the spleens, livers, and lungs of BALB/c and SCID mice after 30 days of infection. The results are shown as the geometric means ± 1 SD of the CFU values from three or four mice. The degree of macrophage activation of the peritoneal cells from the same animals as evaluated from the production of LPS-stimulated nitrite secretion is shown on the right. In vitro production of NO₂⁻ by peritoneal macrophages from uninfected BALB/c mice (open bar), infected BALB/c mice (hatched bar), or infected SCID mice (solid bar) is shown. The data represent means ± 1 SD of triplicates from a pool of three or four mice. ∗, P < 0.05; ∗∗, P < 0.01.