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

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.

Mycobacterium tuberculosis-activated dendritic cells induce protective immunity in mice.

Activated dendritic cells are critically important in the priming of T-cell responses. In this report we show that the infection of a conditionally immortalized dendritic cell line (tsDC) with Mycobacterium tuberculosis resulted in the up-regulation of B7-1 and B7-2 co-stimulatory molecules and the induction of several inflammatory cytokines, including tumour necrosis factor-alpha and interleukin-6, -1beta and -12. In addition, we show that these activated dendritic cells were capable of eliciting antigen-specific T-cell responses and potent anti-mycobacterial protective immunity in a murine model of experimental tuberculosis infection.

Immunostimulatory bacterial DNA sequences activate dendritic cells and promote priming and differentiation of CD8+ T cells.

CD8+ T lymphocytes producing high levels of interferon-gamma (IFN-gamma) and expressing antigen specific cytotoxic activity are effectively induced after plasmid DNA vaccination and mediate protection against several intracellular micro-organisms. Recent evidence suggests that the priming of CD8+ T-cell responses following DNA injection involves antigen presentation mediated by dendritic cells. Here, we show that bacterial DNA and synthetic oligonucleotides containing dinucleotide (CpG) motifs activate cytokine expression in dendritic cells and modulate in vivo CD8+ T-cell priming and differentiation.

Regulation of macrophage gene expression by Mycobacterium tuberculosis: down-regulation of mitochondrial cytochrome c oxidase.

We have investigated changes in gene expression in mouse peritoneal macrophages following infection with virulent Mycobacterium tuberculosis. Using differential-display reverse transcription-PCR (RT-PCR), we have identified a gene that was markedly down-regulated within 6 h of infection and remained so for the duration of the experiment (5 days). On sequencing, this gene was found to encode the murine cytochrome c oxidase subunit VIIc (COX VIIc). Down-regulation of COX VIIc during M. tuberculosis infection was confirmed by three independent techniques: limiting-dilution RT-PCR, RNase protection assay, and Northern analysis. Limiting-dilution RT-PCR and Northern analysis were also used to analyze the specificity of this regulation; heat-killed M. tuberculosis, Mycobacterium bovis BCG, and latex beads had no effect on expression of COX VIIc. Down-regulation of this enzyme was also confirmed by using adherent cells isolated from spleens of M. tuberculosis-infected mice. These ex vivo macrophages showed apoptotic features, suggesting a possible involvement of cytochrome c oxidase in the programmed cell death of the host cells.

Regulation of macrophage gene expression following invasion by Mycobacterium tuberculosis.

INTRODUCTION: Mycobacteria are intracellular pathogens which survive and grow in host macrophages. M. tuberculosis bacilli enter the macrophage via binding to several distinct cell surface molecules. Following phagocytosis, sustained intracellular bacterial growth depends on the ability to avoid destruction by macrophage-mediated host defences such as lysosomal enzymes, reactive oxygen and the reactive nitrogen intermediates. We used differential display reverse transcription polymerase chain reaction (DD RT-PCR) to identify host genes which are regulated during infection and hence which might be involved in the host-parasite cross talk. RESULTS: Live M. tuberculosis (strain H37Rv) was used to infect Balb/c peritoneal murine macrophages. mRNA from infected and uninfected macrophages was isolated at different time intervals after phagocytosis and subjected to DD RT-PCR. Oligo dT12NV and random 10mer primers were used for PCR amplification of cDNA. Macrophage genes which appeared to be differently regulated during infection were subjected to further reamplification by PCR in order to clone and sequence them. The differential expression of the selected bands was further analysed by an RNA protection assay and a Northern blot. RESULTS: Several differentially regulated bands were identified. One band, of 158 bp, was down regulated after infection. Sequencing of this band revealed a high level of homology (95% identity) to mouse cytochrome c oxidase subunit VIIc. The downregulation was specific for live virulent Mtb, while live BCG, heat killed Mtb and latex beads-mediated phagocytosis did not affect the transcriptional level of this enzyme. CONCLUSIONS: The cytochrome oxidase enzyme complex of the inner mytochondrial membrane catalyzes the reaction between ferrocytochrome c and oxygen. The reaction is the terminal event in the electron transport scheme. Downregulation of cytochrome c oxidase subunit VIIc could interfere with: (1) the host apoptotic programme; or (2) the host respiratory burst.

Protection against tuberculosis by a plasmid DNA vaccine.

Past attempts to use fractions of mycobacteria as an alternative to BCG have given disappointing results. The availability of cloned genes and suitable vectors has now opened a new avenue in which individual mycobacterial protein antigens are synthesised within transfected mammalian cells. In an ex vivo transfection approach with a retroviral vector we found that even a single antigen (hsp65) could evoke strong protection when expressed as a transgene and that expression of protection was largely a function of antigen specific cytotoxic T cells. We now find that intramuscular injection of plasmid DNA expressing the antigen from either a viral or a murine promoter can also give protection equivalent to Bacillus Calmette-Guérin (BCG). Plasmids expressing some other mycobacterial antigens, hsp70, 36 kDa and 6 kDa, are also effective, suggesting that this approach may lead to a new vaccine.

Protection of rats from adjuvant arthritis by immunization with naked DNA encoding for mycobacterial heat shock protein 65.

