Determinants of natural immunity against tuberculosis in an endemic setting: factors operating at the level of macrophage-Mycobacterium tuberculosis interaction

Abstract

We aimed to delineate factors operating at the interface of macrophage-mycobacterium interaction which could determine the fate of a 'subclinical' infection in healthy people of a tuberculosis-endemic region. Ten study subjects (blood donors) were classified as 'high' or 'low' responders based on the ability of their monocyte-derived macrophages to restrict or promote an infection with Mycobacterium tuberculosis. Bacterial multiplication between days 4 and 8 in high responder macrophages was significantly lower (P < 0.02) than low responders. All donor sera were positive for antibodies against cell-membrane antigens of M. tuberculosis and bacilli opsonized with heat-inactivated sera were coated with IgG. In low responder macrophages, multiplication of opsonized bacilli was significantly less (P < 0.04) than that of unopsonized bacilli. The levels of tumour necrosis factor (TNF)-alpha and interleukin (IL)-12 produced by infected high responder macrophages was significantly higher (P < 0.05) than low responders. However, infection with opsonized bacilli enhanced the production of IL-12 in low responders to its level in high responders. The antibody level against membrane antigens was also significantly higher (P < 0.05) in high responders, although the antigens recognized by two categories of sera were not remarkably different. Production of certain other cytokines (IL-1beta, IL-4, IL-6 and IL-10) or reactive oxygen species (H2O2 and NO) by macrophages of high and low responders did not differ significantly. The study highlights the heterogeneity of Indian subjects with respect to their capability in handling subclinical infection with M. tuberculosis and the prominent role that TNF-alpha, opsonizing antibodies and, to a certain extent, IL-12 may play in containing it.

Fig. 1

Determination of exposure of the donors (n = 10) to Mycobacterium tuberculosis: (a) T cell proliferation, (b) interferon-γ production and (c) serum antibody levels against cytosol and membrane antigens of the pathogen. Statistically significant differences were noted with respect to (a) and (c).

Fig. 2

Fold multiplication [from day 4 to day 8) of Mycobacterium tuberculosis in macrophages of low (LR) and high responders (HR), five donors each]. Multiplication of live or live-opsonized bacilli in low responders (LR-L/LR-LO) was significantly higher than the corresponding values in high responders (HR-L/HR-LO). Within the LR group, multiplication of live-opsonized bacilli was significantly lower than that of live bacilli.

Fig. 3

(a) Antibody levels (mean OD ± standard error of the mean) against Mycobacterium tuberculosis membrane antigens in low responders (LR) were significantly lower than high responders (HR). (b) Immunoblotting of membrane antigens with (i) monoclonal antibody ML09 (lane 1) shows presence of lipoarabinomannan and (ii) donor sera (lanes 2–6, HR; 7–10, LR) shows antigens which were recognized commonly by most sera (result with one LR serum is not available). The strip (MR) containing electroblotted molecular mass markers (kDa) was stained with amido black.

Fig. 4

Tumour necrosis factor (TNF)-α (a) and interleukin (IL)-12 (b) levels in day 8 culture supernatants of infected macrophages. TNF-α levels of high responder (HR) macrophages infected with live (L) or live-opsonized (LO) Mycobacterium tuberculosis were significantly higher than corresponding values for low responders (LR). Interleukin (IL)-12 level of HR macrophages infected with L bacilli were also significantly higher than corresponding values for LR. However, following infection with LO bacilli, there was an increase in IL-12 levels of LR macrophages.

Fig. 5

Relationship between tumour necrosis factor-α levels and bacterial multiplication in the donor macrophages (n = 10). Statistically significant inverse correlations were seen with respect to infection with live bacilli (a) as well as live-opsonized bacilli (b). Regression lines (non-linear) are shown.