Comparing Responses of Ursolic Acid in Murine Macrophages Infected with Mycobacterium smegmatis and Mycobacterium avium
Abstract
Mycobacterium smegmatis and Mycobacterium avium offer an advantage in examining tuberculosis-like effects and host immune defense. Therefore, the study aims to examine the effect of ursolic acid (UA) on the host immune system by analyzing cytokines concentration, such as TNF-α, IL-6, IL-1β, and nitrite oxide produced by murine macrophages infected with Mycobacterium smegmatis and Mycobacterium avium. Femurs of female C57BL/6 mice aged 6–8 weeks were used to culture the Bone marrow-derived macrophages (BMDM). On day 10, BMDM was infected with Mycobacterium smegmatis and Mycobacterium avium using a multiplicity of infection (MOI) amounting to 8:1, then TNF-α, IL-6, and IL-1β were analyzed using ELISA and nitrite oxide with Griess reagent. The results showed that UA decreased the production of three respective pro-inflammatory cytokines used in the study, both in BMDM infected by Mycobacterium smegmatis and Mycobacterium avium. For TNF-α, the reduction was nearly 65%-90% compared to the control. The decrease in the production of IL-6 occurred from 2700 pg/ml to 750 pg/ml for BMDM infected with Mycobacterium smegmatis, while the reduction was more significant in those infected using Mycobacterium avium with approximately 150 pg/ml compared to the control. Moreover, UA reduced by over 90% of IL-1β and this result was in line with the reduction of nitrite. UA decreases the production of pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β, and nitrite. This result is preliminary but supports further study on the role of UA in immune defense from pathogenic and non-pathogenic mycobacterial infections.
References
Biet, F., Bay, S., Thibault, V.C., Euphrasie, D., Grayon, M., Ganneau, C., Lanotte, P., Daffé, M., Gokhale, R., Etienne, G., and Reyrat, J.-M. 2008. Lipopentapeptide induces a strong host humoral response and distinguishes Mycobacterium avium subsp. Paratuberculosis from M. avium subsp. Avium. Vaccine. 26(2): 257–268.
Bohsali, A., Abdalla, H., Velmurugan, K., and Briken, V. 2010. The non-pathogenic mycobacteria M. smegmatis and M. fortuitum induce rapid host cell apoptosis via a caspase-3 and TNF dependent pathway. BMC Microbiology. 10. 237.
Danelishvili, L., Young, L.S., and Bermudez, L.E. 2006. In vivo efficacy of phage therapy for Mycobacterium avium infection as delivered by a nonvirulent mycobacterium. Microbial Drug Resistance. 12(1): 1–6.
Drapal, M., Wheeler, P.R., and Fraser, P.D. 2018. The assessment of changes to the nontuberculous mycobacterial metabolome in response to anti-TB drugs. FEMS Microbiology Letters. 365(15).
Gao, N., Cheng, S., Budhraja, A., Gao, Z., Chen, J., Liu, E.-H., Huang, C., Chen, D., Yang, Z., Liu, Q., Li, P., Shi, X., and Zhang, Z. 2012. Ursolic acid induces apoptosis in human leukaemia cells and exhibits anti-leukaemic activity in nude mice through the PKB pathway. British Journal of Pharmacology. 165(6): 1813–1826.
González-Pérez, M., Mariño-Ramírez, L., Parra-López, C. A., Murcia, M. I., Marquina, B., Mata-Espinoza, D., Rodriguez-Míguez, Y., Baay-Guzman, G.J., Huerta-Yepez, S., and Hernandez-Pando, R. 2013. Virulence and immune response induced by Mycobacterium avium complex strains in a model of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice. Infection and Immunity. 81(11): 4001–4012.
Jyoti, Md. A., Zerin, T., Kim, T.-H., Hwang, T.-S., Jang, W. S., Nam, K.-W., and Song, H.-Y. 2015. In vitro effect of ursolic acid on the inhibition of Mycobacterium tuberculosis and its cell wall mycolic acid. Pulmonary Pharmacology & Therapeutics. 33. 17–24.
