Low-dose decitabine promotes M2 macrophage polarization in patients with primary immune thrombocytopenia via enhancing KLF4 binding to PPARγ promoter

Abstract

Background: The first-line therapy is effective for the treatment of primary immune thrombocytopenia (ITP); however, maintaining the long-term responses remains challenging. Low-dose decitabine (DAC) has been adopted to treat refractory ITP, while its role in macrophage polarization has not been fully understood. We aimed to investigate the mechanistic role of DAC in M2 macrophage polarization and evaluated its therapeutic effect in ITP.

Methods: The M2 monocytes were identified by flow cytometry from peripheral blood mononuclear cells in healthy controls (HCs) and ITP patients. The expression of PPARγ, Arg-1, DNMT3b and NLRP3, together with IL-10 plasma levels was measured to examine its function. Bisulfite-sequencing PCR was used to evaluate the methylation status of PPARγ promoter, and the binding affinity of KLF4 was measured by Cut&Tag. A sh-PPARγ THP-1 cell line was created to verify if low-dose DAC-modulated M2 macrophage polarization was PPARγ-dependent. The passive ITP models were used to investigate the therapeutic effects of low-dose DAC and its role in modulating polarization and immunomodulatory function of macrophages. NLRP3 inflammasome and reactive oxygen species were also tested to understand the downstream of PPARγ.

Results: The M2 monocytes with impaired immunoregulation were observed in ITP. After high-dose dexamethasone (HD-DXM) treatment, M2 monocytes increased significantly with the elevated expression of PPARγ, Arg-1 and IL-10 in CR patients. Low-dose DAC promoted M2 macrophage polarization in a PPARγ-dependent way via demethylating the promoter of PPARγ, especially the KLF4 binding sites. Low-dose DAC alleviated ITP mice by restoring the M1/M2 balance and fine-tuning immunomodulatory function of macrophages. The downstream of the PPARγ modulation of M2 macrophage polarization might physiologically antagonize NLRP3 inflammasome.

Conclusions: Low-dose DAC promoted M2 macrophage polarization due to the demethylation within the promoter of PPARγ, thus enhanced the KLF4 binding affinity in ITP.

Keywords: decitabine; krüppel-like factor 4; m2 macrophages; peroxisome proliferator-activated receptor-γ; primary immune thrombocytopenia.

FIGURE 1

M2 monocytes expanded after HD‐DXM treatment in the CR group.(A) Representative dot plots of CX3CR1⁺ CD163⁺ M2 in the PBMCs of HC (n = 20) and ITP patients (n = 36).(B) Changes in the M2 monocytes among the CR group (n = 26) and PR+NR group (n = 10) monitored before and after treatment.(C) The mRNA expression level of PPARγ, IL‐10 and Arg‐1 in CX3CR1⁺ cells from HC, ITP patients, CR and PR+NR group (before and after treatment). (D) Representative immunoblots for PPARγ, Arg‐1 and GAPDH in CX3CR1⁺ cells from HC, ITP patients, CR and PR+NR group (before and after treatment).(E) Plasma cytokine IL‐10 is measured by Elisa. Data represent the mean ± SD. All experiments were repeated three times. ^∗∗ p < .01; ^∗∗∗ p < .001.

FIGURE 2

Low‐dose DAC promoted M2 macrophage polarization in a PPARγ‐dependent manner. (A) The CX3CR1⁺ monocytes derived from HC and ITP patients are stimulated with recombinant human M‐CSF (10 ng/mL) for 5 days and IL‐4/IL‐13 for 3 days to induce M2 macrophages. (B) Sh‐PPARγ and Scr THP‐1 cells are bulk differentiated to macrophages using 50 ng/ml PMA for 2 days. DAC (5 μM) or PBS (used as solvent control) was added during the differentiation period. Then add 20 ng/ml IL‐4/IL‐13 for further 3 days to induce M2 macrophages. The M2 macrophage polarization is determined by double positive CD68 (green) and CD163 (red). DAPI (blue) represents the nuclei. (C) The expression of CD14 and CD163 of sh‐PPARγ and Scr THP‐1 cells under low‐dose DAC treatment was measured by FCM. (D) The mRNA expression level of Arg‐1 and DNMT3b in sh‐PPARγ and Scr THP‐1 cells in low‐dose DAC treatment for 3 days. (E) Representative immunoblots for PPARγ, Arg‐1, DNMT3b and GAPDH in sh‐PPARγ and Scr THP‐1 cells under low‐dose DAC treatment for 3 days. Data represent the mean ± SD of three replicates. ^∗∗∗ p < .001; compared to Con; ^### p < .001, compared to IL‐4/IL‐13 group.

