|
Mononuclear phagocytes are not only important immune cells but also the principal host cells of Mycobacterium tuberculosis (M.tuberculosis) and play a key role in its development. Following activation by lymphocytes, mononuclear phagocytes serve as the final effectors in the killing of intracellular M.tuberculosis. Nevertheless, the pathogen can survive, and even thrive, in the intracellular environment of human blood monocytes and tissue macrophages.［1］
At present, various mechanisms have been suggested to contribute to the survival of M.tuberculosis within macrophages, including inhibition of phagosome-lysosome fusion, inhibition of the acidification of the phagosome, resistance to killing by reactive oxygen intermediates and reactive nitrogen intermediates.［2］ The molecular mechanisms through which M.tuberculosis produces these effects and by which host cells attempt to counter this pathogenic strategy remain most unknown.
In order to gain additional insights into the mycobacterium-macrophage interaction, we used a cDNA microarray to observe changes in macrophage gene expression following invasion by M.tuberculosis and to identify host-defense strategies. This information will provide a foundation for understanding the infection process of M.tuberculosis and for identifying new strategies to prevent and treat tuberculosis.
METHODS
Bacterial culture
M.tuberculosis H37Ra provided by the Shanghai Pulmonary Hospital was grown in a liquid Middlebrook 7H9 medium (Difco) supplemented with 0.05% Tween 80, 0.2% glycerol, and 10% oleic acid- albumin-dextrose-catalase (OADC) (Difco) at 37℃ for 2 weeks before infecting some macrophage U937.
Cell infection and mRNA preparation
U937 cells were cultured in a RPMI 1640 medium (GIBCO/BRL), supplemented with 10% fetal calf serum in 75 cm2 cell culture flasks under 5% CO2 at 37℃ (approximately 1×10(6)/ml), and infected with M.tuberculosis at a 10∶1 multiplicity of infection. After 24 hours, acid-fast staining showed that approximately 70% of the U937 cells contained at least one case of M.tuberculosis, and total cellular RNA was prepared from uninfected and infected U937 cells using the TRIZOL Reagent(GIBCO/BRL). The mRNA was purified using an Oligotex mRNA Midi Kit (Qiagen) according to the manufacturer's instructions.
Probe preparation
The fluorescent cDNA probes were prepared through reverse transcription and then purified, according to the protocol of Schena.［3］ The probes from uninfected cells were labeled with Cy3-dUTP, while those from infected cells were labeled with Cy5-dUTP. The probes were then mixed, precipitated and resolved in a hybridization buffer.
Hybridization and washing
The BioDoor 12 800 microarray provided by Shanghai United Gene Holdings, Ltd. consists of a total of 12 788 known and new genes that include 88 positive and negative control spots. Before hybridization, the cDNA microarray was prehybridized and the probes' mixture was denatured. Then, the probes' mixture was added to the microarray and incubated at 42℃ for 15-17 hours. After incubation, the microarray was washed and dried at room temperature.［4］
Detection and analysis
The microarray was scanned at two wavelengths using a ScanArray 5000 laser scanner (General Scanning, Inc.). The acquired image was analyzed using ImaGene 3.0 software (BioDiscovery, Inc.). The intensity of each spot at the two wavelengths represented the quantity of Cy3-dUTP and Cy5-dUTP respectively. Each ratio of Cy5 to Cy3 was computed. Two standards were defined to screen out each differentially expressed genes: firstly, the absolute value of the natural logarithm of the ratio is greater than 0.69; secondly, at least one of both the raw intensity values of Cy3 and Cy5 were larger than 600.
RT-PCR
One-step RT-PCR was conducted using the SUPE-RSCRIPT One-Step RT-PCR system (GIBCO/BRL). According to the manufacturer's instruction, each 50 μl reaction contained 25 μl of 2× reaction mix, 1 μl of enzyme mix, 200 nmol/L of each primer and the appropriate amount of purified total RNA. The reactions were incubated at 50℃ for 30 minutes, 94℃ for 2 minutes; then followed by amplification of 28 cycles of 94℃ for 15 seconds, 55℃ for 30 seconds, and 68℃ for 1 minute followed by 72℃ for 5 minutes. A RT-PCR negative control reaction, containing the appropriate primer pairs but no added template, was performed with each experiment. PCR products were analysed by electrophoresis in 2% agarose gels containing ethidium bromide, and images of the stained bands were obtained and analysed using Molecular Imager FX.
