Critical Role of Methylglyoxal and AGE in Mycobacteria-Induced Macrophage Apoptosis and Activation

Apoptosis and activation of macrophages play an important role in the host response to mycobacterial infection involving TNF-α as a critical autocrine mediator. The underlying mechanisms are still ill-defined. Here, we demonstrate elevated levels of methylglyoxal (MG), a small and reactive molecule that is usually a physiological product of various metabolic pathways, and advanced glycation end products (AGE) during mycobacterial infection of macrophages, leading to apoptosis and activation of macrophages. Moreover, we demonstrate abundant AGE in pulmonary lesions of tuberculosis (TB) patients. Global gene expression profiling of MG-treated macrophages revealed a diverse spectrum of functions induced by MG, including apoptosis and immune response. Our results not only provide first evidence for the involvement of MG and AGE in TB, but also form a basis for novel intervention strategies against infectious diseases in which MG and AGE play critical roles

Unique Transcriptome Signature of Mycobacterium tuberculosis in Pulmonary Tuberculosis

Although tuberculosis remains a substantial global threat, the mechanisms that enable mycobacterial persistence and replication within the human host are ill defined. This study represents the first genome-wide expression analysis of Mycobacterium tuberculosis from clinical lung samples, which has enabled the identification of M. tuberculosis genes actively expressed during pulmonary tuberculosis. To obtain optimal information from our DNA array analyses, we analyzed the differentially expressed genes within the context of computationally inferred protein networks. Protein networks were constructed using functional linkages established by the Rosetta stone, phylogenetic profile, conserved gene neighbor, and operon computational methods. This combined approach revealed that during pulmonary tuberculosis, M. tuberculosis actively transcribes a number of genes involved in active fortification and evasion from host defense systems. These genes may provide targets for novel intervention strategies

<em>Mtb</em>-Specific CD27^low CD4 T Cells as Markers of Lung Tissue Destruction during Pulmonary Tuberculosis in Humans

<div><h3>Background</h3><p>Effector CD4 T cells represent a key component of the host’s anti-tuberculosis immune defense. Successful differentiation and functioning of effector lymphocytes protects the host against severe <em>M. tuberculosis</em> (<em>Mtb</em>) infection. On the other hand, effector T cell differentiation depends on disease severity/activity, as T cell responses are driven by antigenic and inflammatory stimuli released during infection. Thus, tuberculosis (TB) progression and the degree of effector CD4 T cell differentiation are interrelated, but the relationships are complex and not well understood. We have analyzed an association between the degree of <em>Mtb</em>-specific CD4 T cell differentiation and severity/activity of pulmonary TB infection.</p> <h3>Methodology/Principal Findings</h3><p>The degree of CD4 T cell differentiation was assessed by measuring the percentages of highly differentiated CD27^low cells within a population of <em>Mtb</em>- specific CD4 T lymphocytes (“CD27^lowIFN-γ⁺” cells). The percentages of CD27^lowIFN-γ+ cells were low in healthy donors (median, 33.1%) and TB contacts (21.8%) but increased in TB patients (47.3%, p<0.0005). Within the group of patients, the percentages of CD27^lowIFN-γ⁺ cells were uniformly high in the lungs (>76%), but varied in blood (12–92%). The major correlate for the accumulation of CD27^lowIFN-γ⁺ cells in blood was lung destruction (r = 0.65, p = 2.7×10⁻⁷⁾. A cutoff of 47% of CD27^lowIFN-γ⁺ cells discriminated patients with high and low degree of lung destruction (sensitivity 89%, specificity 74%); a decline in CD27^lowIFN-γ⁺cells following TB therapy correlated with repair and/or reduction of lung destruction (p<0.01).</p> <h3>Conclusions</h3><p>Highly differentiated CD27^low Mtb-specific (CD27^lowIFN-γ⁺) CD4 T cells accumulate in the lungs and circulate in the blood of patients with active pulmonary TB. Accumulation of CD27^lowIFN-γ⁺ cells in the blood is associated with lung destruction. The findings indicate that there is no deficiency in CD4 T cell differentiation during TB; evaluation of CD27^lowIFN-γ⁺ cells provides a valuable means to assess TB activity, lung destruction, and tissue repair following TB therapy.</p> </div

TB patients have increased percentages of CD27^lowIFN-γ⁺ CD4 T cells in their blood.

