Alpha/beta interferon impairs the ability of human macrophages to control growth of Mycobacterium bovis BCG

Abstract

Administration of alpha/beta interferon (IFN-alpha/beta) to mice infected with Mycobacterium tuberculosis has been shown to increase mycobacterial growth. Because IFN-alpha/beta has direct pleiotropic effects on the differentiation and functional activities of macrophages, we evaluated the effect of IFN-alpha/beta on mycobacterial growth in human monocytes/macrophages in vitro. Monocytes cultured at optimal cell density could control the growth of M. bovis BCG, as assessed both by measurement of luciferase activity expressed by a mycobacterial reporter strain and by counting of CFU. In contrast, unrestrained mycobacterial growth was observed when monocytes were treated with alpha interferon (IFN-alpha) 3 days prior to or concomitant with infection. This striking loss of mycobacteriostatic activity was observed with IFN-alpha and IFN-beta and was induced in both freshly isolated monocytes and culture-derived macrophages. Pretreatment of monocytes with IFN-alpha modified cellular morphology and reduced viability following culture, but neither was observed for culture-derived macrophages, indicating that the effects of IFN-alpha on mycobacteriostatic activity and cell differentiation and death could be dissociated. These results are compatible with the possibility that the secretion of IFN-alpha/beta could directly promote mycobacterial growth in patients harboring these organisms.

FIG. 1.

Effect of pretreatment of human macrophages with IFN-α on intracellular growth of M. bovis BCG. (A) Human monocytes were cultured at 2 × 10⁵ cells/well in the presence of 3,000 U of recombinant human IFN-α per ml (▪) or culture medium only (□) for 3 days and infected with M. bovis BCG. At the indicated times after infection, culture medium was removed and the luciferase activity (RLU) expressed by the mycobacterial reporter strain was measured by luminometry. Results are means ± SD for 12 experiments using monocytes from different individuals. Numbers of RLU for unpretreated and IFN-α-pretreated monocytes were significantly different at 4, 7, and 10 days (P < 0.01). (B) Human monocytes were cultured at 2 × 10⁵ cells/well in the presence of 3,000 U of IFN-α (• and ○) per ml or culture medium only (▪ and □) for 3 days and infected with the M. bovis BCG reporter strain. After the indicated times in culture, the number of mycobacteria present was assessed by measuring luciferase activity (RLU; ○ and □) and CFU (• and ▪). Results are means ± SD for three independent experiments (days 1, 4, and 7) using cells from different individuals. Data for cells cultured for 10 days were obtained in only one of the experiments. RLU for unpretreated and IFN-α-pretreated monocytes were significantly different at 4 and 7 days (P < 0.01 for both comparisons). Numbers of CFU for unpretreated and IFN-α-pretreated monocytes were significantly different at 4 and 7 days (P < 0.05 and P < 0.01, respectively).

FIG. 2.

Effect of pretreating monocytes with various doses of IFN-α and IFN-β on their ability to control mycobacterial growth. Monocytes were cultured at 2 × 10⁵ cells/well in the presence of the indicated concentrations of recombinant human IFN-α (▪) or IFN-β (□) for 3 days and infected with the M. bovis BCG reporter strain. Seven days after infection, culture medium was removed, and the luciferase activity (RLU) was determined. The results are means ± SD for three independent experiments using monocytes from different individuals. The data for each IFN were fitted to a sigmoidal dose response curve to determine the 50% effective concentration (70 and 248 U/ml, respectively, for IFN-α and IFN-β). R² values were 0.88 and 0.92, respectively.

FIG. 3.

Cell loss in cultures containing IFN-α-pretreated monocytes does not account for the reduced mycobactericidal activity of these cells. Human monocytes were cultured in the presence of 3,000 U of IFN-α per ml at 4 × 10⁵ (•) or 2 × 10⁵ (▪) cells/well or in culture medium only at 2 × 10⁵ cells/well (□) for 3 days and infected with the M. bovis BCG reporter strain. At the indicated times after infection, culture medium was removed, and the DNA content of adherent cells (A) and luciferase activity (B) was determined. The results are means ± SEM of four independent experiments using cells from different individuals. RLU for unpretreated macrophages were significantly lower than those of IFN-α-pretreated cells at 4 days (P < 0.01 for both comparisons) and 7 days (P < 0.001 for both comparisons) by repeated-measures ANOVA. RLU for IFN-α-pretreated macrophages cultured at 2 × 10⁵ and 4 × 10⁵ cells/well were not significantly different at any time point (P > 0.05).

FIG. 4.

IFN-α exposure prior to or concomitant with infection impairs the mycobacteriostatic activity of fresh monocytes and cultured macrophages. Human monocytes were cultured at 2 × 10⁵ cells/well for 3 days (A) or at 5 × 10⁴ cells/well for 10 days (B) prior to infection with the M. bovis BCG reporter strain. Cultures were pretreated with 3,000 U of IFN-α per ml for 3 days prior to infection (gray bars), cultured in the presence of 3,000 U of IFN-α per ml starting on the day of infection (black bars), or not exposed to IFN-α (white bars). At the indicated times after infection, culture medium was removed, and the number of mycobacteria present was assessed by measuring luciferase activity (RLU). The results are means ± SEM for four independent experiments, except for cells pretreated with IFN-α in panel B (n = 2). The hatched rectangle represents the mean ± 1 SEM for cells not exposed to IFN-α and evaluated 1 day after infection. ∗, ∗∗, and ∗∗∗, P < 0.05, P < 0.01, and P < 0.001, respectively, compared to unpretreated macrophages.