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Lee, Oh, Son, Song, Kim, Park, Paik, and Jo: Identification and Functional analysis of Gene Expression in Mycobacterium tuberculosis-infected Human Monocytic Cells Under Hypoxic Conditions
Original Article

J Bacteriol Virol 2007;37(2):91-103.

Published online: 18 February 2007

DOI: https://doi.org/10.4167/jbv.2007.37.2.91

Identification and Functional analysis of Gene Expression in Mycobacterium tuberculosis-infected Human Monocytic Cells Under Hypoxic Conditions

Ji-Sook Lee1, 2, Jae-Hee Oh1, Ji Woong Son3, Chang-Hwa Song1, Hwa-Jung Kim1, Jung-Kyu Park1, Tae-Hyun Paik2, Eun-Kyeong Jo1

1Department of Microbiology, College of Medicine, Chungnam National University, Daejeon 301-747, Korea

2Department of Microbiology, Konyang University, Daejeon 302-718, Korea

3Internal Medicine, College of Medicine, Konyang University, Daejeon 302-718, Korea

Received 28 March 2007       Accepted 5 May 2007

Copyright © 2007 Journal of Bacteriology and Virology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Mycobacterium tuberculosis-induced granulomatous lesions, particularly those undergoing central caseation, are known as hypoxic. To analyze the host genes associated with hypoxic conditions from cells infected with M. tuberculosis, we performed GeneChip analyses on mRNA from M. tuberculosis H37Rv-treated human monocytic THP-1 cells cultured in oxygen-depleted status for 18 h. The expression of 99 genes was altered, including those involved in intracellular signaling, energy production, and protein metabolism, as revealed by stringent microarray data analysis. Most notably, mRNA expression of chemokine macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20) was significantly up-regulated in M. tuberculosis-infected cells under hypoxic conditions. We further analyzed the CCL20 expression in peripheral blood mononuclear cells (PBMCs) and monocyte derived macrophages (MDMs) from healthy controls and TB patients. A comparative analysis has revealed that the mRNA and protein expression of CCL20 were prominently up-regulated in PBMCs, and MDMs from TB patients, compared with healthy controls. Collectively, these data show that the gene expression of CCL20 was up-regulated in M. tuberculosis H37Rv-infected human monocytic THP-1 cells cultured in hypoxic conditions. In addition, the production of CCL20 is substantially increased in cells from TB patients than in healthy controls, suggesting an important role of CCL20 in the immunopathogenesis during TB infection.