<p>A–C, Strategies for determining percentages of CD27^low (A), IFN-γ⁺ (B) and CD27^lowIFN-γ⁺ (C) CD4 T cells. A, CD27^low cells were gated within the total population of CD4⁺ T cells. B, To identify IFN-γ⁺ CD4 T cells, an aliquot of blood was stimulated with <i>Mtb</i> sonicate; another aliquote was left un-stimulated. During the analysis, the gates for IFN-γ⁺ cells in <i>Mtb</i>-stimulated samples were plotted based on <i>Mtb</i> un-stimulated samples (Fig. B, dotted line). To identify CD27^lowIFN-γ⁺ cells, the expression of CD27 was first analyzed in IFN-γ⁻ population. Because this population was always numerous, CD27^low and CD27^hi cells could be easily separated. The gates for CD27^low cells were then applied to IFN-γ⁺ population (C, dotted line). D–F, Percentages of CD27^low (D), IFN-γ⁺ (E), and CD27^lowIFN-γ⁺ (F) cells in TB patients (n = 50), TB contacts (n = 21) and <i>Mtb</i>-unexposed individuals (n = 15). G, Lack of correlation between the percentages of IFN-γ⁺ and CD27^lowIFN-γ⁺ cells in TB patients, TB contacts and <i>Mtb</i>-unexposed individuals (n = 86). H, ROC-curve of CD27^lowIFN- _γ⁺ cell percentages for discriminating TB patients from healthy individuals (TB contacts and <i>Mtb</i>-unexposed). I, Percentages of CD27^lowIFN-γ⁺ cells in TB contacts with positive and negative results of QFT assay *p<0.0005 compared to TB patients.</p

Selection of minimal model to explain variability in the percentages of CD27^lowIFN-γ⁺ cells between TB patients.

1<p>Best minimal models are those that differ insignificantly from the full model (highlighted in bold).</p>2<p>Analysis was initially performed in 50 patients. Subsequently, 12 patients from validation cohort were added (n = 62), mainly to check the consistency of the results. In both cohorts, lung destruction and clinical disease severity predicted best the accumulation of CD27^lowIFN-γ⁺ cells.</p>3<p>In Akaike Information Criterion, this combination was the best minimal model to predict the accumulation of CD27^lowIFN-γ⁺ cells in the blood ( Δ_n = 50 = 4.7; Δ_n = 62 = 4.8).</p

Correlation between TB manifestations and percentages of CD27^lowIFN-γ⁺ CD4 T cells in the blood of TB patients.

1<p>Analysis was initially performed in 50 patients. Subsequently, 12 patients from validation cohort were added (n = 62), mainly to check the consistency of the results. The results obtained in both cohorts are shown.</p>2<p>Simple correlation analysis selects five major predictors for the accumulation of IFN-γ⁺CD27^low cells in the blood of TB patients (highlighted in bold). For TB duration and <i>Mtb</i> multi-drug resistance p-values were >0.007 (insignificant for multiple (seven) parameter testing); these factors were not included in multiple linear regression analysis. rho, Spearman coefficient, p, significance value of the test.</p>3<p>Multiple linear regression identified lung tissue destruction and clinical TB severity as the main correlates for the accumulation of CD27^lowIFN-γ⁺ cells in the blood of TB patients (highlighted in bold).</p>4<p>NA, not included in multiple linear regression analysis.</p

Characterization of groups included in the analysis.

1<p>Indicated are numbers (%).</p>2<p>NA, not applicable.</p