Z. Toossi

Depressed T-Cell Interferon-γ Responses in Pulmonary Tuberculosis: Analysis of Underlying Mechanisms and Modulation with Therapy

Immunological and clinical profiles were evaluated in 2 groups: human immunodeficiency virus (HIV)-uninfected and HIV-infected patients, with newly diagnosed pulmonary tuberculosis (TB), and tuberculin-skin-test-reactive healthy control subjects. HIV-uninfected patients with TB were also followed up longitudinally during and after chemotherapy. At the time of diagnosis, purified protein derivative (PPD)-stimulated production of interferon (IFN)-gamma by peripheral blood mononuclear cells from TB patients was depressed, compared with that of healthy control subjects, whereas levels of transforming growth factor (TGF)-beta and interleukin (IL)-10 were increased. In longitudinal studies, PPD stimulated production of IL-10 and TGF-beta returned to baseline by 3 months, whereas IFN-gamma production remained depressed for at least 12 months. These data indicate that the immunosuppression of TB is not only immediate and apparently dependent (at least in part) on immunosuppressive cytokines early during the course of Mycobacterium TB infection but is also long lasting, presumably relating to a primary abnormality in T-cell function.

Impact of tuberculosis (TB) on HIV-1 activity in dually infected patients

Active TB in HIV‐1‐infected subjects is associated with increased HIV‐1‐related immunodeficiency and mortality. We assessed plasma viral load in HIV‐1‐infected patients with pulmonary TB (HIV/TB) and non‐TB symptomatic HIV‐1‐infected patients (HIV). HIV‐1 load was higher in HIV/TB compared with HIV at higher CD4 counts (> 500/μl) (P < 0·01), but not at lower CD4 counts (< 500/μl). We also evaluated the status of HIV‐1 gene expression in peripheral blood mononuclear cells (PBMC) and serum from HIV/TB and CD4‐matched healthy HIV‐infected patients (HIV/C) by reverse transcriptase‐polymerase chain reaction over a range of CD4 (> 900/μl to < 200/μl). HIV‐1 RNA in serum and PBMC correlated to one another, and both were markedly higher in HIV/TB compared with HIV/C with higher CD4 counts. Also, during a longitudinal study of anti‐tuberculous chemoprophylaxis in HIV‐1‐infected patients, 10 subjects who developed TB had serologies before, at the time, and after the diagnosis of TB. These HIV/TB patients had an increase in viral load (average 2·5‐fold) at the time of diagnosis of TB (P < 0·05). Overall, these data indicate that the transcriptional activity of HIV‐1 is enhanced in HIV‐1‐infected patients with active TB, especially during early HIV‐1 disease. As TB often is an early HIV‐1 opportunistic infection, it may particularly favour early viral replication and dissemination, and therefore contribute to progression of HIV‐1 disease.

Augmentation of Apoptosis and Interferon-γ Production at Sites of Active Mycobacterium tuberculosis Infection in Human Tuberculosis

Pleural tuberculosis (TB) was employed as a model to study T cell apoptosis at sites of active Mycobacterium tuberculosis (MTB) infection in human immunodeficiency virus (HIV)-coinfected (HIV/TB) patients and patients infected with TB alone. Apoptosis in blood and in pleural fluid mononuclear cells and cytokine immunoreactivities in plasma and in pleural fluid were evaluated. T cells were expanded at the site of MTB infection, irrespective of HIV status. Apoptosis of CD4 and non-CD4 T cells in the pleural space occurred in both HIV/TB and TB. Interferon (IFN)-gamma levels were increased in pleural fluid, compared with plasma. Spontaneous apoptosis correlated with specific loss of MTB-reactive, IFN-gamma-producing pleural T cells. Immunoreactivities of molecules potentially involved in apoptosis, such as tumor necrosis factor-alpha, Fas-ligand, and Fas, were increased in pleural fluid, compared with plasma. These data suggest that continued exposure of immunoreactive cells to MTB at sites of infection may initiate a vicious cycle in which immune activation and loss of antigen-responsive T cells occur concomitantly, thus favoring persistence of MTB infection.

An increase in expression of a Mycobacterium tuberculosis mycolyl transferase gene ( fbpB) occurs early after infection of human monocytes

Changes in the mRNA levels of two Mycobacterium tuberculosis genes (fbpB known as antigen 85B, and hspX known as Acr) were studied in infected human monocytes. Antigen 85B is an enzyme involved in cell wall biosynthesis and is also a major target of the immune response. Acr is a stress protein believed to be involved in the bacillary response to adverse conditions and in non‐replicating persistence. During the first 24 h of intracellular infection, the intramonocyte 85B mRNA level increased 54‐fold (P = 0.00001) and 14.6 times in comparison with the 16S ribosomal rRNA. In contrast, the Acr mRNA fell 14.3 times. Although monocyte cytokine production was very variable, the 24 h secretion of tumour necrosis factor (TNF)‐α correlated with the 85B−16S RNA ratio at 24 h (r = 0.77, Pcorr < 0.01). Furthermore, the addition of exogenous TNF‐α to cultures was associated with a twofold increase in the 85B−16S ratio and, conversely, neutralization of endogenous TNF‐α reduced the ratio. As antigen 85B also induces TNF‐α, the positive feedback implied by our findings suggests a previously unsuspected role for this protein in the immunopathogenesis of tuberculosis.

