Immune response to Mycobacterium tuberculosis and identification of molecular markers of disease

Abstract

The complex molecular events that occur within the host during the establishment of a Mycobacterium tuberculosis infection are poorly defined, thus preventing identification of predictive markers of disease progression and state. To identify such molecular markers during M. tuberculosis infection, global changes in transcriptional response in the host were assessed using mouse whole genome arrays. Bacterial load in the lungs, the lesions associated with infection, and gene expression profiling was performed by comparing normal lung tissue to lungs from mice collected at 20, 40, and 100 days after aerosol infection with the H37Rv strain of M. tuberculosis. Quantitative, whole lung gene expression identified signature profiles defining different signaling pathways and immunological responses characteristic of disease progression. This includes genes representing members of the interferon-associated gene families, chemokines and cytokines, MHC, and NOS2, as well as an array of cell surface markers associated with the activation of T cells, macrophages, and dendritic cells that participate in immunity to M. tuberculosis infection. More importantly, several gene transcripts encoding proteins that were not previously associated with the host response to M. tuberculosis infection, and unique molecular markers associated with disease progression and state, were identified.

Figure 1.

Development of pulmonary granulomatous lesions in mice exposed to Mycobacterium tuberculosis. Growth curve of M. tuberculosis in C57BL/6 mice after low dose aerosol exposure. (A) Bacterial load in the lungs was monitored at Day 1, Day 20, Day 40, and Day 100 of infection by plating and enumeration of colony-forming units (CFU). Sections of formalin-fixed and paraffin-embedded lung tissue of C57BL/6 mice exposed to M. tuberculosis on Days 20, 40, and 100 after M. tuberculosis challenge were visualized with hematoxylin and eosin (H&E) staining. (B) Mild interstitial pneumonia and moderately sized lesions observed at Day 20 of the infection with M. tuberculosis. (C) Large aggregates of lymphocytes were seen within the epithelioid macrophage and increasing numbers of foamy highly vacuolated cells at Day 40. (D) Development of organized inflammatory multifocal granulomas containing lymphocytes and macrophages and large numbers of foamy cells by Day 100 of infection. Pictures were taken with an IX70 Olympus microscope with an attached ZP70 digital camera. Total magnification: A–C, ×4; insets, ×2.

Figure 2.

Real-time PCR of IFN-γ and TNF-α at different times of infection. (A) IFN-γ expression at Day 0 (D0), Day 20 (D20), Day 40 (D40), and Day 100 (D100). (B) TNF-α expression at Day 0 (D0), Day 20 (D20), Day 40 (D40), and Day 100 (D100). The primer and probe sequences for murine IFN-γ and TNF-α were previously published (18, 19). Data are presented using the mean values (n = 5) using replicated samples and duplicate or triplicate assays. A parametric method, the Student t test, was used to assess statistical significance between groups of data.

Figure 3.

Analysis of gene expression ontology of global response and physiology of immune responses. (A) Principal component analysis and scatter plot of the transcriptional response of uninfected mice (D0) and of mice at Day 20 (D20), Day 40 (D40), and Day 100 (D100) of infection with M. tuberculosis. (B) Global ontology profile and (C) ontology of the immune associated genes. Data displayed are (A) based on 1,310 genes and (B) based on 183 immune discriminant genes (1.5-fold or greater alteration; P values < 0.01).

Figure 4.

Identification of molecular markers of disease state and progression. Tandem self-organizing mapping (tandem-SOM) analysis was performed to categorize genes and identify discriminant groups of disease state and progression. (A) gSOM analysis of transcriptional active genes differentially regulated > 1.5-fold (P < 0.01). This analysis distributed genes into 20 groups (0–19). (B) Discriminant groups identified from sSOM analysis. Discriminant groups correspond to Day 20 (discriminant group 1; mean expression = 2.3), Day 40 (discriminant group 2; mean expression = 2.4), Day 100 (discriminant group 3; mean expression = 2.7), Days 40 and 100 (discriminant group 4; mean expression = 2.6 [D40], 2.6 [D100]), and Days 20, 40, and 100 (discriminant group 5; mean expression = 5.4 [D20], 14.1 [D40], 20.8 [D100]).