CHBP induces stronger immunosuppressive CD127+ M-MDSC via erythropoietin receptor

Abstract

Erythropoietin (EPO) is not only an erythropoiesis hormone but also an immune-regulatory cytokine. The receptors of EPO (EPOR)₂ and tissue-protective receptor (TPR), mediate EPO's immune regulation. Our group firstly reported a non-erythropoietic peptide derivant of EPO, cyclic helix B peptide (CHBP), which could inhibit macrophages inflammation and dendritic cells (DCs) maturation. As a kind of innate immune regulatory cell, myeloid-derived suppressor cells (MDSCs) share a common myeloid progenitor with macrophages and DCs. In this study, we investigated the effects on MDSCs differentiation and immunosuppressive function via CHBP induction. CHBP promoted MDSCs differentiate toward M-MDSCs with enhanced immunosuppressive capability. Infusion of CHBP-induced M-MDSCs significantly prolonged murine skin allograft survival compared to its counterpart without CHBP stimulation. In addition, we found CHBP increased the proportion of CD11b⁺Ly6G^-Ly6C^high CD127⁺ M-MDSCs, which exerted a stronger immunosuppressive function compared to CD11b⁺Ly6G^-Ly6C^high CD127^- M-MDSCs. In CHBP induced M-MDSCs, we found that EPOR downstream signal proteins Jak2 and STAT3 were upregulated, which had a strong relationship with MDSC function. In addition, CHBP upregulated GATA-binding protein 3 (GATA-3) protein translation level, which was an upstream signal of CD127 and regulator of STAT3. These effects of CHBP could be reversed if Epor was deficient. Our novel findings identified a new subset of M-MDSCs with better immunosuppressive capability, which was induced by the EPOR-mediated Jak2/GATA3/STAT3 pathway. These results are beneficial for CHBP clinical translation and MDSC cell therapy in the future.

Fig. 1. CHBP induced M-MDSCs differentiation and enhanced M-MDSCs immunosuppressive function.

A Detection of CD11b⁺ myeloid cells, CD11b⁺Ly6G⁻Ly6C^high M-MDSCs, and CD11b⁺Ly6G⁺Ly6C^low G-MDSCs by flow cytometry. B Quantification analysis of CD11b⁺ myeloid cells, M-MDSCs and G-MDSCs. C T-cell proliferation detected by CFSE. The ratio of G-MDSCs, M-MDSCs, and T cells was 1:1.

Fig. 2. Infusion of CHBP-induced M-MDSCs prolonged skin allograft survival.

A Skin pictures at different days post transplantation. B The survival rates. C H&E staining of the skin (POD 14). Scale bars: 1st row 1 mm, 2nd row 250 μm, and 3rd row 100 μm.

Fig. 3. Infusion of CHBP-induced M-MDSCs decreased CD4⁺, CD8⁺ T cells, and increased Tregs in peripheral and skin.

A Detection of the proportion of CD4⁺, CD8⁺ T cells, and Foxp3⁺ Tregs in the blood. B Detection the proportion of CD4⁺, CD8⁺ T cells, and Foxp3⁺ Tregs in the spleen. C CD4, CD8, and Foxp3 immunohistochemistry staining in the alloskin with semi-quantification analysis (POD 14). Scale bar: 100 μm.

Fig. 4. The effects of CHBP on MDSCs were dependent on EPOR.

A Detection of CD11b⁺Ly6G⁻Ly6C^high M-MDSCs and CD11b⁺Ly6G⁺Ly6C^low G-MDSCs by flow cytometry. B Quantification analysis of the G-MDSCs and M-MDSCs proportions. C T-cell proliferation detected by CFSE. The ratios of G-MDSCs and T cells were 1:2, 1:4, 1:8, 1:16, and 1:32. D T-cell proliferation detected by CFSE. The ratios of M-MDSCs and T cells were 1:2, 1:4, 1:8, 1:16, and 1:32. E The expressions of iNOS and Arg-1 by qPCR. F The arginase-1 activity. G The protein expressions of iNOS and Arg-1 by western blot.

Fig. 4. The effects of CHBP on MDSCs were dependent on EPOR.

A Detection of CD11b⁺Ly6G⁻Ly6C^high M-MDSCs and CD11b⁺Ly6G⁺Ly6C^low G-MDSCs by flow cytometry. B Quantification analysis of the G-MDSCs and M-MDSCs proportions. C T-cell proliferation detected by CFSE. The ratios of G-MDSCs and T cells were 1:2, 1:4, 1:8, 1:16, and 1:32. D T-cell proliferation detected by CFSE. The ratios of M-MDSCs and T cells were 1:2, 1:4, 1:8, 1:16, and 1:32. E The expressions of iNOS and Arg-1 by qPCR. F The arginase-1 activity. G The protein expressions of iNOS and Arg-1 by western blot.

Fig. 5. CD127⁺ M-MDSCs induced by CHBP exerted stronger immunosuppressive function.

A RNA-seq detected the different RNA expressions in M-MDSCs with or without CHBP treatment in vitro. B The String analysis of mRNA sequencing. C The Jak2 and GATA3 protein expression by western blot. D The p-STAT3 and STAT3 protein expression by western blot. E Protein array compared the difference in M-MDSCs with or without CHBP treatment in vitro. F The molecular function enrichment of protein array. G Detection and quantification of the CD127-positive cells in M-MDSCs by flow cytometry. H T-cell proliferation detected by CFSE. The ratio of G-MDSCs and T cells was 1:1.