OBJECTIVE: To assess the feasibility of vaccination with naked DNA encoding for mycobacterial heat shock protein 65 (hsp65) in the modulation of experimental arthritis. METHODS: Adjuvant arthritis (AA) was induced in Lewis rats preimmunized, intramuscularly, with a plasmid encoding for hsp65 (pCMV3.65). Clinical scores were recorded for 3-4 weeks, and histologic and radiologic parameters were evaluated. Cellular and antibody reactivity to hsp65 was assessed. The expression of the hsp65 gene was investigated in injected muscles. RESULTS: The pCMV3.65-treated rats were significantly protected from disease development in comparison with the control groups. This finding correlated with the results of histologic and radiologic examinations of the involved joints. The message for hsp65 was detected in injected muscles. T cell proliferation and antibodies to this protein were found to be elevated in pCMV3.65-treated rats when compared with both the arthritic control (AA-induced) and the naive (did not receive adjuvant) animals. CONCLUSION: We have demonstrated for the first time that naked DNA delivery is feasible in controlling experimental autoimmune disease. Although the actual mechanism of protection has not been fully elucidated, this simple and versatile approach could represent a useful tool in dissecting basic autoimmune mechanisms.

Vaccination against tuberculosis by DNA injection.

There are 3 million deaths per annum worldwide due to tuberculosis, and AIDS is compounding the problem. A better vaccine than the live mycobacterium currently in use, bacillus Calmette-Guérin (BCG), is needed. When mice were injected with plasmid DNA encoding a single mycobacterial antigen (65-kDa heat shock protein, hsp65) they made specific cellular and humoral responses to the protein and became immune to subsequent challenge with Mycobacterium tuberculosis. Protection was equivalent to that obtained by vaccinating with live BCG, whereas immunizing with the protein was ineffective. Protection was also obtained with DNA encoding another mycobacterial antigen (36-kDa proline-rich antigen). These results suggest that DNA vaccination might yield improved vaccines to replace BCG.

A synthetic 10-kD heat shock protein (hsp10) from Mycobacterium tuberculosis modulates adjuvant arthritis.

The heat shock protein, hsp10, is an abundant protein in Mycobacterium tuberculosis (Mtb), its nucleotide sequence encoding a protein of 99 amino acids with a molecular mass of 10.7 kD. This sequence is phylogenetically conserved, being represented by the GroES homologue of Escherichia coli. Hsp10 and GroES are members of the chaperonin 10 family of molecular chaperones, and GroEs is necessary for the optimal activity of GroEL, a member of the chaperonin 60 family and the E. coli homologue of mycobacterial hsp65. Since hsp65 has been implicated in both experimental and human rheumatoid arthritis, we aimed to assess the immunomodulatory effects of its co-chaperonin, hsp10, in experimental arthritis. Our results show that an aqueous solution of a mycobacterial hsp10 delayed the onset and severity of adjuvant-induced arthritis in rodents when administered after disease induction but before joint involvement occurred. This biological activity was specific for the hsp10 of Mtb, since neither GroES not the rat homologue was effective. Using synthetic hsp10 fragments, the activity was localized to the N-terminal region of the molecule. Assessment of circulating antibody levels to mycobacterial hsp10 and hsp65 indicated that all arthritic rats had increased titres to both hsp10 and hsp65: hsp10-treated rats showed further elevation of this humoral response not only to hsp10 but also to hsp65 when compared with the untreated arthritic control. This is the first report of the immunomodulatory activity of mycobacterial hsp10 in experimental arthritis, and exhibits a potential role for this co-chaperonin in pathophysiological situations.

PPD and hsp65 induced monoarthritis initiates spontaneous recurrent flares in Lewis rats.

OBJECTIVES: To investigate the time course of a monoarthritis induced with the purified protein derivative of tuberculin (PPD) or a recombinant 65 kDa heat shock protein (rhsp65) in two different strains of sensitised rats. METHODS: Wistar and Lewis rats, sensitised with heat killed Mycobacterium tuberculosis in oil, were challenged intraarticularly with PPD or rhsp65 and monitored for six weeks. Inflammation was assessed by joint swelling, histology, and radiographic studies. RESULTS: Sensitised Lewis rats injected with PPD or rhsp65 showed a chronic response with recurrent spontaneous flares, while Wistar rats showed one inflammatory episode. CONCLUSIONS: The Wistar strain response to PPD resembles a transient reactive arthritis, while the response of the Lewis strain mimics the relapsing nature of rheumatoid synovitis, providing an interesting model to determine dominant immunopathogenic mechanisms.

Arthritogenic potential of the 65 kDa stress protein--an experimental model.

OBJECTIVES: To assess the effect of an intra-articular presentation of stress (heat shock) proteins (hsp) on joint inflammation. METHODS: Wistar rats were sensitised with a suspension of heat killed Mycobacterium tuberculosis in oil in the scruff of the neck and challenged intra-articularly with stress protein or M tuberculosis preparations. Inflammation was assessed by joint swelling and, using immunohistology, cellular infiltration of the synovium and antibody induction by an enzyme-linked immunosorbent method. RESULTS: It was shown, for the first time, that the intra-articular administration of a recombinant myobacterial 65 kDa hsp can induce joint inflammation in M tuberculosis sensitised recipients; both powdered M tuberculosis and the purified protein derivative of tuberculin (PPD) produced a similar response, with T cell infiltration of the synovium and a time course typical of delayed type hypersensitivity. This response was specific to the 65 kDa protein as another immunodominant mycobacterial stress protein of 10 kDa was ineffective. Furthermore, intra-articular injection of the 65 kDa hsp induced an antibody response against both the 65 kDa and 10 kDa proteins and the antibody titres continued to rise when knee swelling had subsided. CONCLUSIONS: These results support the hypothesis that 60 kDa proteins are a relevant arthritogenic stimulus in an M tuberculosis background. Moreover, when antigen presentation occurs in the synovium of previously sensitised individuals, circulating antibodies are generated which persist and recognise cross-reactive epitopes on several stress proteins.