The Inflammatory Response in Mycobacterium Tuberculosis Infection

Infection with Mycobacterium tuberculosis (MTB) is accompanied by an intense local inflammatory response which may be critical to the pathogenesis of tuberculosis. Activation of components of the innate immune response, such as recruitment of polymorphonuclear (PMN) and mononuclear phagocytes and induction of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), by MTB occurs early after MTB infection, however, may persist as the organism establishes itself within granulomas. MTB and its protein and non-protein components are potent in induction of cytokines and chemokines from PMN and monocytes. This review focuses on the interaction of MTB and the host with regard to activation of the innate immune response. It also attempts to identify the potential impact of this early response on the subsequent pathogenesis of MTB, and its role in development and extent of tuberculosis. Insights into the initiation and persistent of the inflammatory response may allow the application of anti-inflammatory agents as adjuncts in the treatment of tuberculosis.

Bioactivation of Latent Transforming Growth Factor β1 by Mycobacterium tuberculosis in Human Mononuclear Phagocytes

Biologically active transforming growth factor beta 1 (TGFβ1) has been identified at sites of Mycobacterium tuberculosis (MTB) infection in the lung; however, the underlying mechanism(s) for its activation is not clear. Here using an enzyme‐linked immunospot assay for TGFβ1, we show that human blood monocytes (MN) and alveolar macrophages (AM) produce bioactive TGFβ1 upon stimulation by MTB. However, only MTB‐stimulated MN increased TGFβ1 production on a per cell basis. The frequency of TGFβ1‐producing MN was reduced by an inhibitor of plasmin, bdellin, indicating a role for plasmin pathways in the bioactivation of cytokine. The expression of urokinase plasminogen activator receptor (uPAR) mRNA and both surface and soluble uPAR (CD87) was increased in MTB‐activated MN. However, antibody neutralization of uPAR suppressed bioactive TGFβ1 in MN alone. Thus, the more immature MN, which are continuously recruited to the lung during tuberculosis (TB), have a higher capacity to bioactivate TGFβ1 by expression of components of the plasmin pathway. Excess production and bioactivation of TGFβ1 at sites of MTB infection may undermine host immune responses during TB.

Protective Responses in Tuberculosis: Induction of Genes for Interferon-γ and Cytotoxicity by Mycobacterium tuberculosis and During Human Tuberculosis

The host effector mechanisms against Mycobacterium tuberculosis infection are not well understood, and this remains a problem in the development of new vaccines and immunotherapies in tuberculosis (TB). Here, we studied the expression of genes for interferon gamma (IFN‐γ) and molecules involved in lymphocyte‐mediated cytotoxicity [granzyme B (grzB), perforin, granulysin and Fas ligand (FasL)] against M. tuberculosis‐infected macrophages. The kinetics of expression of these molecules were first established in peripheral blood mononuclear cells (PBMC) of healthy donors, and then investigated in TB patients with and without HIV‐1 coinfection and appropriate control groups. We found that only IFN‐γ and grzB were induced by M. tuberculosis in PBMC from healthy purified protein derivative skin test reactive subjects. However, expression of neither gene nor IFN‐γ protein correlated with intracellular M. tuberculosis growth containment by macrophages. Mycobacterium tuberculosis induction of IFN‐γ, but not grzB, mRNA expression was significantly lower (P < 0.03) in TB patients as compared with healthy subjects.

Analysis of transforming growth factor-beta 1 (TGF-β1) expression in human monocytes infected with Mycobacterium avium at a single cell level by ELISPOT assay

Transforming growth factor beta 1 (TGF-beta1) has been implicated in the pathogenesis of a number of diseases including infection with intracellular pathogens such as Mycobacterium avium complex (MAC). In this study, we developed an ELISPOT assay for measurement of active TGF-beta1 produced by peripheral blood mononuclear cells (PBMC) from healthy individuals in response to LPS or MAC. The frequency of TGF-beta1 producing cells was significantly (p<0.04) higher in response to LPS (10 microg/ml) as compared to unstimulated cells (n=4). Moreover, the frequency of TGF-beta1 producing cells was threefold higher in monocyte (MN)-enriched cell population than those in PBMC indicating that the source of TGF-beta1 producing cells in PBMC was MN. In addition, the frequency of TGF-beta1 producing cells in response to MAC (10:1, cfu:MN) was significantly higher (p<0.03) than unstimulated cells. However, the frequency of TGF-beta1 producing cells in response to MAC (10:1) was eight to ninefold lower than that by LPS (10 microg/ml). Moreover, there was a correlation between the level of total TGF-beta1 in 24-h culture supernatants and the number of TGF-beta1 producing cells upon MAC stimulation. TGF-beta1 ELISPOT-assay may be a sensitive and a powerful tool for detection of TGF-beta1 producing cells, and may be helpful in elucidation of the nature of TGF-beta1 production at sites of diseases.

Inhibition of Mycobacterium tuberculosis-Induced Signalling by Transforming Growth Factor-β in Human Mononuclear Phagocytes: Inhibitors of TGF-β Signalling in TB

Tuberculosis (TB) is associated with excessive production and bioactivation of transforming growth factor bets (TGF‐β) in situ. Here, modification of expression of components of plasminogen/plasmin pathway in human monocytes (MN) by inhibitors of TGF‐β signalling was examined. Smad3 siRNA effectively inhibited TGF‐β‐induced urokinase plasminogen activator receptor (uPAR). Agents known to interfere with TGF‐β signalling, including the Smad inhibitors SIS3 and erythromycin derivatives, and ALK5 receptor inhibitor (SB 431542) in inhibition of uPAR expression in response to Mycobacterium tuberculosis (MTB) were examined. Inhibition by SIS3 only inhibited uPAR mRNA significantly. SIS3 may prove to be an effective adjunct to TB therapy.