Currently, confirmation of a diagnosis of pulmonary TB requires either isolation of Mtb
or identification of specific Mtb
DNA from respiratory specimens. Both tests have important drawbacks: culture requires 2–3 wk, and nucleic acid amplification tests, although faster, are not as sensitive as culture. Commonly, treatment for active TB is begun before there is bacteriologic confirmation of the diagnosis, and it is not unusual for clinical criteria to be met without bacteriologic proof, leading to empiric treatment and its attendant risks if the person does not actually have active TB. Two diagnostic tests, the TST and the expression of IFN-γ by PBMC in response to ESAT-6 and CFP-10, are widely used to identify Mtb
-infected individuals (10
), but neither differentiates individuals with LTBI from those with active TB. In the present study, we found that the measurement of the combination of ESAT-6-stimulated IL-8, FOXP3, and IL-12β mRNA in PBMC distinguishes individuals with active TB from latent Mtb
Although the results presented in this study may lead to potentially useful new diagnostic tests, much additional work is needed. For this initial survey, we excluded TB patients with HIV/AIDS, reasoning that various degrees of immunosuppression might complicate data interpretation. However, patients with HIV/AIDS contribute most of the cases with culture-negative TB for whom a new diagnostic test is urgently needed (16
). Studies on newly diagnosed TB patients before treatment are also planned. Lastly, a less expensive, technically demanding test must be developed, such as a multiplex or dipstick format in which the cytokines themselves are measured in response to stimulation.
Our findings provide new insights into the immune responses to Mtb
. The patterns of cytokine expression imply that the transition from latent infection to active disease is not merely a “switch” from a Th1 to Th2 response. Expression of FOXP3, a transcription factor specific for Tregs, correlates with the suppressive activity of these cells, and activation of human CD4+
T cells leads to expression of FOXP3 and acquisition of regulatory function (17
). There have been reports that Tregs are increased in patients with active TB or H. pylori
), suggesting that Tregs may underlie the suppressed cellular immunity described in these chronic infections. The mechanism by which Tregs suppress other T cell responses is controversial, but requires either cell-cell contact in vitro or the release of soluble factors such as IL-10 and TGF-β (19
). In the patients with active TB in our cohort, FOXP3+
cells were induced in an Ag-specific manner because there was no difference in FOXP3 expression in unstimulated PBMC between the TB and LTBI groups (data not shown), but there was a significant increase in FOXP3 expression in ESAT-6-stimulated PBMC from TB patients.
IL-8 (CXCL8) induces chemotaxis of inflammatory cells and directs immune responses. Multiple stimuli induce the secretion of IL-8, including TNF-α and IL-1 (20
). Granulocytes from patients with TB produce IL-8 in response to lipoarabinomannan isolated from Mtb
). Elevated levels of IL-8 in bronchoalveolar lavage fluid have been described in patients with TB compared with uninfected controls (22
). We show in this study that the fold increase in IL-8 mRNA in ESAT-6-stimulated PBMC is significantly elevated in LTBI compared with active TB (), and a similar increase was also observed for IL-1α and IL-1β (). Of note, neutralization of IL-1 blocks IL-8 production in Mtb
), suggesting that the enhanced IL-8 expression in LTBI subjects may be controlled by IL-1.
IL-12, a heterodimer composed of two subunits encoded by IL-12α and IL-12β, is a major inducer of IFN-γ. IL-12p40-deficient children suffer from mycobacterial infection primarily because their IFN-γ-mediated immunity is impaired (25
). In the present study, expression of IL-12β was significantly decreased in patients with TB compared with the LTBI group, indicating that low expression of IFN-γ in TB patients might be due to the reduction of IL-12β. Indeed, addition of exogenous IL-12 enhanced the IFN-γ response of ESAT-6 stimulated PBMC from TB patients (data not shown), underscoring the importance of IL-12β in the induction of IFN-γ.
In summary, our study suggests that measuring the ESAT-6-stimulated expression of mRNA of immune related genes in PBMC can be used to identify Mtb-infected individuals. Expression of IL-8/FOXP3/IL-12β can distinguish latent infection from active TB regardless of anti-TB treatment time. In newly diagnosed or short-term treated TB patients, decreased IL-8 and IL-12β expression may reflect impairment of immune cell recruitment and Th1 responses, while increased levels of FOXP3 suggest involvement of regulatory T lymphocyte-mediated immunosuppression. Our findings imply that the cellular immune response changes as latent infection transitions to active disease and that monitoring expression of the genes identified here may allow identification of individuals needing anti-TB treatment, particularly in developing countries.