Combination of G-CSF and AMD3100 Improves the Anti-inflammatory Effect of Mesenchymal Stem Cells on Inducing M2 Polarization of Macrophages Through NF-κB-IL1RA Signaling Pathway

Abstract

Mobilized peripheral blood-derived mesenchymal stem cells (PB-MSCs) mainly derived from bone marrow-derived MSCs (BM-MSCs) exert a similar anti-inflammatory effect. However, the mechanism of anti-inflammatory effect of mobilized PB-MSCs by a combination of G-CSF and AMD3100 remains unclear. Cultured rat PB-MSCs mobilized by G-CSF/AMD3100 have shown typical surface markers and potential for multiple differentiations, similar to non-mobilized BM-MSCs. In a co-culture system, rat M0-type macrophages co-cultured with PB-MSCs have shown higher expression of M2 markers including CD206, Arg-1, IL-10, and CCL-22 than BM-MSCs, indicating that PB-MSCs induced greater M0 polarization to M2. Furthermore, compared with BM-MSCs, PB-MSCs in a co-culture system with lipopolysaccharide-induced M1-type macrophages more efficiently promoted M1 polarization to M2, accompanied by increasing expression of CD206, Arg-1, IL-10, and CCL-22 while decreasing expression of M1 markers including iNOS, TNF-α, IL-1β and IL-6, indicating that PB-MSCs triggered greater M1 polarization to M2. Subsequently, polymerase chain reaction arrays showed higher expressions of both IL1rn and Tnfrsf11b in PB-MSCs versus BM-MSCs. In response to an inflammatory niche, such as TNF-α, PB-MSCs have shown higher expression and release of IL1RA, causing greater M2 polarization of macrophages, and the special effects may be almost entirely abolished through the neutralization antibody of IL1RA. Mechanistic studies determined that PB-MSCs showed higher levels NF-κBp65 and NF-κBp-p65 than BM-MSCs, which could be obviously enhanced by TNF-α. And the increased IL1RA expression by TNF-α in PB-MSCs could be markedly canceled by an NF-κB inhibitor PDTC. Interestingly, mimicking the mobilized PB-MSCs by a combination of G-CSF and AMD3100 in vivo, BM-MSCs were treated with G-CSF and/or AMD3100 in vitro, showing the increased expressions of NF-κBp65 and IL1RA, which could be prominently abolished by PDTC. Therefore, targeting IL1rn, gene modification or drug intervention for MSCs may provide a novel therapeutic strategy for human diseases, especially inflammatory diseases.

Keywords: IL1rn; NF-κB; anti-inflammatory; macrophage; peripheral blood-derived mesenchymal stem cells; polarization.

FIGURE 1

The morphologic and phenotypic characteristics and multiple-differentiation potential of BM-MSCs and PB-MSCs. (A) The typical image of third generation PB-MSCs and BM-MSCs with fibroblast-like shape. Osteogenesis of PB-MSCs and BM-MSCs was determined by Alizarin Red staining 21 days after osteogenic induction. Adipogenesis of PB-MSCs and BM-MSCs was detected via the formation of neutral lipid vacuoles stainable with Oil Red O staining 21 days after induction. Chondrogenesis of PB-MSCs and BM-MSCs was evaluated through Alician Blue staining 21 days after induction. (B) BM-MSCs and PB-MSCs were positive for CD44, CD29, and CD90, and negative for CD34, CD11b, and CD45.

FIGURE 2

PB-MSCs efficiently promoted M0-type polarization toward M2 type. (A–E) M1 (i.e., iNOS and TNFα) or M2 (i.e., CD206 and Arg-1) type typical markers were evaluated by flow cytometry in M0-type macrophages following co-culture with either PB-MSCs or BM-MSCs using the Transwell system for 3 days. (F–I) The expression of pro-inflammatory (e.g., IL-6 and IL-1β) and anti-inflammatory (e.g., IL-10 and CCL-22) factors was determined by using a real-time PCR in M0 macrophage co-cultured with either PB-MSCs or BM-MSCs for 3 days. (J,K) The levels of pro-inflammatory cytokine (e.g., TNFα) and anti-inflammatory cytokine (e.g., IL-10) factors were detected by using ELISA in supernatants of M0 macrophages co-cultured with either PB-MSCs or BM-MSCs for 3 days. n = 5, ^#p < 0.05, vs. M0 co-cultured without either PB-MSCs or BM-MSCs, ^∗p < 0.01, vs. M0 co-cultured with either PB-MSCs.

FIGURE 3

PB-MSCs triggered M1-type polarization toward M2 type. (A–E) PB-MSCs more effectively increased the expression of CD206 and Arg-1 in LPS-induced M1-type macrophages co-cultured with BM-MSCs using the Transwell system for 3 days, as determined by flow cytometry for M1 (i.e., iNOS and TNFα) or M2 (i.e., CD206 and Arg-1) typical markers. (F–I) The expression of pro-inflammatory (e.g., IL-6 and IL-1β) and anti-inflammatory (e.g., IL-10 and CCL-22) factors was determined using a real-time PCR in M1 macrophages co-cultured with either PB-MSCs or BM-MSCs for 3 days. (J,K) The levels of pro-inflammatory cytokine (e.g., TNFα) and anti-inflammatory cytokine (e.g., IL-10) factors were determined by using ELISA in supernatants of M1-type macrophages co-cultured with either PB-MSCs or BM-MSCs for 3 days. n = 5, ^#p < 0.05, vs. M1 co-cultured without either PB-MSCs or BM-MSCs, ^∗p < 0.01, vs. M1 co-cultured with either PB-MSCs.