Keywords: Mycobacterium tuberculosis, Hypoxic, CCL20, 30-kDa antigen

References

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Figure 1.
Comparative analysis of human cathepsin D and CCL20 mRNA expression in M. tuberculosis-infected THP-1 cells under normoxic and hypoxic conditions. (A) RT-PCR analysis was used to determine the differential expression of cathepsin D and CCL20 genes in THP-1 cells cultured under normoxic and hypoxic conditions. normoxic, aerobically incubated (20% O2); Hypoxic, incubated in hypoxic conditions (2% O2); Un, unstimluated. Rv, M. tuberculosis H37Rv-infected; Ra, M. tuberculosis H37Ra-infected. (B) Values are ratios of band density to band density of GAPDH mRNA at each condition and are mean ± SEM. ∗∗, p<0.01; ∗∗∗, p<0.001
jbv-37-91f1.tif
Figure 2.
Kinetics of CCL20 induction in response to 30-kDa Ag. PBMCs and MDMs were incubated with different doses of 30-kDa Ag (A) and different time point (B). Supernatants were harvested after the times indicated and CCL20 levels were measured by ELISA.
jbv-37-91f2.tif
Figure 3.
CCL20 expressions in PBMCs and MDMs from healthy controls (HC) and TB patients (E-TB and Cr-TB) in response to 30-kDa Ag. (A) The 30-kDa Ag was added to PBMCs at 1.0 μg/ml for 6 hr. RT-PCR analysis was done to determine the comparative expression of CCL20 mRNA from PBMCs from HCs and TB patients. PBMCs (B) and MDMs (C) from HCs and TB patients were stimulated by 30-kDa Ag. After 18-hr stimulation, the supernatants were harvested and CCL20 protein levels were measured by ELISA. Closed circle, unstimulated; open circle, stimulated with the 30-kDa Ag. E-TB, early diagnosed pulmonary tuberculosis patients; Cr-TB, chronic tuberculosis patients. ∗, p<0.05; ∗∗, p<0.01; ∗∗∗, p<0.001
jbv-37-91f3.tif
Table 1.
Down-regulated genes in THP-1 cells infected with M. tuberculosis H37Rv under hypoxic conditions
Genbank accession no. Symbol Name of gene Fold changea
    1. Binding  
R43015 SF3A3 Splicing factor 3a, subunit 3, 60kDa 2.14
AA047478 CORO1A Coronin, actin binding protein, 1A 2.57
AA281733 EIF1AX Eukaryotic translation initiation factor 1A, X-linked 2.30
AA460286 GNG10 Guanine nucleotide binding protein (G protein), gamma 10 2.10
AA133577 SNRPG Small nuclear ribonucleoprotein polypeptide G 2.57
W72693 HNRPAB Heterogeneous nuclear ribonucleoprotein A/B 2.92
AA454585 SFRS2 Splicing factor, arginine/serine-rich 2 2.31
AA453749 HDGF Hepatoma-derived growth factor 2.82
AA464731 S100A11 S100 calcium binding protein A11 (calgizzarin) 2.02
AA598526 HIF1A Hypoxia-inducible factor 1, alpha subunit 3.41
AW075424 SFRS3 Splicing factor, arginine/serine-rich 3 2.60
AA086471 S100A8 S100 calcium binding protein A8 (calgranulin A) 5.78
AW087572 CALM2 Calmodulin 2 (phosphorylase kinase, delta) 2.65
    2. Catalytic activity  
T72398 TDO2 Tryptophan 2,3-dioxygenase 3.58
AW028938 PPM1B Protein phosphatase 1B (formerly 2C) 4.23
H65395 PSME2 Proteasome (prosome, macropain) activator subunit 2 (PA28 beta) 2.38
AA863149 PSMA7 Proteasome (prosome, macropain) subunit, alpha type, 7 2.01
T71606 HMOX1 Heme oxygenase (decycling) 1 2.17
AI375353 SGK Serum/glucocorticoid regulated kinase 2.21
N20475 CTSD Cathepsin D (lysosomal aspartyl protease) 7.23
AI361530 ACSL1 Acyl-CoA synthetase long-chain family member 1 2.55
AA708298 ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex 2.54
AA160913 PDE9A Phosphodiesterase 9A 2.39
AI828190 PLAUR Plasminogen activator, urokinase receptor 2.15
AW087572 CALM2 Calmodulin 2 (phosphorylase kinase, delta) 2.65
AW078798 UBB Ubiquitin B 2.38
    3. Chaperone activity  
AA448396 HSPE1 Heat shock 10 kDa protein 1 (chaperonin 10) 2.40
AW075457 CCT3 Chaperonin containing TCP1, subunit 3 (gamma) 2.70
AW004895 HSPD1 Heat shock 60 kDa protein 1 (chaperonin) 3.40
    4. Defense immunity protein activity  
AA865464 LY6E Lymphocyte antigen 6 complex, locus E 2.35
H29485 SSB Sjogren syndrome antigen B (autoantigen La) 2.13
AI362062 NOVA1 Neuro-oncological ventral antigen 1 2.06
AA775355 XRCC5 X-ray repair complementing defective repair in CHO cells 5 2.26
AW075443 G22P1 Thyroid autoantigen 70 kDa (Ku antigen) 2.25
    5. Signal transducer activity  
AW058480 LBR Lamin B receptor 2.47
AA133212 NCOA4 Nuclear receptor coactivator 4 2.21
AI828190 PLAUR Plasminogen activator, urokinase receptor 2.15
    6. Transporter activity  
H85355 ATP2A ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 2.26
AI815076 2SLC7A7 Solute carrier family 7 (cationic amino acid transporter) 2.17
R52654 CYCS Cytochrome c, somatic 2.99
AA598759 PGD Phosphogluconate dehydrogenase 2.27
AI969670 LDHB Lactate dehydrogenase B 2.68
AA708298 ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex 2.54
AA865265 CYCS Cytochrome c, somatic 2.47
AA463492 CYBB Cytochrome b-245, beta polypeptide 2.07
    7. Others  
AA608568 CCNA2 Cyclin A2 2.65
H99170 CALR Calreticulin 2.70
AA699697 TNF Tumor necrosis factor (TNF superfamily, member 2) 2.02
AA644088 CTSC Cathepsin C 2.00
AA406020 G1P2 Interferon, alpha-inducible protein (clone IFI-15K) 2.13
AA600177 CALR Calreticulin 2.08
AA489087 KPNA2 Karyopherin alpha 2 (RAG cohort 1, importin alpha 1) 3.82
W73874 CTSL Cathepsin L 2.70
AW087897 OK/SW-cl.56 Beta 5-tubulin 2.22
AW071125 PRDX1 Peroxiredoxin 1 3.39
W73144 LCP1 Lymphocyte cytosolic protein 1 (L-plastin) 2.42
N90109 NCL Nucleolin 4.84

