MicroRNAs can regulate inflammatory responses by modulating macrophage polarization. Although microRNA miR-21 is linked to crucial processes involved in inflammatory responses, its precise role in macrophage polarization is controversial. In this study, we investigated the functional relevance of endogenous miRNA-21 and the role of exosomes. RAW 264.7 macrophages were transfected with miR-21 plasmid, and the inflammatory response was evaluated by flow cytometry, phagocytosis, and real-time PCR analysis of inflammatory cytokines. To understand the signaling pathways’ role, the cells were treated with inhibitors specific for PI3K or NFĸB. Exosomes from transfected cells were used to study the paracrine action of miR-21 on naive macrophages. Overexpression of miR-21 resulted in significant upregulation of pro-inflammatory cytokines, pushing the cells towards a pro-inflammatory phenotype, with partial involvement of PI3K and NFĸB signal pathways. The cells also secreted miR-21 rich exosomes, which, on delivery to naive macrophages, caused them to exhibit pro-inflammatory activity. The presence of miR-21 inhibitor quenched the inflammatory response. This study validates the pro-inflammatory property of miR-21 with a tendency to foster an inflammatory milieu. Our findings also reinforce the dual importance of exosomal miR-21 as a biomarker and therapeutic target in inflammatory conditions.
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The data used to support the findings of this study are available from the corresponding authors upon reasonable request.
Locati, M., G. Curtale, and A. Mantovani. 2020. Diversity, mechanisms, and significance of macrophage plasticity. Annu Rev Pathol 15: 123–147.
Murray, P.J., and T.A. Wynn. 2011. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11: 723–737.
Novak, M.L., and T.J. Koh. 2013. Phenotypic transitions of macrophages orchestrate tissue repair. Am J Pathol 183: 1352–1363.
Porta, C., E. Riboldi, A. Ippolito, and A. Sica. 2015. Molecular and epigenetic basis of macrophage polarized activation. Semin Immunol 27: 237–248.
Koh, T.J., and L.A. DiPietro. 2011. Inflammation and wound healing: the role of the macrophage. Expert Rev Mol Med 13: e23.
O'Connell, R.M., D.S. Rao, and D. Baltimore. 2012. microRNA regulation of inflammatory responses. Annu Rev Immunol 30: 295–312.
Cunha, C., C. Gomes, A.R. Vaz, and D. Brites. 2016. Exploring new inflammatory biomarkers and pathways during LPS-induced M1 polarization. Mediators Inflamm 2016: 6986175.
Self-Fordham, J.B., A.R. Naqvi, J.R. Uttamani, V. Kulkarni, and S. Nares. 2017. MicroRNA: dynamic regulators of macrophage polarization and plasticity. Front Immunol 8: 1062.
Roy, S. 2016. miRNA in macrophage development and function. Antioxid Redox Signal 25: 795–804.
Sil, S., R.S. Dagur, K. Liao, E.S. Peeples, G. Hu, P. Periyasamy, and S. Buch. 2020. Strategies for the use of extracellular vesicles for the delivery of therapeutics. J Neuroimmune Pharmacol 15: 422–442.
Kosaka, N., H. Iguchi, Y. Yoshioka, F. Takeshita, Y. Matsuki, and T. Ochiya. 2010. Secretory mechanisms and intercellular transfer of microRNAs in living cells. J Biol Chem 285: 17442–17452.
Takahashi, R.U., M. Prieto-Vila, A. Hironaka, and T. Ochiya. 2017. The role of extracellular vesicle microRNAs in cancer biology. Clin Chem Lab Med 55: 648–656.
Ghai, V., and K. Wang. 2016. Recent progress toward the use of circulating microRNAs as clinical biomarkers. Arch Toxicol 90: 2959–2978.
Doyle, L.M., and M.Z. Wang. 2019. Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells 8.
Bui, T.M., L.A. Mascarenhas, and R. Sumagin. 2018. Extracellular vesicles regulate immune responses and cellular function in intestinal inflammation and repair. Tissue Barriers 6: e1431038.
McDonald, M.K., Y. Tian, R.A. Qureshi, M. Gormley, A. Ertel, R. Gao, et al. 2014. Functional significance of macrophage-derived exosomes in inflammation and pain. Pain 155: 1527–1539.
Alexander, M., R. Hu, M.C. Runtsch, D.A. Kagele, T.L. Mosbruger, T. Tolmachova, M.C. Seabra, J.L. Round, D.M. Ward, and R.M. O’Connell. 2015. Exosome-delivered microRNAs modulate the inflammatory response to endotoxin. Nat Commun 6: 7321.
Shi, Y., P. Luo, W. Wang, K. Horst, F. Bläsius, B. Relja, D. Xu, F. Hildebrand, and J. Greven. 2020. M1 but not M0 extracellular vesicles induce polarization of RAW264.7 macrophages via the TLR4-NFκB pathway in vitro. Inflammation 43: 1611–1619.