RESULTS
We found that these positive control spots showed a high intensity of signal, while the negative control spots showed a low intensity of signal after the hybridization process, thus proving statistical reliability. After infected with M.tuberculosis H37Ra, the altered gene expression is monitored by the microarray.According to the standards described in the methods, there were 463 differentially expressed genes amongst the infected and non-infected cells, of which 366 genes were known genes registered in the Gene Bank. There were 25 known genes showing up-regulated expression and 341 known genes showing down-regulated expression in infected cells. In addition, 97 new genes were also influenced by M.tuberculosis infection.To test the validity of the microarray results further, we assessed the levels of specific mRNA for bcl-2, p0071 protein, VCP, p65, adaptor protein p150, and syntenin from infected and uninfected cells. As an internal control we analyzed the relative level of GAPD mRNA using the same technique, because the array results showed that its expression was not changed after infection. The RT-PCR results support the microarray-based observation that the expression of bcl-2 and p0071 protein was up-regulated, and that the expression of VCP, p65, adaptor protein p150, and syntenin was down-regulated ( Fig. ).
DISCUSSION
High density DNA microarray analysis of host gene expression in response to pathogen infection provides a powerful approach to examining microbial pathogens from a new perspective. The ability to survey the responses of a large subset of the host genome, and to find patterns among the profiles from many different microorganisms and hosts, allows fundamental questions to be addressed (or re-addressed) concerning the basis of pathogen recognition, the features of the interaction between host and pathogen, and the mechanisms of host defense and microbial virulence.［5］In this study, we used a high-density cDNA microarray to identify 463 genes from macrophages that change in expression level in response to infection with M.tuberculosis H37Ra strains. The 366 known genes have been reported to function in a variety of cellular processes, including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity and cellular metabolic pathways. Presumably, these genes must play a role in the host cell defense mechanisms and/or the facilitation of the bacterial life cycle.There is evidence that apoptosis plays important roles in modulating the pathogenesis of a variety of infectious diseases. M.tuberculosis can not only induce but also inhibit apoptosis in macrophages via different pathways.［6,7］ The modulation of apoptosis by M.tuberculosis and its direct and overlapping effects on the immune system are likely to play key roles in pathogenesis. In this study, we found that several apoptosis-associated genes, when infected with H37Ra for 24 hours, displayed an altered expression. Therefore, uncovering the mechanisms of the pro- and anti-apoptosis activities induced by M.tuberculosis and their roles in pathogenesis of tuberculosis, is significant for elucidating complex host-pathogen interaction.
Cellular receptors are highly important for the process of M.tuberculosis infection. M.tuberculosis enters macrophages through receptor-mediated pathways while macrophage receptors are involved in host resistance to M.tuberculosis. For instance, CD14 is a 55 kDa glycosylphosphatidyl-instiol (GPI) -anchored cell surface molecule. Peterson et al［8］ showed that microglial cells can make use of CD14 to recognize M.tuberculosis (H37Rv), and facilitate entry of bacteria into cells. As CD14 is involved in the uptake of M.tuberculosis, the down-regulation of them (by 2.3 fold) in this study means that the pathogen limits M.tuberculosis uptake in the infected cells. Another receptor, vitamin D receptor (down-regulated by 3.3 fold), has been linked to the antimycobacterial activity of a host. Several studies have shown that susceptibility to tuberculosis may be increased by deficiency of vitamin D (25-hydroxycholecalciferol) and that the active metabolite of Vitamin D3, 1,25-dihydroxyvitamin D3, helps mononuclear phagocytes to suppress the intracellular growth of M.tuberculosis.［9,10］ This effect is influenced by the vitamin D receptor.
