Interferon-γ loaded dextran-based nanoparticles for the polarization of macrophages
Loading...
Date issued
Authors
Editors
Journal Title
Journal ISSN
Volume Title
Publisher
Reuse License
Abstract
The immune system, comprising cellular and humoral elements, is crucial in both malignancy progression and regression. Macrophage progenitors develop in the bone marrow and differentiate into various subtypes with diverse target tissues and functions. In the context of malignant tumours, two major subclasses are distinguished: TAMs (Tumour associated macrophages), which resemble the M2 phenotype and tumouricidal proinflammatory M1 macrophages. TAMs contribute to tumour-promoting immunosuppression and vascularization in the tumour microenvironment. This negatively affects primary tumour growth, metastasis, and therapy response. Regarding macrophage differentiation, interferon-γ (IFN-γ) is a potent cytokine to support M1 polarization and is relevant to immunological tumour defence. Nanoparticles can serve as carriers to reduce the side effects of cytokine therapy by enhancing the uptake of the active substance in the target tissue, while sparing unrelated tissues. Dextran nanoparticles (DNPs) are taken up by macrophages in vitro without any toxic impact. In vivo, DNP accumulate mainly in the liver, where they are prominently taken up by macrophages.
Until now, the efficacy of interferon-γ (IFN-y) loaded DNPs to induce M1-type polarization/repolarization of TAMs in vitro and a possible therapeutic effect in vivo has not been studied. The aim of this thesis was to study if IFN-γ-DNP affect the polarization of bone marrow derived macrophages of Balb/c and C57BL/6 mice in vitro and whether they have a positive therapeutic effect in a melanoma metastasis model in vivo.
Toxicity of IFN-γ-DNP was excluded in in vitro experiments with primary macrophages derived from wild-type mice. Using quantitative real-time polymerase chain reaction (qPCR), the genetic profiles of native and in vitro M2 differentiated macrophages subsequently treated with IFN-γ and IFN-γ-DNP were analysed. Despite different assumptions, both IFN-γ and IFN-γ-DNP required costimulation with lipopolysaccharide (LPS) to significantly increased the expression of tumour necrosis factor α (tnfa), cluster of differentiation 38 (cd38), class II major histocompatibility complex molecules (mhc2),inducible nitric oxide synthase (inos) and lymphocyte antigen 6 complex (ly6c), all M1 marker genes, and upregulated the expression of early growth response protein (egr2) and mannose receptor, cluster of differentiation 206 (cd206/mrc1), both M2 marker genes. Macrophages treated with IFN-γ-DNP and LPS-loaded dextran nanoparticles (LPS-DNP) transcribed significantly more mRNA of specific M1 marker genes and reduced the expression of M2-specific genes compared to macrophages treated with IFN-γ and LPS alone. Therefore, it may be concluded that IFN-γ (IFN-γ-DNP) and LPS have a synergistic effect towards M1-type polarization due to the priming ability of IFN-γ. This effect could be increased with enhanced delivery and uptake of IFN-γ loaded into DNP. Furthermore, an increased metabolic activity due to dextran as an additional source of energy may also contribute. However, empty DNP (eDNP) alone had no polarizing effect. Single IFN-γ-DNP treatment induced an M1 phenotype also as characterized by fluorescence-activated cell sorting (FACS)-analysis showed an increased MHCII and decreased CD206 expression. In contrast, coactivation with LPS was required to detect a significant change in the mRNA expression of mhc2 and mrc1 by qPCR.
In addition, the combination of IFN-γ-DNP and LPS, LPS as well as LPS-DNP, led to an increased tumour necrosis factor α (TNFα) production, as determined by enzyme-linked immunosorbent assay (ELISA), while the increased interleukin-10 (previous described as M2-type marker) content in the cell culture supernatant can presumably be explained by an increased production of this cytokine that can occur initially during M1 macrophage polarization.
The DNPs were successfully tested for tolerability in a mouse (C57BL/6) model of liver metastasis using B16F10 melanoma cells. Although IFN-γ-DNPs generated a MHCII high CD206 low phenotype in vitro, as detected by FACS, no therapeutic effect was observed in vivo. Treatment with IFN-γ-DNP was neither able to inhibit the growth of liver metastases nor to induce a polarization of macrophages/TAMs in vivo towards a tumouricidal M1 type in healthy or metastatic livers.
Based on the observed results, further research may investigate the potential therapeutic effect of longer lived and shielded PEGylated LPS-DNP, especially combined with IFN-γ-DNP, in vitro and in vivo.
In conclusion, although IFN-γ-DNP alone did not have a therapeutic anti-tumour effect in vivo, IFN-γ-DNP, particularly in combination with LPS, demonstrated promising results in polarizing and repolarizing bone marrow-derived macrophages to a tumouricidal phenotype in vitro.