PRDX6-iPLA2 aggravates neuroinflammation after ischemic stroke via regulating astrocytes-induced M1 microglia

Abstract

The crosstalk between astrocytes and microglia plays a pivotal role in neuroinflammation following ischemic stroke, and phenotypic distribution of these cells can change with the progression of ischemic stroke. Peroxiredoxin (PRDX) 6 phospholipase A2 (iPLA2) activity is involved in the generation of reactive oxygen species(ROS), with ROS driving the activation of microglia and astrocytes; however, its exact function remains unexplored. MJ33, PRDX6^D140A mutation was used to block PRDX6-iPLA2 activity in vitro and vivo after ischemic stroke. PRDX6^T177A mutation was used to block the phosphorylation of PRDX6 in CTX-TNA2 cell lines. NAC, GSK2795039, Mdivi-1, U0126, and SB202190 were used to block the activity of ROS, NOX2, mitochondrial fission, ERK, and P38, respectively, in CTX-TNA2 cells. In ischemic stroke, PRDX6 is mainly expressed in astrocytes and PRDX6-iPLA2 is involved in the activation of astrocytes and microglia. In co-culture system, Asp140 mutation in PRDX6 of CTX-TNA2 inhibited the polarization of microglia, reduced the production of ROS, suppressed NOX2 activation, and inhibited the Drp1-dependent mitochondrial fission following OGD/R. These effects were further strengthened by the inhibition of ROS production. In subsequent experiments, U0126 and SB202190 inhibited the phosphorylation of PRDX6 at Thr177 and reduced PRDX6-iPLA2 activity. These results suggest that PRDX6-iPLA2 plays an important role in the astrocyte-induced generation of ROS and activation of microglia, which are regulated by the activation of Nox2 and Drp1-dependent mitochondrial fission pathways. Additionally, PRDX6-iPLA2 activity is regulated by MAPKs via the phosphorylation of PRDX6 at Thr177 in astrocytes.

Keywords: Astrocytes; Drp1‑ dependent mitchondrial fission; Microglia; Nox2; PRDX6-iPLA2.

Fig. 1

Schematics of ischemic stroke model and experimental design

Fig. 2

Inhibition of PRDX6-iPLA2 reduces ischemic brain injury. a iPLA2 activity and GPx activity in rats. b Western blot analysis of PRDX6. c TTC staining and Infarct volume of the brain. d Neurological deficit scores. e Brain water content. f HE and Nissl staining (× 400) and histograms showing the number of intact neurons. g Mitochondrial ultrastructure. Values are mean ± SEM, * p  < 0.05, ** p  < 0.01, *** p  < 0.001. n  = 6 per group

Fig. 3

Inhibition of PRDX6-iPLA2 influences astrocyte and microglia activation in the ischemic penumbra. a Immunocytochemistry of Iba-1 staining for microglia. Immunocytochemistry of GFAP staining for astrocytes. PRDX6 expression and location was measured by immunohistochemistry in rats after treatment with MJ33. Cell nuclei were stained with DAPI (× 400). b Histogram reveals the number of the activated GFAP + astrocytes. c Histogram shows PRDX6 + astrocytes. d Histogram reveals the amoebodilike microglia. e Immunocytochemistry of Iba-1 staining for microglia. CD16 and CD206 expression were detected by immunohistochemistry in microglia after treatment with MJ33. Cell nuclei were stained with DAPI (× 400). f Histogram reveals the number of CD16 + microglia and CD206 + microglia. g Quantification of IL-1α, IL-1β, IL-6, and TNF-α in rats by ELISA. Values are mean ± SEM, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001. n  = 6 per group

Fig. 4

Inhibition of astrocytic PRDX6-iPLA2 influences microglia/infiltrated macrophages polarization. a Western blot analysis of PRDX6 in CTX-TNA2 cells or BV2 cells. b iPLA2 activity in CTX-TNA2 cells or BV2 cells. GPx activity in CTX-TNA2 cells or BV2 cells. c Immunocytochemistry of Iba-1 staining for microglia. CD16 and CD206 expression were detected by immunohistochemistry in BV2 cells. Cell nuclei were stained with DAPI (× 400). d Histogram reveals fluorescence intensity of CD16 and CD206. e Quantification of iNOS, IL-1β, TNF-α, Arg-1, IL10 and IL-4 in BV2 cells by ELISA. Values are mean ± SEM, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001

Fig. 5

Inhibition of astrocytic PRDX6-iPLA2 influences microglia/infiltrated macrophages polarization through mediating the generation of ROS. a DCFH-DA staninig. b Quantification of ROS. c Immunocytochemistry of Iba-1 staining for microglia. CD16 and CD206 expression were detected by immunohistochemistry in BV2 cells. Cell nuclei were stained with DAPI (× 400). d Histogram reveals fluorescence intensity of CD16 and CD206. e Quantification of iNOS, IL-1β, TNF-α, Arg-1, IL10 and IL-4 in BV2 cells by ELISA. Values are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

Inhibition of astrocytic PRDX6-iPLA2 influences Drp1-dependent mitochondrial fission through mediating the generation of ROS. a Mitochondrial morphology. b Mitochondrial membrane potential. c Western blot analysis of Drp1, cyto-Drp1, p-Drp1, mitochondrial Drp1 in CTX-TNA2 cells. d Western blot analysis of fission-related proteins and fusion-related factors in CTX-TNA2 cells. e DCFH-DA staninig and quantification of ROS. Values are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 7

Phosphorylation of PRDX6 mediates NOX2 activation by regulating its translocation and gaining iPLA2 activity in astrocytes. a Quantification of ROS. b DCFH-DA staninig. c Western blot analysis of membranous PRDX6, NOX2, P22^phox, P47^phox, Rac1, and total PRDX6 levels. d Immunoprecipitation and immunoblot analyses of the interaction between P22^phox, P47^phox, Rac1 and P67^phox. e PRDX6 peptides. f Immunoprecipitation and immunoblotting analyses of PRDX6 phosphorylation. g Western blot analysis of membranous PRDX6, total PRDX6, and p-PRDX6 levels. Values are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 8

MAPK activity mediates phosphorylation of PRDX6 and its translocation to the cell membrane in astrocytes after OGD/R. a Western blot analysis of ERK, p-ERK, P38 and p-P38 in CTX-TNA2 cells. b iPLA2 activity in CTX-TNA2 cells. C Molecular docking of ERK with PRDX6, P38 with PRDX6. d, f Western blot analysis of PRDX6, p-PRDX6, membranous PRDX6. e Immunoprecipitation and immunoblot analyses of ERK-PRDX6 in CTX-TNA2 cells. g Immunoprecipitation and immunoblot analyses of P38-PRDX6 in CTX-TNA2 cells. h Immunoprecipitation and immunoblot analyses of ERK-PRDX6 or P38-PRDX6 in HEK293T cells. Values are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 6 per group

Fig. 9

Mechanisms of a novel role for PRDX6 and iPLA2 in regulating astrocyte-induced ROS production and ROS-induced microglia/infiltrated macrophage polarization by activating the NOX2 and Drp1‑mitochondrial fission pathways. Further, PRDX6-iPLA2 activity is regulated by ERK and P38 via phosphorylation of PRDX6 at Thr177 in astrocytes