It has been reported that cholesterol is essential for the uptake of mycobacteria by macrophages: cholesterol accumulated at the site of mycobacterial entry and depleting plasma membrane cholesterol specifically inhibited mycobacterial uptake. Cholesterol also mediated the phagosome association with tryptophane aspartate-containing coat protein (TACO), which prevents the fusion of a phagosome with lysosomes.［11,12］ Mycobacteria that enter macrophages at cholesterol-rich domains of plasma membrane may ensure their intracellular survival in TACO-coated phagosomes. In this study, the mRNAs for enzymes participating in cholesterol synthesis and protein regulating cholesterol synthesis including squalene synthase, the phosphomevalonate kinase and oxysterol-binding protein, were down-regulated in infected cells. Since cholesterol is important in the uptake and intracellular survival of mycobacteria, the down-regulation of those enzymes and above-mentioned protein, when infected is worth further investigation.
The expression of other genes associated with intracellular signalling, cytoskeletal rearrangement, transcriptional regulation, and cell-mediated immunity were also changed in infected macrophages. The significance of the altered expression of these genes needs to be researched and analyzed further.
Our results show that the monitoring of gene expression profiling using a cDNA microarray is a powerful approach to characterizing and understanding host-pathogen interaction. Gene expression profiling analysis of macrophages infected with different strains of M.tuberculosis has helped to reveal the mechanisms by which the host responds to M.tuberculosis and some of the mechanisms by which the pathogens avoid host defense thereby surviving and growing in host cells. This information may help to identify new therapeutic targets.
REFERENCES
1. Sliver RF, Li Q, Ellner JJ. Expression of virulence of Mycobacterium tuberculosis within human monocytes: virulence correlates with intracellular growth and induction of tumor necrosis factor Alpha but not with evasion of lymphocyte-dependent monocyte effector functions. Infect Immun 1998;66:1190-1199.
2. Wei J, Dahl JL, Moulder JW, et al. Identification of a Mycobacterium tuberculosis gene that enhances Mycobacterial survival in macrophages. J Bacteriol 2000;182:377-384.
3. Schena M, Shalon D, Heller R, et al. Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. Proc Natl Acad Sci U S A 1996;93:10614-10619.
4. Cheng JX, Tang R, Ying K, et al. Study on metastasis-associated gene in carcinoma by cDNA microarray. Acda J Sec Mil Med Univ 2000;21:806-811.
5. Manger ID, Relman DA. How the host ‘sees' pathogens: global gene expression responses to infection. Curr Opin Immunol 2000;12:215-218.
6. Keane J, Balcewicz-Sablinska MK, Remold HG， et al. Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis. Infect Immun 1997;65:298-304.
7. Balcewicz-Sablinska MK, Keane J, Kornfeld H, et al. Pathogenic Mycobacterium tuberculosis evades apoptosis of host macrophages by release of TNFR2, resulting in inactivation of TNF-α. J Immunol 1998; 161:2636-2641.
8. Peterson PK, Gekker G, Hu S, et al. CD14 receptor-mediated uptake of nonopsonized Mycobacterium tuberculosis by human microglia. Infect Immun 1995;63:1598-1602.
9. Rockett KA, Brookes R, Udaloval I, et al. 1,25-dihydroxyvitamin D3 induces nitric oxide synthase and suppresses growth of Mycobacterium tuberculosis in a human macrophage-like cell line. Infect Immun 1998;66:5314-5321.
10. Sly LM, Lopez M, Nauseef WM, et al. 1, 25-dihydroxyvitamin D3-induced monocyte antimycobacterial activity is regulated by phosphatidylinositol 3-kinase and mediated by the NADPH-dependent phagocyte oxidase. J Biol Chem 2001;276:35482-35493.
11. Gatfield J, Pieters J. Essential role for cholesterol in entry of mycobacteria into macrophages. Science 2000;288:1647-1650.
12. Ferrari G, Langen H, Naito M, et al. A coat protein on phagosomes involved in the intracellular survival of mycobacteria. Cell 1999;97:435-447.
|
PDF(-)
Rapid Response |