Immunomodulatory Macrophages Enable E-MNC Therapy for Radiation-Induced Salivary Gland Hypofunction

Abstract

A newly developed therapy using effective-mononuclear cells (E-MNCs) is reportedly effective against radiation-damaged salivary glands (SGs) due to anti-inflammatory and revascularization effects. However, the cellular working mechanism of E-MNC therapy in SGs remains to be elucidated. In this study, E-MNCs were induced from peripheral blood mononuclear cells (PBMNCs) by culture for 5–7 days in medium supplemented with five specific recombinant proteins (5G-culture). We analyzed the anti-inflammatory characteristics of macrophage fraction of E-MNCs using a co-culture model with CD3/CD28-stimulated PBMNCs. To test therapeutic efficacy in vivo, either E-MNCs or E-MNCs depleted of CD11b-positive cells were transplanted intraglandularly into mice with radiation-damaged SGs. Following transplantation, SG function recovery and immunohistochemical analyses of harvested SGs were assessed to determine if CD11b-positive macrophages contributed to tissue regeneration. The results indicated that CD11b/CD206-positive (M2-like) macrophages were specifically induced in E-MNCs during 5G-culture, and Msr1- and galectin3-positive cells (immunomodulatory macrophages) were predominant. CD11b-positive fraction of E-MNCs significantly inhibited the expression of inflammation-related genes in CD3/CD28-stimulated PBMNCs. Transplanted E-MNCs exhibited a therapeutic effect on saliva secretion and reduced tissue fibrosis in radiation-damaged SGs, whereas E-MNCs depleted of CD11b-positive cells and radiated controls did not. Immunohistochemical analyses revealed HMGB1 phagocytosis and IGF1 secretion by CD11b/Msr1-positive macrophages from both transplanted E-MNCs and host M2-macrophages. Thus, the anti-inflammatory and tissue-regenerative effects observed in E-MNC therapy against radiation-damaged SGs can be partly explained by the immunomodulatory effect of M2-dominant macrophage fraction.

Recent progress in regenerative therapy for damaged salivary glands: From bench to bedside

“…In mice, E‐MNC transplantation was shown to reduce the expression of inflammatory genes and increase factors associated with tissue regeneration, such as stem cell marker expression, cell proliferation, and blood vessel formation. Thus, E‐MNCs could be used to regenerate radiation‐injured SGs and ameliorate salivary secretory function (Honma et al, 2023; I et al, 2019). As we describe in more detail below, these promising cell‐based therapies using MSCs or E‐MNCs have proceeded to clinical studies.…”

“…Finally, our group conducted the first clinical study of a regenerative therapy using E‐MNCs for patients with severe radiation‐induced xerostomia (Sumita et al, 2020). As mentioned above, we recently developed a novel culture method for PBMNCs using serum‐free medium containing five recombinant growth factors (stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin‐6 [IL‐6], and fms‐like tyrosine kinase [Flt‐3 ligand]) (Honma et al, 2023; I et al, 2019). E‐MNCs acquire anti‐inflammatory and vasculogenic characteristics during 5G‐culture.…”