Abstract
Biglycan, a small leucine-rich proteoglycan (SLRP), is a crucial component of the extracellular matrix (ECM) associated with the maintenance of tissue homeostasis. In response to tissue damage, the ECM-derived soluble form of biglycan acts as a danger signal by triggering an inflammatory response via the toll-like receptor (TLR)2/TLR4 in macrophages and dendritic cells. The impact and signaling mechanism of biglycan in innate immunity is better understood with the use of specific and reliable research tools and investigation techniques. Accordingly, our lab has established explicit and detailed experimental protocols to examine the in vitro and in vivo effects of biglycan in cellular immune responses. To evaluate the in vitro effects of biglycan on macrophage activation, a comprehensive protocol that makes use of murine peritoneal macrophages has been described. Further, to study the in vivo effects of biglycan, a method that uses a pLIVE vector to generate transgenic mice transiently overexpressing human biglycan is detailed. A step-by-step protocol for analyzing the effects of soluble biglycan overexpression in transgenic mice is explained under the following sections: (1) construction of pLIVE-hBGN plasmid, (2) intravenous delivery of transgenic vector, (3) identification of hBGN transgene in hepatocytes (4) detection of transgenic biglycan protein in the plasma of transgenic mice, and (5) evaluation of the presence and pro-inflammatory effects of transgenic biglycan in extrahepatic mouse tissues.
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Acknowledgments
This work was supported by the German Research Council (SFB 1039, project B02, SFB 1177, 259130777, project E02 (both to LS)); the Cardio-Pulmonary Institute ((CPI), EXC 2026, Project ID: 390649896 (to LS and MW)); the Hessian Ministry within the LOEWE Research Initiative ACLF-I (To LS and JT); European Union’s Horizon 2020 Research And Innovation Program’s MICROB-PREDICT study (No 825694); European Union’s Horizon 2020 Research and Innovation Program’s GALAXY (No. 668031) and Societal Challenges LIVERHOPE (health, demographic change, and well-being, No. 731875); the German Research Council (SFB TRR57, CRC1382); and Cellex Foundation (PREDICT) all to JT.
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Zeng-Brouwers, J., Huber, L.S., Merline, R., Trebicka, J., Wygrecka, M., Schaefer, L. (2023). Evaluation of the In Vitro and In Vivo Effects of Biglycan in Innate Immunity. In: Karamanos, N.K. (eds) Proteoglycans. Methods in Molecular Biology, vol 2619. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2946-8_9
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DOI: https://doi.org/10.1007/978-1-0716-2946-8_9
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