Dynamic changes in pro- and anti-inflammatory cytokine profiles and gamma interferon receptor signaling integrity correlate with tuberculosis disease activity and response to curative treatment

Abstract

Pro- and anti-inflammatory cytokines and their signaling pathways play key roles in protection from and pathogenesis of mycobacterial infection, and their balance and dynamic changes may control or predict clinical outcome. Peripheral blood cells' capacity to produce proinflammatory (tumor necrosis factor alpha [TNF-alpha], interleukin-12/23p40 [IL-12/23p40], and gamma interferon [IFN-gamma]) and anti-inflammatory (IL-10) cytokines in response to Mycobacterium tuberculosis or unrelated stimuli (lipopolysaccharide, phytohemagglutinin) was studied in 93 pulmonary tuberculosis (TB) patients and 127 healthy controls from Indonesia. Their cells' ability to respond to IFN-gamma was examined to investigate whether M. tuberculosis infection can also inhibit IFN-gamma receptor (IFN-gammaR) signaling. Although there was interindividual variability in the observed responses, the overall results revealed that M. tuberculosis-induced TNF-alpha and IFN-gamma levels showed opposite trends. Whereas TNF-alpha production was higher in active-TB patients than in controls, IFN-gamma production was strongly depressed during active TB, correlated inversely with TB disease severity, and increased during therapy. By contrast, mitogen-induced IFN-gamma production, although lower in patients than in controls, did not change during treatment, suggesting an M. tuberculosis-specific and reversible component in the depression of IFN-gamma. Depressed IFN-gamma production was not due to decreased IL-12/IL-23 production. Importantly, IFN-gamma-inducible responses were also significantly depressed during active TB and normalized during treatment, revealing disease activity-related and reversible impairment in IFN-gammaR signaling in TB. Finally, IFN-gamma/IL-10 ratios significantly correlated with TB cure. Taken together, these results show that M. tuberculosis-specific stimulation of IFN-gamma (but not TNF-alpha) production and IFN-gammaR signaling are significantly depressed in active TB, correlate with TB disease severity and activity, and normalize during microbiological TB cure. The depression of both IFN-gamma production and IFN-gammaR signaling may synergize in contributing to defective host control in active TB.

FIG. 1.

(a) M. tuberculosis-induced TNF-α production in the cross-sectional analysis was highest in untreated TB patients, decreased during therapy, and was lowest at the end of TB therapy, closely approaching the values measured in control subjects, correlating with the TB disease-related inflammatory process. A smaller set of patients was followed up in a longitudinal-observation study. No significant differences in TNF-α production were observed between the mild-to-moderate TB (b) and advanced-TB (c) groups before, during, and at the end of the therapy. Each dot in the cross-sectional study represents one individual. A horizontal bar indicates the median of each group. A dashed line indicates the lower detection limit of the assay. A statistically significant difference (P < 0.05) is indicated by the asterisk (Mann-Whitney U test).

FIG. 2.

M. tuberculosis-induced IL-12/23p40 production tended to be slightly increased after 2 months of TB therapy but decreased at the end of anti-TB therapy in the cross-sectional analysis. This trend was most evident in the longitudinal study (see text; not shown). Each dot in the cross-sectional study represents one individual. A horizontal bar indicates the median of each group. A dashed line indicates the lower detection limit of the assay. A statistically significant difference (P < 0.05) is indicated by the asterisk (Mann-Whitney U test).

FIG. 3.

LPS-induced IL-10 production of pulmonary TB patients was slightly but significantly increased in active TB and normalized at the end of therapy compared to that of the control group. In the longitudinal study, no significant differences were detectable between the mild-to-moderate TB and advanced-TB subgroups (see text; not shown). Each dot in the cross-sectional study represents one individual. A horizontal bar indicates the median of each group. A dashed line indicates the lower detection limit of the assay. A statistically significant difference (P < 0.05) is indicated by the asterisk (Mann-Whitney U test).

FIG. 4.

(a) M. tuberculosis-induced IFN-γ production of PBMCs was strongly suppressed in untreated TB but increased during the course of therapy, approaching values measured in control subjects at the end of therapy, correlating inversely with TB disease activity. Overall, IFN-γ production was higher in mild-to-moderate TB patients (b) than in advanced-TB patients (c) at all of the time points studied, showing that, in contrast to TNF-α, IL-10, and IL-12/23p40, the depression in IFN-γ production correlates not only with TB disease activity but also with TB disease severity. Each dot in the cross-sectional study represents one individual. A horizontal bar indicates the median in each group. A dashed line indicates the lower detection limit of the assay. Statistically significant differences (P < 0.05) are indicated by the symbol # between median values before, during, and at the end of the therapy (Wilcoxon signed-rank test); by the symbol * compared to control subjects (Mann-Whitney U test); and by the symbol [o] between mild-to-moderate TB and advanced-TB patients (Mann-Whitney U test).

FIG. 5.

PHA-induced IFN-γ production of PBMCs was strongly suppressed in patients with untreated TB compared to that in controls. In contrast to the results from M. tuberculosis-specific stimulation, no increased IFN-γ production during anti-TB therapy was observed. There was overall no significant difference between mild-to-moderate TB patients and advanced-TB patients (see text; not shown). Each dot in the cross-sectional study represents one individual. A horizontal bar indicates the median in each group. A dashed line indicates the lower detection limit of the assay. Statistically significant differences (P < 0.05) are indicated by asterisks (Mann-Whitney U test).

FIG. 6.

IFN-γ responsiveness as measured by synergy in TNF-α induction upon stimulation with M. tuberculosis sonicate (ratio of M. tuberculosis plus IFN-γ to M. tuberculosis only). IFN-γ significantly enhanced M. tuberculosis-driven production of TNF-α. IFN-γ responsiveness was decreased significantly in active-TB patients compared to that of controls and normalized during therapy to levels comparable to those of the control group. Each dot represents the IFN-γ response ratio of one individual. A dashed line indicates a ratio of 1. A horizontal bar indicates the median level of each group. Statistically significant differences (P < 0.05) are indicated by the symbol # between median values before, during, and at the end of the therapy (Wilcoxon signed-rank test) and by the symbol * compared to control subjects (Mann-Whitney U test).

FIG. 7.

TNF-α/IL-10 ratios were significantly increased in TB patients before, during, and following completion of therapy compared to those of control subjects. By contrast, IL-12/23p40/IL-10 ratios were only temporarily increased during anti-TB therapy and decreased at the end of therapy. The ratio of M. tuberculosis-specific IFN-γ to IL-10 production showed a strong increase during the course of therapy and was slightly increased at the end of therapy compared to that of control subjects, suggesting a shift toward a proinflammatory host immune phenotype during control of infection. Each dot represents one individual. A horizontal bar indicates the median level of each group. A dashed line indicates a ratio of 1. A statistically significant difference (P < 0.05) is indicated by the symbol # (Wilcoxon signed-rank test) between median values before, during, and at the end of therapy or by the symbol * (Mann-Whitney U test) compared to control subjects.