Iron Released after Cryo-Thermal Therapy Induced M1 Macrophage Polarization, Promoting the Differentiation of CD4+ T Cells into CTLs

Abstract

Macrophages play critical roles in both innate and adaptive immunity and are known for their high plasticity in response to various external signals. Macrophages are involved in regulating systematic iron homeostasis and they sequester iron by phagocytotic activity, which triggers M1 macrophage polarization and typically exerts antitumor effects. We previously developed a novel cryo-thermal therapy that can induce the mass release of tumor antigens and damage-associated molecular patterns (DAMPs), promoting M1 macrophage polarization. However, that study did not examine whether iron released after cryo-thermal therapy induced M1 macrophage polarization; this question still needed to be addressed. We hypothesized that cryo-thermal therapy would cause the release of a large quantity of iron to augment M1 macrophage polarization due to the disruption of tumor cells and blood vessels, which would further enhance antitumor immunity. In this study, we investigated iron released in primary tumors, the level of iron in splenic macrophages after cryo-thermal therapy and the effect of iron on macrophage polarization and CD4⁺ T cell differentiation in metastatic 4T1 murine mammary carcinoma. We found that a large amount of iron was released after cryo-thermal therapy and could be taken up by splenic macrophages, which further promoted M1 macrophage polarization by inhibiting ERK phosphorylation. Moreover, iron promoted DC maturation, which was possibly mediated by iron-induced M1 macrophages. In addition, iron-induced M1 macrophages and mature DCs promoted the differentiation of CD4⁺ T cells into the CD4 cytolytic T lymphocytes (CTL) subset and inhibited differentiation into Th2 and Th17 cells. This study explains the role of iron in cryo-thermal therapy-induced antitumor immunity from a new perspective.

Keywords: CD4 CTL; CD4+ T cell differentiation; M1 macrophages; cryo-thermal therapy; iron.

Figure 1

Iron released from tumor after cryo-thermal therapy. (A,B) Representative H&E staining of the tumors before and after cryo-thermal therapy. (C) Heme levels in tumor interstitial fluid 6 h, 12 h and 24 h after cryo-thermal therapy. (D) Iron quantification in tumor interstitial fluid 6 h, 12 h and 24 h after cryo-thermal therapy. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3 for each group.

Figure 2

Splenic macrophages taken up the iron after cryo-thermal therapy. (A) Schematic of experimental design. 4T1 tumor-bearing mice were injected with DFO introtumoral 30 min before cryo-thermal therapy and spleens were harvested for analyzing the changes in iron. (B) Representative Perls’ staining of the spleen after cryo-thermal therapy with or without DFO injection. (C) Representative DAB-enhanced Perls’ staining (middle) and immunohistochemistry staining using an F4/80 antibody (right). (D,E) Splenic macrophages were sorted and the mRNA levels and protein levels (fold change) of FtH were determined by RT-qPCR and western blot, respectively. (F) F4/80⁺ macrophages were sorted by flow cytometer and iron contents were quantified by ICP-MS. (G) RAW264.7 cells were treated with tumor interstitial fluid from control or cryo-thermal treated mice. DFO was added to chelate iron in the medium. After 24 h of cultivation, protein levels of FtH were determined by western blot. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3 for each group.

Figure 3

DFO treatment inhibited the M1 polarization of macrophages. Mice were sacrificed and spleen were harvested 12 h, 24 h and 72 h after cryo-thermal therapy. (A) The percentages of M1 macrophages (CD86⁺MHC II⁺) were measured by using flow cytometry. (B) Macrophages were sorted and the expression levels of CXCL10, TNF, IL-6, IL-12 and IL-10 were determined by using RT-qPCR. (C) Intracellular IL-12 and IL-10 in macrophages were determined by using flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3 for each group.

Figure 4

Iron promoted M1 polarization of macrophages in vitro. (A) Schematic of experimental design. Tumor interstitial fluid was havested from tumor-bearing mice (CIF) or treated mice (AIF). Splenic macropahges sorted from tumor-bearing mice were treated with AIF, CIF or combined with DFO treatment. (B) The percentages of M1 macrophages (CD86⁺MHC II⁺) were measured by flow cytometry and (C) the expression levels of TNF, IL-6, IL-12 and IL-10 were determined by RT-qPCR. ** p < 0.01, n = 3 for each group.

Figure 5

Iron promoted M1 polarization of macrophages by inhibiting the phosphorylation of ERK. (A) RAW264.7 cells were treated with AIF with or without DFO treatment for 24 h, the MEK/ERK pathway was analyzed by western blot. (B) Cells were pretreated with U0126, an inhibitor of MEK/ERK pathway, for 30 min to inhibit the phosphorylation of ERK. The phosphorylation of ERK was analyzed by Western blot and (C) the expression levels of TNF, IL-6, IL-12 and IL-10 were measured by Real-Time qPCR. Experiments were performed in duplicate. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Iron promotes DCs maturation. Mice were sacrificed and spleens were harvested 24 h and 72 h after cryo-thermal therapy. (A,B) The percentages of mature DCs (CD86⁺MHC II⁺) were measured by flow cytometry. (C) DCs were sorted and the expression levels of CXCL10, TNF, IL-6, IL-12 and IL-10 were determined by RT-qPCR. (D) Intracellular IL-12 and IL-10 in DCs were determined by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3 for each group.

Figure 7

Iron promoted the differentiation of CD4 CTL and Tfh. Mice were sacrificed and spleens were harvested 72 h after cryo-thermal therapy. (A,B) The percentages of CD4⁺ and CD8⁺ T cells were measured by flow cytometry. (C) CD4 CTL (Thpok^-), Th1 (IFN-γ⁺), Tfh (Bcl6⁺), Th2 (IL-4⁺), Th17 (IL-17⁺) and Treg (Foxp3⁺) subsets in CD4⁺ T cells were measured by flow cytometry. (D) Intracellular granzyme B, perforin and IFN-γ⁺ in CD4⁺ and CD8⁺ T cells were determined by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, n = 4 for each group.

Figure 8

Iron-induced macrophages and DCs after cryo-thermal therapy promoted the differentiation of CD4 CTL. Mice were sacrificed and spleens were harvested 72 h after cryo-thermal therapy. Macrophages and DCs were sorted from tumor-bearing mice, cryo-thermal group and DFO treated cryo-thermal group mice and cocultured with CD4⁺ T cells for 24 h. (A,B) CD4 CTL (Thpok⁻), Th1 (IFN-γ⁺), Tfh (Bcl6⁺), Th2 (IL-4⁺), Th17 (IL-17⁺) and Treg (Foxp3⁺) subsets in CD4⁺ T cells were measured by using flow cytometry. (C,D) Intracellular granzyme B and perforin in CD4⁺ T cells were determined by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3 for each group.