a The indicated values represent the ratio of normoxic/hypoxic signals. A mean ratio ≥2.0 refers to a decrease in hypoxic relative to normoxic expression.

Table 2.
Up-regulated genes in THP-1 cells infected with M. tuberculosis H37Rv under hypoxic conditions
Genbank accession no. Symbol Name of gene Fold changea
    1. Binding  
AA936135 Gemin7 Gem (nuclear organelle) associated protein 7 2.40
AA278764 ZNF406 Zinc finger protein 406 3.24
AI800882 NRL Neural retina leucine zipper 2.41
AA457153 ZNF282 Zinc finger protein 282 2.09
    2. Catalytic activity  
AA644211 PTGS2 Prostaglandin-endoperoxide synthase 2 6.93
AA459292 CKS1B CDC28 protein kinase regulatory subunit 1B 2.74
AI961583 MAPK10 Mitogen-activated protein kinase 10 2.15
AA496013 NEK4 NIMA (never in mitosis gene a)-related kinase 4 2.05
R61229 GATM Glycine amidinotransferas 3.57
AA427725 CPZ Carboxypeptidase Z 2.10
AA481562 DARS Aspartyl-tRNA synthetase 2.07
AA457671 P4HA1 Procollagen-proline, 2-oxoglutarate 4-dioxygenase 4.35
AA630620 PIK3R4 Phosphoinositide-3-kinase, regulatory subunit 4, p150 2.59
AA457700 SCD Stearoyl-CoA desaturase (delta-9-desaturase) 3.34
AA279533 HMGCS1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 3.61
AA461478 RNGTT RNA guanylyltransferase and 5′-phosphatase 2.04
AA866043 CBS Cystathionine-beta-synthase 2.01
N63567 PDK3 Pyruvate dehydrogenase kinase, isoenzyme 3 2.00
AI335255 NEK6 NIMA (never in mitosis gene a)-related kinase 6 2.05
    3. Cell adhesion molecule  
AA922832 ICAM3 Intercellular adhesion molecule 3 2.13
    4. Signal transducer activity  
AA453774 RGS16 Regulator of G-protein signalling 16 4.95
T62636 CXCR4 Chemokine (C-X-C motif) receptor 4 3.06
AA017544 RGS1 Regulator of G-protein signalling 1 5.08
AI380755 PVR Poliovirus receptor 2.08
    5. Transcription regulator activity  
AI418194 SOX21; SOX25LD Homo sapiens cDNA clone IMAGE: 2105121 3′, mRNA sequence 2.25
    6. Transporter activity  
AA453467 LDHC Lactate dehydrogenase C 2.22
AA157955 SC4MOL Sterol-C4-methyl oxidase-like 2.40
AI160757 KCNJ10C Potassium inwardly-rectifying channel, subfamily J, member 10 2.73
    7. Others  
AI285199 CL20 Chemokine (C-C motif) ligand 20 5.54
AA857944 CSPG2 Chondroitin sulfate proteoglycan 2 (versican) 7.76
AA450123 ENO2 Enolase 2 (gamma, neuronal) 4.90
AI951114 ALDOC Aldolase C, fructose-bisphosphate 4.43
AI656802 OLFM1 Olfactomedin 1 2.12
AA872001 ANXA6 Annexin A6 2.21
AA620859 SSPN Sarcospan (Kras oncogene-associated gene) 2.01
AI362949 SYN2 Synapsin II 2.36
AI056417 MSLN Mesothelin 2.12
AI015641 SDC1 Syndecan 1 2.27

a The indicated values represent the ratio of hypoxic/normoxic signals. A mean ratio ≥2.0 refers to an increase in hypoxic relative to normoxic expression.

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