Citation: Halimani, N.; Nesterchuk, M.; Andreichenko, I.N.; Tsitrina, A.A.; Elchaninov, A.; Lokhonina, A.; Fatkhudinov, T.; Dashenkova, N.O.; Brezgina, V.; Zatsepin, T.S.; et al. Phenotypic Alteration of BMDM In Vitro Using Small Interfering RNA. Cells 2022, 11, 2498. https://doi.org/ 10.3390/cells11162498 Academic Editors: Maciej Lech and Stefanie Steiger Received: 14 July 2022 Accepted: 9 August 2022 Published: 11 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Phenotypic Alteration of BMDM In Vitro Using Small Interfering RNA Noreen Halimani 1, * , Mikhail Nesterchuk 1 , Irina N. Andreichenko 1 , Alexandra A. Tsitrina 2 , Andrey Elchaninov 3,4 , Anastasia Lokhonina 3,5 , Timur Fatkhudinov 4,6 , Nataliya O. Dashenkova 2 , Vera Brezgina 1 , Timofei S. Zatsepin 1 , Arsen S. Mikaelyan 2,† and Yuri V. Kotelevtsev 1, * ,† 1 Vladimir Zelman Center for Neurobiology and Brain Rehabilitation and Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow 143025, Russia 2 Koltzov Institute of Developmental Biology of Russian Academy of Sciences, 26 Vavilov Street, Moscow 119334, Russia 3 National Medical Research Center for Obstetrics, Gynecology and Perinatology Named after Academician V.I. Kulakov, Russian Federation, 4 Oparina Street, Moscow 117997, Russia 4 Department of Histology, Pirogov Russian National Research Medical University, Ministry of Healthcare of The Russian Federation, 1 Ostrovitianov Street, Moscow 117997, Russia 5 Department of Histology, Cytology and Embryology, Peoples’ Friendship University of Russia, 6 Miklukho-Maklaya Street, Moscow 117198, Russia 6 Scientific Research Institute of Human Morphology, 3 Tsurupa Street, Moscow 117418, Russia * Correspondence: noreen.halimani@skoltech.ru (N.H.); y.kotelevtsev@skoltech.ru (Y.V.K.) † These authors contributed equally to this work. Abstract: Autologous macrophage transfer is an emerging platform for cell therapy. It is anticipated that conventional macrophage reprogramming based on ex vivo polarization using cytokines and ligands of TLRs may enhance the therapeutic effect. We describe an alternative approach based on small interfering RNA (siRNA) knockdown of selected molecular cues of macrophage polarization, namely EGR2, IRF3, IRF5, and TLR4 in Raw264.7 monocyte/macrophage cell line and mouse-bone- marrow-derived macrophages (BMDMs). The impact of IRF5 knockdown was most pronounced, curtailing the expression of other inflammatory mediators such as IL-6 and NOS2, especially in M1-polarized macrophages. Contrary to IRF5, EGR2 knockdown potentiated M1-associated markers while altogether abolishing M2 marker expression, which is indicative of the principal role of EGR2 in the maintenance of alternative phenotypes. IRF3 knockdown suppressed M1 polarization but upregulated Arg 1, a canonical marker of alternative polarization in M1 macrophages. As anticipated, the knockdown of TLR4 also attenuated the M1 phenotype but, akin to IRF3, significantly induced Arginase 1 in M0 and M1, driving the phenotype towards M2. This study validates RNAi as a viable option for the alteration and maintenance of macrophage phenotypes. Keywords: macrophages; polarization; siRNA; IRF5; IRF3; EGR2; TLR4 1. Introduction Recent progress in CAR-T therapy warrants further developments based on the mod- ification and transplantation of myeloid cells for the treatment of cancer, liver and lung fibrosis, and many other pathologies [1–3]. The feasibility and efficacy of allogeneic bone-marrow-derived macrophage (BMDM) transplantation were demonstrated in animal models of pulmonary alveolar proteinosis [4], visceral leishmaniasis [5], and liver and idiopathic pulmonary fibrosis [6,7]. The first phase one in-human trial of autologous macrophage transplantation in patients with cirrhosis has already been completed successfully. Each subject received a single peripheral injection containing 10 7 , 10 8 , or up to 10 9 cells. The procedure did not cause any dose-limiting toxicities, transfusion reactions, or macrophage-activation syndrome [7]. Cells 2022, 11, 2498. https://doi.org/10.3390/cells11162498 https://www.mdpi.com/journal/cells