Changes in gene expression in macrophages infected with Mycobacterium tuberculosis: a combined transcriptomic and proteomic approach

Abstract

We investigated the changes which occur in gene expression in the human macrophage cell line, THP1, at 1, 6 and 12 hr following infection with Mycobacterium tuberculosis. The analysis was carried out at the transcriptome level, using microarrays consisting of 375 human genes generally thought to be involved in immunoregulation, and at the proteomic level, using two-dimensional gel electrophoresis and mass spectrometry. The analysis of the transcriptome using microarrays revealed that many genes were up-regulated at 6 and 12 hr. Most of these genes encoded proteins involved in cell migration and homing, including the chemokines interleukin (IL)-8, osteopontin, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha), regulated on activation, normal, T-cell expressed and secreted (RANTES), MIP-1beta, MIP-3alpha, myeloid progenitor inhibitory factor-1 (MPIF-1), pulmonary and activation regulated chemokine (PARC), growth regulated gene-beta (GRO-beta), GRO-gamma, MCP-2, I-309, and the T helper 2 (Th2) and eosinophil-attracting chemokine, eotaxin. Other genes involved in cell migration which were up-regulated included the matrix metalloproteinase MMP-9, vascular endothelial growth factor (VEGF) and its receptor Flk-1, the chemokine receptor CCR3, and the cell adhesion molecules vesicular cell adhesion molecule-1 (VCAM-1) and integrin a3. In addition to the chemokine response, genes encoding the proinflammatory cytokines IL-1beta (showing a 433-fold induction), IL-2 and tumour necrosis factor-alpha (TNF-alpha), were also found to be induced at 6 and/or 12 hr. It was more difficult to detect changes using the proteomic approach. Nevertheless, IL-1beta was again shown to be strongly up-regulated. The enzyme manganese superoxide dismutase was also found to be strongly up-regulated; this enzyme was found to be macrophage-, rather than M. tuberculosis, derived. The heat-shock protein hsp27 was found to be down-regulated following infection. We also identified a mycobacterial protein, the product of the atpD gene (thought to be involved in the regulation of cytoplasmic pH) in the infected macrophage extracts.

Figure 1

Microarray analysis of Mycobacterium tuberculosis-infected THP1 cells. Gene expression 6 hr following infection (b) was compared with that in uninfected cells (a). The most strongly up-regulated genes are labelled.

Figure 2

Analysis by two-dimensional gel electrophoresis of Mycobacterium tuberculosis-infected THP1 cells. Protein extracts of THP1 cells infected with M. tuberculosis (b) were compared with those of uninfected THP1 cells (a). A non-linear, pH 3–10 IEF first-dimension and 12% second-dimension gel was used. Two up-regulated proteins and one down-regulated protein were identified (inserts) and analysed by MALDI-TOF.

Figure 3

Analysis by two-dimensional gel electrophoresis of Mycobacterium tuberculosis-infected THP1 cells. The experiment is identical to that described in Figure 2, except that a linear pH 4–7 IEF first-dimension and 7·5% second-dimension gel was used. The up-regulated protein shown in the insert was found to be the ATP synthase β chain, the product of the M. tuberculosis atpD gene (c).