FIGURE 3

Higher methylation status of the KLF4 binding site of the PPARγ promoter could be reversed by DAC.(A) Bisulfite‐sequencing PCR (BSP) analysis of PPARγ methylation. The 161‐bp region (−1625 to −1785) containing 10 CpG residues in the CpG rich area upstream of transcriptional start site (TSS) was amplified and sequenced.(B) The representative plots of BSP analysis of DNA methylation (left) from HC and ITP patients. The percentage of methylated residues in the PPARγ promoter of CX3CR1⁺ cells of all HCs and ITP patients. (C) Combine trends manifested by Cut&Tag in THP‐1 cells under different treatments (control, IL‐4 + IL‐13, IL‐4 + IL‐13 combined with DAC). ^∗ p < .05; ^∗∗ p < .01; ^∗∗∗ p < .001.

FIGURE 4

Low‐dose DAC restored the balance of macrophages in ITP mice. (A) Injecting C57B/6 mice with monoclonal antibody anti‐mouse CD41 intraperitoneally to achieve passive ITP mice model; low‐dose DAC treatment offset the decreased platelets in ITP mice on day 1. (B) On day 3, mice from different groups were executed and spleens were harvested for further analysis. Confocal fluorescence microscope at 400× magnification of macrophage (M1:F4/80⁺ CD86⁺, M2: F4/80⁺ CD206⁺) in spleen (blue, DAPI‐stained nuclei; green, F4/80‐stained; red, CD86‐stained, CD206‐stained). (C) Representative dot plots of M1 (F4/80⁺ CD86⁺) and M2 (F4/80⁺ CD206⁺) in the splenocyte of different groups (Control, ITP group, DAC treated group) and (D) the corresponding statistical graph. (E) The gating strategy of F4/80⁺ macrophages in spleen of mice among different groups (Control, ITP group, DAC treated group). (F) Mean fluorescence intensity of CD64 (FcγI), CD16 (FcγIII) and CD32 (FcγII) of F4/80⁺ macrophages in the splenocyte among different groups (Control, ITP group, DAC treated group) and the corresponding statistical graph. Data represent the mean ± SD. ^∗∗ p < .01; ^∗∗∗ p < .001, compared to control group; ^# p < .05; ^## p < .01; ^### p < .001, compared to ITP group.

FIGURE 5

The effect of low‐dose DAC on the immunomodulatory function of macrophages. (A) BMDM derived from different groups under the same M2‐polarized microenvironment for 2 days. Phase contrast microscope is used to distinguish M1 and M2. (B) Representative immunoblots for PPARγ, Arg‐1, DNMT3b and GAPDH in bone marrow derived M2 from different groups (control, ITP group, DAC treated group). (C) Plasma cytokine IL‐10 is measured by Elisa. (D) Representative dot plots of Th17 (IL‐17⁺) and Treg (CD25⁺ Foxp3⁺) in the co‐culture system of CD4⁺ T cells with bone marrow derived M2 of different groups (control, ITP group, DAC treated group) and (E) the corresponding statistical graph.(F) CD4⁺ T cells were seeded in 96 well‐plate with bone marrow‐derived M2 of different groups. The cell division index is calculated based on the dilution of CFSE fluorescence measured by flow cytometry and represents the average number of cell divisions that CD4⁺ T cells in the original population have undergone. (G) Confocal fluorescence microscope at 400× magnification and (H) FCM verified the phagocytosis of bone marrow derived M1macrophages from different groups. (I) The gating strategy of bone marrow derived F4/80⁺ CD86⁺ M1 macrophages among different groups (control, ITP group, DAC treated group). (J) Mean fluorescence intensity of bone marrow derived M1 macrophages among different groups (Control, ITP group, DAC treated group) and the corresponding statistical graph. ^∗∗∗ p < .001, compared to Control group; ^# p < .05; ^## p < .01; ^### p < .001, compared to ITP group.

FIGURE 6

The association of PPARγ ameliorated NLRP3 inflammasome with low‐dose DAC modulation. (A) Representative immunoblots for NLRP3 in sh‐PPARγ and Scr THP‐1 cells under the stimulation of IL4 and IL13 for two days, with or without the treatment of low‐dose DAC. (B) Representative immunoblots for NLRP3 and GAPDH in CX3CR1⁺ cells of HC and ITP patients (upper) and in BMDM of different groups (lower) (control, ITP group, DAC treated group). (C) In BMDM of different groups, cellular mitochondrial ROS are stained with MitoSOX probe. (D) The quantification of ROS production. ^∗∗ p < .01, compared to WT; #p < .05; compared to ITP group.