Kannan, N., Haug, M., Steigedal, M., and Flo, T.H. 2020. Mycobacterium smegmatis Vaccine Vector Elicits CD4+ Th17 and CD8+ Tc17 T Cells With Therapeutic Potential to Infections With Mycobacterium avium. Frontiers in Immunology. 11. 1116.
Kellogg, J.A., Seiple, J.W., Klinedinst, J.L., and Stroll, E. 1996. Diff-Quik stain as a simplified alternative to Papanicolaou stain for determination of quality of endocervical specimens submitted for PCR detection of Chlamydia trachomatis. Journal of Clinical Microbiology. 34(10). 2590–2592.
Lee, K.-I., Whang, J., Choi, H.-G., Son, Y.-J., Jeon, H.S., Back, Y.W., Park, H.-S., Paik, S., Park, J.-K., Choi, C.H., and Kim, H.-J. 2016. Mycobacterium avium MAV2054 protein induces macrophage apoptosis by targeting mitochondria and reduces intracellular bacterial growth. Scientific Reports. 6. 37804.
Lelovic, N., Mitachi, K., Yang, J., Lemieux, M.R., Ji, Y., and Kurosu, M. 2020. Application of Mycobacterium smegmatis as a surrogate to evaluate drug leads against Mycobacterium tuberculosis. The Journal of Antibiotics. 73(11): 780–789.
López-García, S., Castañeda-Sanchez, J.I., Jiménez-Arellanes, A., Domínguez-López, L., Castro-Mussot, M.E., Hernández-Sanchéz, J., and Luna-Herrera, J. 2015. Macrophage Activation by Ursolic and Oleanolic Acids during Mycobacterial Infection. Molecules. 20(8): 14348–14364.
Mondino, S., Gago, G., and Gramajo, H. 2013. Transcriptional regulation of fatty acid biosynthesis in mycobacteria. Molecular Microbiology. 89(2): 372–387.
Pitaloka, D.A.E., Cooper, A.M., Artarini, A.A., Damayanti, S., and Sukandar, E. Y. 2020. Regulation of mitogen-activated protein kinase signaling pathway and proinflammatory cytokines by ursolic acid in murine macrophages infected with Mycobacterium avium. Infectious Disease Reports. 12. 8717.
Seo, D.Y., Lee, S.R., Heo, J.-W., No, M.-H., Rhee, B.D., Ko, K.S., Kwak, H.-B., and Han, J. 2018. Ursolic acid in health and disease. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology 22(3): 235–248.
Sharbati, S., Ravon, F., Einspanier, R., and zur Bruegge, J. 2019. Mycobacterium smegmatis But Not Mycobacterium avium subsp. Hominissuis Causes Increased Expression of the Long Non-Coding RNA MEG3 in THP-1-Derived Human Macrophages and Associated Decrease of TGF-β. Microorganisms. 7(3): 63.
T, J.A.S., J, R., Rajan, A., and Shankar, V. 2020. Features of the biochemistry of Mycobacterium smegmatis, as a possible model for Mycobacterium tuberculosis. Journal of Infection and Public Health. 13(9): 1255–1264.
World Health Organization. (2021, October 14). Tuberculosis. https://www.who.int/news-room/fact-sheets/detail/tuberculosis
Yone, É. W.P., Kuaban, C., and Kengne, A. P. 2012. Impact of HIV infection on the evolution of tuberculosis among adult patients in Yaounde, Cameroon. Revue De Pneumologie Clinique. 68(6): 338–344.
Zerin, T., Lee, M., Jang, W.S., Nam, K.-W., and Song, H.-Y. 2015. Ursolic Acid Reduces Mycobacterium tuberculosis-Induced Nitric Oxide Release in Human Alveolar A549 cells. Molecules and Cells. 38(7): 610–615.
Zhang, X., Goncalves, R., and Mosser, D.M. 2008. The Isolation and Characterization of Murine Macrophages. Current Protocols in Immunology.14.1.
Zhao, J., Zheng, H., Sui, Z., Jing, F., Quan, X., Zhao, W., and Liu, G. 2019. Ursolic acid exhibits anti-inflammatory effects through blocking TLR4-MyD88 pathway mediated by autophagy. Cytokine,.123. 154726.