FIGURE 4

Differences between PB-MSCs and BM-MSCs analyzed using an inflammatory Cytokines and Receptors RT² Profiler PCR Array. (A) The expressions of interleukin 1 receptor antagonist (IL1rn) and tumor necrosis factor receptor superfamily member 11b (Tnfrsf11b) genes were upregulated (fold change > 2.5, p < 0.001) while the 22 genes’ expressions were downregulated as analyzed by the PCR array. (B) The analysis of the gene network was performed using the STRING database.

FIGURE 5

PB-MSCs-released IL1RA was involved in the M1-type switch into M2 type. (A) The typical results of M1 (i.e., iNOS and TNFα) or M2 (i.e., CD206 and Arg-1) type by flow cytometry in M1 macrophages treated with condition medium (CM) from TNFα-stimulated PB-MSCs (^TNFαCM) with or without the presence of a neutralizing anti-IL1RA antibody for 3 days. (B–E) Quantitative analysis of results in A showed that CM promoted the polarization of the M1 type toward M2 type, which could be almost completely abolished by the anti-IL1RA antibody. n = 5, ^#p < 0.05, vs. CM, ^&p < 0.01, vs. ^TNFαCM.

FIGURE 6

Increased IL1RA expression in PB-MSCs by TNFα was involved in NF-κB signaling pathway. (A) TNFα (50 ng/mL) increased the levels of NF-κB p-p65 and p65 in both BM-MSCs and PB-MSCs as detected by Western blot, especially in PB-MSCs. (B) Semi-quantitative analysis of results in A showed a higher ratio of NF-κB p-p65/p65 in PB-MSCs treated with TNFα (50 ng/ml) compared to BM-MSCs. n = 5, ^&p < 0.05, vs. PB-MSCs; ^#p < 0.05, vs. PB-MSCs; ^∗p < 0.05, vs. BM-MSCs. (C) Typical results of IL1RA expression in PB-MSCs as determined by flow cytometry. (D) Semi-quantitative analysis of results in C showed that TNFα (50 ng/mL) induced the expression of IL1RA in PB-MSCs, which could be obviously abolished by NF-κB inhibitor PDTC (100 μM). n = 5, ^&p < 0.05, vs. PB-MSCs treated without TNFα and PDTC;^#p < 0.05, vs. PB-MSCs treated without TNFα and PDTC; ^∗p < 0.05, vs. BM-MSCs treated with TNFα.

FIGURE 7

BM-MSCs treated with G-CSF/AMD3100 showed stronger migration potential. (A) Typical image of BM-MSCs migration treated with G-CSF and/or AMD3100 using the Transwell system. (B) Combined treatment of AMD3100 and G-CSF promoted greater BM-MSCs migration than single treatment of AMD3100 or G-CSF. n = 5, ^@p < 0.05, vs. BM-MSCs treated with or without G-CSF; ^#p < 0.05, vs. BM-MSCs treated with G-CSF. (C) Combined treatment of AMD3100 and G-CSF induced IL-1RA expression in BM-MSCs as detected by ELISA. n = 3, ^@p < 0.05, vs. BM-MSCs treated with or without G-CSF; ^#p < 0.05, vs. BM-MSCs treated with either G-CSF or AMD3100. (D,E) Combined treatment of AMD3100 and G-CSF increased IL-1RA protein levels in BM-MSCs as analyzed by Western blot (D) and semi-quantitative analysis (E). n = 3, ^∗p < 0.05, vs. BM-MSCs treated without either AMD3100 or G-CSF; ^%p < 0.05, vs. BM-MSCs treated without either AMD3100 or G-CSF; ^#p < 0.05, vs. BM-MSCs treated with either AMD3100 or G-CSF.

FIGURE 8

Induced IL1RA expression in BM-MSCs by a combination of G-CSF and AMD3100 was involved in NF-κB signaling pathway. (A) Western blot was used to detect the indicated proteins in BM-MSCs treated with G-CSF/AMD3100. (B–G) Semi-quantitative analysis of results in A showed higher levels of NF-κB protein and IL-1RA in BM-MSCs following the combined treatment of AMD3100 and G-CSF. n = 3, ^∗p < 0.05, vs. BM-MSCs treated without either AMD3100 or G-CSF; ^%p < 0.05, vs. BM-MSCs treated without either AMD3100 or G-CSF; ^#p < 0.05, vs. BM-MSCs treated with either AMD3100 or G-CSF. (H,I) NF-κB inhibitor PDTC (100 μM) abolished the effects of AMD3100 and G-CSF on inducing IL-1RA expression in BM-MSCs as determined by Western blot (H) and semi-quantitative assay (I). n = 3, ^∗p < 0.05, vs. BM-MSCs treated without either AMD3100 or G-CSF; ^#p < 0.05, vs. BM-MSCs treated with combination of AMD3100 and G-CSF.

FIGURE 9

Schematic diagram of the interaction between PB-MSCs and macrophages. Quiescent MSCs inhabit the hematopoietic niche, which is mainly composed of osteoblasts and the vascular network. In the process of mobilization by G-CSF combined with AMD3100, BM-MSCs detached from the niche because of block of SDF-1α/CXCR4 axis by AMD3100 to cut off SDF-1α binding into CXCR4, and G-CSF to reduce SDF-1α levels in the niche (Petit et al., 2002; Tang et al., 2011; Hoggatt et al., 2018). Mobilized BM-MSCs enter peripheral blood, functioning as PB-MSCs. PB-MSCs contributed to the polarization of M1 toward M2 type in the inflammatory sites though IL1RA, which were associated with NF-κB signaling pathway.