Sheedy, F.J. 2015. Turning 21: Induction of miR-21 as a key switch in the inflammatory response. Front Immunol 6: 19.
Madhyastha, R., H. Madhyastha, Y. Pengjam, Q.I. Nurrahmah, Y. Nakajima, and M. Maruyama. 2019. The pivotal role of microRNA-21 in osteoclastogenesis inhibition by anthracycline glycoside aloin. J Nat Med 73: 59–66.
Madhyastha, R., H. Madhyastha, Y. Nakajima, S. Omura, and M. Maruyama. 2012. MicroRNA signature in diabetic wound healing: promotive role of miR-21 in fibroblast migration. Int Wound J 9: 355–361.
Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 2006; Chapter 3:Unit 3.22.
Koyasu, S. 2003. The role of PI3K in immune cells. Nat Immunol 4: 313–319.
Ruse, M., and U.G. Knaus. 2006. New players in TLR-mediated innate immunity: PI3K and small Rho GTPases. Immunol Res 34: 33–48.
Liu, T., L. Zhang, D. Joo, and S.C. Sun. 2017. NF-κB signaling in inflammation. Signal Transduct Target Ther 2.
Mann, M., A. Mehta, J.L. Zhao, K. Lee, G.K. Marinov, Y. Garcia-Flores, L.F. Lu, A.Y. Rudensky, and D. Baltimore. 2017. An NF-κB-microRNA regulatory network tunes macrophage inflammatory responses. Nature Communications 8: 851.
Pengjam, Y., H. Madhyastha, R. Madhyastha, Y. Yamaguchi, Y. Nakajima, and M. Maruyama. 2016. NF-κB pathway inhibition by anthrocyclic glycoside aloin is key event in preventing osteoclastogenesis in RAW264.7 cells. Phytomedicine 23: 417–428.
Essandoh, K., Y. Li, J. Huo, and G.C. Fan. 2016. MiRNA-mediated macrophage polarization and its potential role in the regulation of inflammatory response. Shock 46: 122–131.
Chakraborty, C., A.R. Sharma, G. Sharma, and S.S. Lee. 2020. The interplay among miRNAs, major cytokines, and cancer-related inflammation. Mol Ther Nucleic Acids 20: 606–620.
Banerjee, S., H. Cui, N. Xie, Z. Tan, S. Yang, M. Icyuz, V.J. Thannickal, E. Abraham, and G. Liu. 2013. miR-125a-5p regulates differential activation of macrophages and inflammation. J Biol Chem 288: 35428–35436.
Lippai, D., S. Bala, T. Csak, E.A. Kurt-Jones, and G. Szabo. 2013. Chronic alcohol-induced microRNA-155 contributes to neuroinflammation in a TLR4-dependent manner in mice. PLoS One 8: e70945.
Møller, T., J.P. James, K. Holmstrøm, F.B. Sørensen, J. Lindebjerg, and B.S. Nielsen. 2019. Co-detection of miR-21 and TNF-α mRNA in budding cancer cells in colorectal cancer. Int J Mol Sci 20.
Das, A., K. Ganesh, S. Khanna, C.K. Sen, and S. Roy. 2014. Engulfment of apoptotic cells by macrophages: a role of microRNA-21 in the resolution of wound inflammation. J Immunol 192: 1120–1129.
Melnik, B.C. 2015. MiR-21: an environmental driver of malignant melanoma? J Transl Med 13: 202.
Ando, Y., G.X. Yang, T.P. Kenny, K. Kawata, W. Zhang, W. Huang, P.S.C. Leung, Z.X. Lian, K. Okazaki, A.A. Ansari, X.S. He, P. Invernizzi, W.M. Ridgway, Q. Lu, and M.E. Gershwin. 2013. Overexpression of microRNA-21 is associated with elevated pro-inflammatory cytokines in dominant-negative TGF-β receptor type II mouse. J Autoimmun 41: 111–119.
Loboda, A., M. Sobczak, A. Jozkowicz, and J. Dulak. 2016. TGF-β1/Smads and miR-21 in renal fibrosis and inflammation. Mediators Inflamm 2016: 8319283.
Barnett, R.E., D.J. Conklin, L. Ryan, R.C. Keskey, V. Ramjee, E.A. Sepulveda, S. Srivastava, A. Bhatnagar, and W.G. Cheadle. 2016. Anti-inflammatory effects of miR-21 in the macrophage response to peritonitis. J Leukoc Biol 99: 361–371.
Zamorano, J., A.L. Mora, M. Boothby, and A.D. Keegan. 2001. NF-kappa B activation plays an important role in the IL-4-induced protection from apoptosis. Int Immunol 13: 1479–1487.
Ruiz-Lafuente, N., M.J. Alcaraz-García, S. Sebastián-Ruiz, A.M. García-Serna, J. Gómez-Espuch, J.M. Moraleda, A. Minguela, A.M. García-Alonso, and A. Parrado. 2015. IL-4 up-regulates MiR-21 and the MiRNAs Hosted in the CLCN5 gene in chronic lymphocytic leukemia. PLoS One 10: e0124936.
