Irf5 deficiency in myeloid cells prevents necrotizing enterocolitis by inhibiting M1 macrophage polarization

Abstract

Necrotizing enterocolitis (NEC) is a life-threatening inflammatory disease in newborns, but the mechanisms remain unclear. Interferon regulatory factor 5 (IRF5) is a master regulator of macrophage function and is essential for proinflammatory M1 macrophage polarization. Our previous data indicated that M1 macrophages promote NEC injury. Here, we investigated whether IRF5 is involved in the pathogenesis of NEC. First, we found that IRF5 was upregulated in infiltrated macrophages in human neonates with NEC compared to controls. We further confirmed IRF5 upregulation in macrophages in experimental murine NEC and that the infiltrated macrophages were predominantly polarized into the M1 but not the M2 phenotype. Myeloid-specific deficiency of Irf5, which was associated with reduced M1 macrophage polarization and systematic inflammation, dramatically prevented experimental NEC. Moreover, we found that the ablation of Irf5 in myeloid cells markedly suppressed intestinal epithelial cell apoptosis and further prevented intestinal barrier dysfunction in experimental NEC. Bioinformatic and chromatin immunoprecipitation analysis further showed that IRF5 binds to the promoters of the M1 macrophage-associated genes Ccl4, Ccl5, Tnf, and Il12b. Overall, our study provides evidence that IRF5 participates in the pathogenesis of NEC, while the deletion of Irf5 in myeloid cells prevents NEC via inhibiting M1 macrophage polarization.

Fig. 1

IRF5 is induced in infiltrated macrophages in human NEC neonates. a Representative immunohistochemical staining for IRF5 in the intestines of control and NEC neonates. NEC neonates exhibited a significant elevation in IRF5 expression. b Representative immunofluorescence staining for IRF5 (green) and CD68 (red) in control and NEC intestines. NEC led a significantly increased number of CD68-positive cells in the intestines, and IRF5 was mostly localized in CD68-positive cells. (n = 5 in the control group; n = 10 in the NEC group. Scale bar: 50 μm in (a) and 100 μm in (b). *P < 0.05)

Fig. 2

IRF5 is induced in infiltrated macrophages in murine experimental NEC. a Representative HE staining of samples from breast-fed pups and pups with NEC. Histological injury grade was significantly increased in the NEC group. b Representative immunofluorescence staining for IRF5 (green) and CD68 (red) in the intestines of mouse pups. The pups with NEC exhibited significantly increased IRF5 expression compared to that in the breast-fed pups, and IRF5 was mostly localized in infiltrated CD68-positive cells in the intestines. c Western blotting analysis showed that IRF5 expression in the intestine was significantly induced in the NEC pups compared to the breast-fed pups. (n = 5 in breast fed; n = 10 in NEC. Scale bar: 100 μm. *P < 0.05)

Fig. 3

Infiltrated macrophages are polarized into the M1 phenotype in experimental NEC. a Representative immunofluorescence staining for iNOS (green) and CD68 (red) in the intestine. The pups with NEC exhibited a significant increase in the number of iNOS⁺CD68⁺ cells in the intestines compared to that in breast-fed pups. b Quantitative PCR analysis of the mRNA expression of M1 and M2 macrophage-associated genes in the intestines. The mRNA expression of M1 macrophage-associated genes was significantly increased in NEC, while the mRNA expression of M2 macrophage-associated genes, except that of Retnla, was unchanged. (n = 5 in the breast-fed group; n = 10 in the NEC group. *P < 0.05)

Fig. 4

Myeloid-specific deficiency of Irf5 attenuates experimental NEC. a Representative HE staining of the intestines of the indicated groups. The Irf5^fl/fl pups with NEC exhibited a significantly increased histological injury grade compared to that of the breast-fed pups, while pups with myeloid-specific Irf5 deletion exhibited a markedly reduced histological injury grade compared to that in the Irf5^fl/fl pups with NEC. b The pups with myeloid-specific ablation of Irf5 exhibited a significantly reduced incidence of NEC compared to that in the Irf5^fl/fl pups with NEC (n = 5 in the breast-fed Irf5^fl/fl group; n = 5 in the breast-fed Irf5^ΔMΦ group; n = 13 in the NEC Irf5^fl/fl group; n = 12 in the NEC Irf5^ΔMΦ group. Scale bar: 100 μm. *P < 0.05)

Fig. 5

Irf5 ablation in myeloid cells suppresses M1 macrophage polarization in experimental NEC. a Representative immunofluorescence staining for iNOS (green) and CD68 (red) in the intestines of the indicated groups. The pups with NEC exhibited a significant increase in the number of iNOS⁺CD68⁺ cells in the intestines compared to that in the breast-fed pups, while Irf5 deletion in myeloid cells markedly blocked this effect. b Quantitative PCR analysis of the mRNA expression of M1 and M2 macrophage-associated genes in the intestines of the indicated groups. The myeloid-specific deficiency of Irf5 significantly inhibited the NEC-induced mRNA expression of M1 macrophage-associated genes in the Irf5^fl/fl pups. The mRNA expression of the M2 macrophage-associated genes, except that of Retnla, was unaltered among the groups. (n = 5 in the breast-fed Irf5^fl/fl group; n = 5 in the breast-fed Irf5^ΔMΦ group; n = 13 in NEC Irf5^fl/fl group; n = 12 in the NEC Irf5^ΔMΦ group. Scale bar: 100 μm. *P < 0.05)

Fig. 6

Myeloid-specific deletion of Irf5 inhibits intestinal epithelial cell apoptosis in experimental NEC. a Representative TUNEL (red) staining of the intestines of the indicated groups. Intestinal epithelial cells were stained with EpCAM (green). NEC significantly increased TUNEL⁺EpCAM⁺ cells in the Irf5^fl/fl pups, while Irf5 deletion in myeloid cells markedly blocked this effect. b Western blotting analysis of cleaved caspase-3 expression in the intestines of the indicated groups. The expression of cleaved caspase-3 was significantly increased in the Irf5^fl/fl pups with NEC compared to the breast-fed Irf5^fl/fl pups, while Irf5 deletion in myeloid cells significantly suppressed this effect. (n = 5 in the breast-fed Irf5^fl/fl group; n = 5 in the breast-fed Irf5^ΔMΦ group; n = 13 in the NEC Irf5^fl/fl group; n = 12 in the NEC Irf5^ΔMΦ group. Scale bar: 100 μm. *P < 0.05)

Fig. 7

IRF5 binds with the promoters of M1 macrophage-associated genes. a Representative IGV tracks in the Ccl4, Ccl5, Tnf, and Il12b loci for IRF5 of LPS-stimulated (2 h) WT and Irf5^-/- BMDMs. b ChIP assays were performed to analyze the interaction between IRF5 and the Ccl4, Ccl5, Tnf, Il12b promoters. Quantitative PCR analysis showed that IRF5 binds with the promoter regions of Ccl4, Ccl5, Tnf, and Il12b. LPS significantly promoted these bindings (n = 3, *P < 0.05)