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Mycobacterial Species Differ in Intracellular Survival, Cytokine mRNA Expression, NF-Kappa B Activation, and Macrophage Apoptosis
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A4309 - Mycobacterial Species Differ in Intracellular Survival, Cytokine mRNA Expression, NF-Kappa B Activation, and Macrophage Apoptosis
Author Block: E. D. Chan1, X. Bai2, A. Musheyev1, A. Bai2, L. Li3, J. R. Honda2, Z. Feng1; 1Natl Jewish Med Ctr, Denver, CO, United States, 2National Jewish Health, Denver, CO, United States, 3DEOHS National Jewish Med and Research Ctr, Denver, CO, United States.
RATIONALE: Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) account for all causes of lung disease due to mycobacteria, with MTB generally considered to be more virulent than NTM although host factors also contribute to disease development. We sought to determine if there was differential growth of MTB H37Rv and four different NTM species in differentiated THP-1 macrophages as well as any differences in cytokine expression, nuclear factor-kappa B (NF-kappaB) activation, and apoptosis. METHODS: The monocytic THP-1 cell line was differentiated with PMA, infected with 10:1 multiplicity of infection of either MTB H37Rv, M. avium, M. intracellulare, M. abscessus or M. smegmatis, and cell-associated mycobacteria quantified at various times after infection. Cytokine expression by quantitative RT-PCR, NF-kappaB activation by measuring binding of the p65 subunit of NF-kappaB to its cis-regulatory element, and apoptosis was determined by the TUNEL assay. RESULTS: Bacterial burden at two and four days after infection showed that MTB had greater burden than the four other NTM at four days after infection. There was also differential burden between the NTM infections with greater CFU of M. avium than M. intracellulare for the slow-growing NTM, and greater CFU of M. abscessus than M. smegmatis for the rapidly-growing mycobacteria. There was differential expression of mRNA for various pro-inflammatory cytokines (TNF-alpha, IL-8, IL-1-beta) and anti-inflammatory cytokine IL-10. MTB H37Rv induced greater amount of p65 NF-kappaB binding and lesser number of TUNEL-positive cells compared to the NTM species. CONCLUSION: Differences in the intracellular burden and cytokine gene expression in THP-1 cells infected with the five different species of mycobacteria. In addition, MTB H37Rv had greater NF-kappaB activation and apoptosis compared to NTM species. These findings suggest that differences in virulence among mycobacterial species are closely linked to differences in host and pathogen interactions.
Author Block: E. D. Chan1, X. Bai2, A. Musheyev1, A. Bai2, L. Li3, J. R. Honda2, Z. Feng1; 1Natl Jewish Med Ctr, Denver, CO, United States, 2National Jewish Health, Denver, CO, United States, 3DEOHS National Jewish Med and Research Ctr, Denver, CO, United States.
RATIONALE: Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) account for all causes of lung disease due to mycobacteria, with MTB generally considered to be more virulent than NTM although host factors also contribute to disease development. We sought to determine if there was differential growth of MTB H37Rv and four different NTM species in differentiated THP-1 macrophages as well as any differences in cytokine expression, nuclear factor-kappa B (NF-kappaB) activation, and apoptosis. METHODS: The monocytic THP-1 cell line was differentiated with PMA, infected with 10:1 multiplicity of infection of either MTB H37Rv, M. avium, M. intracellulare, M. abscessus or M. smegmatis, and cell-associated mycobacteria quantified at various times after infection. Cytokine expression by quantitative RT-PCR, NF-kappaB activation by measuring binding of the p65 subunit of NF-kappaB to its cis-regulatory element, and apoptosis was determined by the TUNEL assay. RESULTS: Bacterial burden at two and four days after infection showed that MTB had greater burden than the four other NTM at four days after infection. There was also differential burden between the NTM infections with greater CFU of M. avium than M. intracellulare for the slow-growing NTM, and greater CFU of M. abscessus than M. smegmatis for the rapidly-growing mycobacteria. There was differential expression of mRNA for various pro-inflammatory cytokines (TNF-alpha, IL-8, IL-1-beta) and anti-inflammatory cytokine IL-10. MTB H37Rv induced greater amount of p65 NF-kappaB binding and lesser number of TUNEL-positive cells compared to the NTM species. CONCLUSION: Differences in the intracellular burden and cytokine gene expression in THP-1 cells infected with the five different species of mycobacteria. In addition, MTB H37Rv had greater NF-kappaB activation and apoptosis compared to NTM species. These findings suggest that differences in virulence among mycobacterial species are closely linked to differences in host and pathogen interactions.
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