Phenotypic Alteration of BMDM In Vitro Using Small Interfering RNA

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.

Interleukin 28A aggravates Con A‐induced acute liver injury by promoting the recruitment of M1 macrophages

“…To further investigate the role of IL‐28A in the regulation of M1 macrophages, mouse bone marrow cells were cultured with complete DMEM with GM‐CSF for 7 days. On day 7, the BMDMs were replated, and untreated (M0, CD11b + F4/80 + ) macrophages from WT and IL‐28A −/− mice were stimulated with LPS and IFN‐γ 43,44 . Cells were collected to detect the proportion of F4/80 + CD86 + cells after 24 h, which are indicative of M1 macrophages.…”

“…On day 7, the BMDMs were replated, and untreated (M0, CD11b + F4/80 + ) macrophages from WT and IL-28A −/− mice were stimulated with LPS and IFN-γ. 43,44 Cells were collected to detect the proportion of F4/80 + CD86 + cells after 24 h, which are indicative of M1 macrophages. IL-28A deficiency markedly reduced the proportion of M1 macrophages (Figure 7A).…”

Improving combination cancer immunotherapy by manipulating dual immunomodulatory signals with enzyme-triggered, cell-penetrating peptide-mediated biomodulators

“…As widely documented in the literature, IL-4 stimulation is an effective method to polarize BMDMs to the M2 type. 37,38 Consequently, the macrophage phenotype in our co-culture system predominantly assumes the M2 type. After 20 h of incubation, we observed that the biomodulator BPC3 significantly enhanced the engulfment of the target cells by both M2-type macrophages and dendritic cells in vitro, indicating the favorable immunostimulatory ability of the PP1-disrupting motif (Fig.…”