Nrf2 Promotes Inflammation in Early Myocardial Ischemia-Reperfusion via Recruitment and Activation of Macrophages

Figure 1. Early myocardial ischemia reperfusion promoted myocardial apoptosis and inflammatory response. (A) Heatmap of mouse inflammatory genes between the control group and IR6h group, including Nfe2l2 and Pdcd4. (B) KEGG - enriched signaling pathways according to differentially expressed genes. (C) GO-enriched modules of biological processes. (D) Transcriptional level analysis of caspase family members. (E) GSEA-enriched signaling pathway associated with inflammation or phagocytosis. IR6h, ischemia/reperfusion treatment for 6 h KEGG, Kyoto Encyclopedia of Genes and Genomes. GO, Gene Ontology. GSEA, Gene Set Enrichment Analysis. *P < 0.05, compared to the control group.

Figure 2. Nrf2 cooperated with Pdcd4 to promote infiltration of M1 macrophages. (A) Analysis of immune cell infiltration by ImmuCellAI. (B) Correlation analysis among seven categories of immune cells, Nfe2l2 and Pdcd4. The P-value is in the upper left corner; the corresponding Pearson correlation coefficient is in the lower right corner. (C) Subtype analysis of macrophages using ImmuCellAI. (D) Correlation analysis between M1 macrophages and Nfe2l2 expression. (E) Analysis of infiltrated M1 macrophages between the low Nfe2l2 and high Nfe2l2 groups. (F) Correlation analysis between M1 macrophages and Pdcd4 expression. (G) Analysis of infiltrated M1 macrophages between the low PDCD4 and high PDCD4 groups. ImmuCellAI, immune cell abundance identifier.

Figure 3. Nrf2 cooperated with Pdcd4 to promote transcription of chemokines for macrophages. (A) Correlation analysis between chemokines for macrophages and inflammation factors of M1 macrophages, as well as between Nfe2l2 and PDCD4. (B) Genes enriched in term “Chemokine-mediated signaling pathways”. (C) Venn diagram of transcription factors of Ccl2, Ccl3, or Ccl7, as well as genes enriched in term “Positive regulation of response to wounding”; Nfe2l2 was the only common gene. (D–F) Transcription factor score of Ccl2, Ccl3, or Ccl7. (G–I) Relationship between Nfe2l2 expression and the expression of Ccl2, Ccl3, and Ccl7.

Figure 4. Nrf2 promoted transcription of Ccl3 validated by chromatin immunoprecipitation sequencing (CHIP-seq). (A) anti-NRF2 antibodies could bind to the DNA fragments of Ccl3, rather than to Ccl2 or Ccl7. (B) Heatmap of the peak signal. (C) The peak signal was significantly reduced in the NRF2 silencing group compared with the control group (P < 0.001). (D) The transcription start site (TSS) signal was significantly reduced in NRF2 silencing group, as compared with the control group (P < 0.001). (E) Gene Ontology (GO) analysis of peak id.

Figure 5. Nrf2 promoted the infiltration of M1 macrophages and release of inflammatory factors via initiating the transcription of Ccl3. (A) Correlation analysis between Ccl3 expression and M1 macrophage infiltration. (B) Analysis of infiltrated M1 macrophages between the low Ccl3 and high Ccl3 groups. (C) Correlation analysis between Ccl3 expression and the signature score of Ccr5+ M1 macrophages. (D) signature score of Ccr5+ M1 macrophages between the low Ccl3 and high Ccl3 groups. (E–H) The expression of inflammatory factors II1b, II16, Nfkb1, and macrophage labeling Cd11b between the low-score Ccr5+ M1 macrophage signature group and the high-score Ccr5+ M1 macrophage signature group. (I) Schematic diagram of Nrf2, cooperated with PDCD4, promoting the infiltration of M1 macrophages and release of inflammatory factors by initiating the transcription of Ccl3 in early myocardial ischemia reperfusion.