New molecular diagnostic and immunological tools for tuberculosis research

Tuberculosis (TB) remains one of the world’s leading causes of mortality due to a single infectious agent, with approximately 1.5 million deaths and 9.2 million new cases per year as estimated in 2006. It is assumed that about 5-10% of individuals infected with M. tuberculosis develop TB and the remaining 90-95% contain M. tuberculosis through their immune systems, but have a latent tuberculosis infection (LTBI). To effectively control TB, it is essential to detect individuals with LTBI and to reliably diagnose active TB. Conventional TB diagnosis continues to rely on smear microscopy and culture that have several known limitations in terms of both speed and sensitivity that delay the diagnosis and, consequently, hold-up TB treatment and increase the spread of infection in the community. M. tuberculosis infection remains widespread, but the disease is generally limited to the primary infection stage. Patients with an immune defect or impaired immunity are more prone to develop the disease. In LTBI, the host immune response is capable of controlling the infection by the release of chemokines and cytokines produced by T helper (Th) cells, critical for the outcome of the infection. Several cells of the immune system are involved in the control of TB, from the macrophages and dendritic cells, called antigen presenting cells (APC) to the T cells, CD4, CD8, gamma delta T cells. Activation of these cells with excessive pro inflammatory responses can lead to tissue damage, with the need of mechanisms to counteract this, such as Th2 and T regulatory cells (Treg)-mediated responses. The optimal scenario would therefore seem to have balanced Th1, Th2 and Treg response, suited to the immune challenge. The balance between these types of response is reflected in the resultant host resistance against infection. Therefore the aims of the thesis were to find new approaches for diagnosis of active TB (First Part) and LTBI (Second Part). In this work we wanted to explore the immune mechanisms of TB pathogenesis with particular focus on the impact of Treg on suppressing M. tuberculosis-specific response (Third Part). For the diagnosis of active TB, we describe an alternative PCR methodology based on the amplification of small DNA fragments, originated from cells dying throughout the body (transrenal DNA; Tr-DNA) and detected in urine. It was found that small M. tuberculosis DNA fragments were specifically detected in the cell-free fraction of urine specimens from pulmonary TB patients. To detect LTBI, we compared the performances of two short-incubation interferon (IFN)-g release assays (IGRAs), the commercial QuantiFERON TB-Gold and the in-house whole blood stimulation with region of difference (RD)-1 proteins, with those of a 7-day whole blood stimulation and tuberculin skin test (TST). In an effort to find new markers for LTBI diagnosis, we also evaluated the production of pro-inflammatory cytokines [interleukin (IL)-1, IL-2, IL-6 and Tumor Necrosis Factor (TNF)-alfa], anti-inflammatory cytokines (IL-4, IL-10, IL-13) and chemokines [inducible protein (IP)-10, Macrophage Inflammatory Protein (MIP)-alfa, MIP-1beta, IL-8] after specific stimulation. The results raise the hypothesis that short-incubation IGRAs mainly detect recent or ongoing infection with M. tuberculosis, while prolonged-incubation IGRAs seem to be more sensitive for the diagnosis of past latent infection. Moreover we found that IL-2 and IP-10 may be additional markers for TB infection after RD1 specific stimulation. Finally we wanted to evaluate the impact of Treg on suppressing M. tuberculosis-specific response. Using classical markers for Treg recognition, discordant results were found in terms of Treg expansion during active TB disease. Recently CD39 has been shown to be an accurate marker for Treg detection. Objectives of this part of the thesis were: 1) to identify Treg expressing CD39 in patients with TB and to compare the results with those obtained by the standard phenotypic markers; 2) to evaluate if Treg are expanded in vitro by exogenous IL-2 or by antigen-specific stimulation; 3) to characterize Treg function on the modulation of antigen-specific responses. In this study we demonstrated that CD39 is a useful marker to detect Treg because within CD4+CD25high cells, it identifies a cell subset characterized by high production of transforming growth factor (TGF)-beta1 and the absence of IFN-gamma expression. Moreover, we showed that CD39+ Treg are expanded by M. tuberculosis-specific stimulation in patients with active TB disease.