Luzina, I.G., A.D. Keegan, N.M. Heller, G.A. Rook, T. Shea-Donohue, and S.P. Atamas. 2012. Regulation of inflammation by interleukin-4: a review of “alternatives”. J Leukoc Biol 92: 753–764.
Zhou, R., G. Hu, A.Y. Gong, and X.M. Chen. 2010. Binding of NF-kappaB p65 subunit to the promoter elements is involved in LPS-induced transactivation of miRNA genes in human biliary epithelial cells. Nucleic Acids Res 38: 3222–3232.
Madhyastha, R., H. Madhyastha, Y. Pengjam, Y. Nakajima, S. Omura, and M. Maruyama. 2014. NFkappaB activation is essential for miR-21 induction by TGFβ1 in high glucose conditions. Biochem Biophys Res Commun 451: 615–621.
Wang, Z., S. Brandt, A. Medeiros, S. Wang, H. Wu, A. Dent, and C.H. Serezani. 2015. MicroRNA 21 is a homeostatic regulator of macrophage polarization and prevents prostaglandin E2-mediated M2 generation. PLoS One 10: e0115855.
Kumarswamy, R., I. Volkmann, and T. Thum. 2011. Regulation and function of miRNA-21 in health and disease. RNA Biol 8: 706–713.
Cao, L.Q., X.W. Yang, Y.B. Chen, D.W. Zhang, X.F. Jiang, and P. Xue. 2019. Exosomal miR-21 regulates the TETs/PTENp1/PTEN pathway to promote hepatocellular carcinoma growth. Mol Cancer 18: 148.
Simeoli, R., K. Montague, H.R. Jones, L. Castaldi, D. Chambers, J.H. Kelleher, V. Vacca, T. Pitcher, J. Grist, H. al-Ahdal, L.F. Wong, M. Perretti, J. Lai, P. Mouritzen, P. Heppenstall, and M. Malcangio. 2017. Exosomal cargo including microRNA regulates sensory neuron to macrophage communication after nerve trauma. Nat Commun 8: 1778.
Lakhter, A.J., R.E. Pratt, R.E. Moore, K.K. Doucette, B.F. Maier, L.A. DiMeglio, et al. 2018. Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes. Diabetologia 61: 1124–1134.
Melnik, B.C., S.M. John, P. Carrera-Bastos, and G. Schmitz. 2020. MicroRNA-21-enriched exosomes as epigenetic regulators in melanomagenesis and melanoma progression: the impact of Western lifestyle factors. Cancers (Basel) 12.
Bakirtzi, K., I.K. Man Law, K. Fang, D. Iliopoulos, and C. Pothoulakis. 2019. MiR-21 in Substance P-induced exosomes promotes cell proliferation and migration in human colonic epithelial cells. Am J Physiol Gastrointest Liver Physiol 317: G802–G810.
Cui, H., Y. He, S. Chen, D. Zhang, Y. Yu, and C. Fan. 2019. Macrophage-derived miRNA-containing exosomes induce peritendinous fibrosis after tendon injury through the miR-21-5p/Smad7 pathway. Mol Ther Nucleic Acids 14: 114–130.
Tsukamoto, M., H. Iinuma, T. Yagi, K. Matsuda, and Y. Hashiguchi. 2017. Circulating exosomal MicroRNA-21 as a biomarker in each tumor stage of colorectal cancer. Oncology 92: 360–370.
Harrell, C.R., N. Jovicic, V. Djonov, N. Arsenijevic, and V. Volarevic. 2019. Mesenchymal stem cell-derived exosomes and other extracellular vesicles as new remedies in the therapy of inflammatory diseases. Cells 8.
Soeda, N., H. Iinuma, Y. Suzuki, D. Tsukahara, H. Midorikawa, Y. Igarashi, Y. Kumata, M. Horikawa, T. Kiyokawa, T. Fukagawa, and R. Fukushima. 2019. Plasma exosome-encapsulated microRNA-21 and microRNA-92a are promising biomarkers for the prediction of peritoneal recurrence in patients with gastric cancer. Oncol Lett 18: 4467–4480.
The authors thank the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan, for granting the Monbukagashusho Research Scholarship to Nurrahmah Queen Intan and Bethasiwi Purbasari.
This work was funded by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan, under the Grants-in-aid for Scientific Research (19 K10010) to Madhyastha R.
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Madhyastha, R., Madhyastha, H., Nurrahmah, Q.I. et al. MicroRNA 21 Elicits a Pro-inflammatory Response in Macrophages, with Exosomes Functioning as Delivery Vehicles. Inflammation (2021). https://doi.org/10.1007/s10753-021-01415-0
- macrophage polarization
- microRNA 21, miR-21
- exosomal miRNA