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miR-214-3p Deficiency Enhances Caspase-1-Dependent Pyroptosis of Microglia in White Matter Injury - PubMed

  • ️Sat Jan 01 2022

miR-214-3p Deficiency Enhances Caspase-1-Dependent Pyroptosis of Microglia in White Matter Injury

Liufang He et al. J Immunol Res. 2022.

Abstract

White matter injury (WMI) is the most frequent impairment of neurodevelopment in preterm infants. Here, we report that the caspase-1 inflammasome is abundantly activated in the microglia of WMI mice and results in increased pyroptosis of microglia. Pharmacology inhibition of caspase-1 cleavage alleviated the pathogenesis of WMI mice. The expression of microRNA miR-214-3p was largely reduced in the microglia of WMI mice compared to controls. Compromised expression of miR-214-3p on microglia gives rise to the inflammasome activation and microglial pyroptosis. Treatment with miR-214-3p agomir is sufficient to relieve the white matter lesion and demyelination in WMI mice. miR-214-3p is able to bind to the 3' region of the NLRP-3 inflammasome compartment NEK7, preventing the transcription of NEK7 mRNA. As a result, in WMI mice, the lack of miR-214-3p leads to the accumulation of NEK7 which supports NLRP 3 inflammasome activation, microglial pyroptosis, and white matter pathogenesis.

Copyright © 2022 Liufang He et al.

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Conflict of interest statement

No conflict of interest was declared by the authors.

Figures

Figure 1
Figure 1

Caspase-1-dependent pyroptosis boosted in microglia of white matter injury mice. 300 μg/kg LPS was injected into mice intraperitoneally on postnatal day 8. 14 hours later, mice were anesthetized, and the brain was harvested. (a) H&E staining for brain sections; white matter was imaged. (b) MBP staining for brain sections. Microglia were isolated with a CD11b cell separation kit. (c) LDH is released by microglia after ex vivo culture for 24 hours. (d) FLICA assay staining in microglia for activated caspase-1, caspase-3, caspase-6, and caspase-8. (e) Immunoblot for microglia from control (Ctrl) and WMI mice. (f) Protein level quantification by normalizing with β-actin. (g) IL-1β release by microglia. (h) IL-18 release by microglia. (i) Dual-color staining for lba1 and caspase-1 in the white matter. Data are mean ± SEM. Individual data points are displayed: ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 2
Figure 2

Inactivation of caspase-1 attenuated the pathogenesis of WMI. 25 mg/kg caspase-1 inhibitor Vx-765 injected to the WMI mouse model i.p. Microglia were isolated with a CD11b cell separation kit. (a) FLICA assay staining for caspase 1 (representative dot plot). (b) FLICA assay staining for caspase-1 (statistical data). (c) Immunoblot for procaspase-1 and caspase-1 P20. (d) Relative protein level quantification by normalizing with β-actin. (e) IL-1β release. (f) IL-18 release. (g) Dual-color staining for lba1 and caspase-1 in the white matter. (h) LDH is released by microglia after ex vivo culture for 24 hours. (i) H&E staining for brain sections. (j) MBP staining for brain sections. Data are mean ± SEM. Individual data points are displayed: ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 3
Figure 3

Loss of miR-214-3p in microglia drives the pathogenesis of WMI. (a) Collect data from the GSE49330 database. Different expressed gene numbers between LPS treatment and PBS/IL-4 treatment. Overlapped gene numbers were in red. (b) Relative expression level of miR-214-3p from the GSE49330 database. (c) Relative expression of miR-214-3p in microglia from Ctrl and WMI mice. WMI mice were treated with 40 mg/kg agomiR-214-3p. (d) H&E staining for brain sections. (e) MBP staining for brain sections. Data are mean ± SEM. Individual data points are displayed: ∗P < 0.01.

Figure 4
Figure 4

miR-214-3p mediates caspase-1 inflammasome activation in microglia. BV-2 microglia cell lines were cultured and transfected with agomir or antagomir of miR-214-3p. (a) FLICA assay staining for caspase-1 (representative dot plot). (b) FLICA assay staining for caspase-1 (statistical data). (c) Immunoblot for procaspase-1 and caspase-1 P20. (d) Relative protein level quantification by normalizing with β-actin. (e) LDH is released by microglia after ex vivo culture for 24 hours. (f) IL-1β release in culture medium. (g) IL-18 release in culture medium. Data are mean ± SEM. Individual data points are displayed: ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 5
Figure 5

miR-214-3p regulates microglial pyroptosis in WMI mice. 40 mg/kg agomiR-214-3p was injected into the WMI mouse model i.p. Microglia were isolated with a CD11b cell separation kit. (a) Dual-color staining for lba1 and caspase-1 in the white matter. (b) Immunoblot for procaspase-1 P20. (c) Relative protein level quantification by normalizing with β-actin. (d) FLICA assay staining for caspase-1 (representative dot plot). (e) FLICA assay staining for caspase-1 (statistical data). (f) IL-1β release after 24 hours of culture ex vivo. (g) IL-18 released after 24 hours of culture ex vivo. Data are mean ± SEM. Individual data points are displayed: ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 6
Figure 6

miR-214-3p binds to NEK7 to mediate caspase-1 inflammasome activation in microglia. (a) Scheme graph for the NLRP3 complex. (b) Transcripts of NEK7, NLRP3, and ASC in microglia from Ctrl and WMI mice. (c) Immunoblot for NEK7, NLRP3, and ASC proteins in Ctrl and WMI microglia. (d) Quantification of immunoblot by normalizing with β-actin. (e) BV-2 cells were transfected with miR-214-3p agomir and antagomir. NEK7 transcripts were measured by RT-PCR. (f) Assay design for luciferase reporter assay of miR-214 and NEK7 mRNA. (g) Dual-luciferase reporter assay for the binding of miR-214 and NEK7 mRNA. (h) BV-2 cells were transfected with miR-214-3p agomir. NEK proteins were determined by immunoblot. (i) Quantification of NEK7 protein expression after miR-214-39 agomir and antagomir transfection by normalizing with β-actin. Data are mean ± SEM. Individual data points are displayed. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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