Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke

Abstract

Background: Ischemic stroke is a leading cause of disability worldwide and characteristically accompanied by downregulation of the Wnt/β-catenin signaling. Activation of Wnt/β-catenin signaling emerges to attenuate neuroinflammation after ischemic stroke; however, its effect on modulating microglial polarization is largely unknown. Here, we explored whether Wnt/β-catenin pathway activator TWS119 facilitated long-term neurological recovery via modulating microglia polarization after experimental stroke.

Methods: Ischemic stroke mice model was induced by permanent distal middle cerebral artery occlusion plus 1 h hypoxia. TWS119 was administrated from day 1 to 14 after stroke. Neurological deficits were monitored up to 21 days after stroke. Angiogenesis, neural plasticity, microglial polarization, and microglia-associated inflammatory cytokines were detected in the peri-infarct cortex at days 14 and 21 after stroke. Primary microglia and mouse brain microvascular endothelial cell lines were employed to explore the underlying mechanism in vitro.

Results: TWS119 mitigated neurological deficits at days 14 and 21 after experimental stroke, paralleled by acceleration on angiogenesis and neural plasticity in the peri-infarct cortex. Mechanistically, cerebral ischemia induced production of microglia-associated proinflammatory cytokines and priming of activated microglia toward pro-inflammatory polarization, whereas TWS119 ameliorated microglia-mediated neuroinflammatory status following ischemic stroke and promoted angiogenesis by modulating microglia to anti-inflammatory phenotype. The beneficial efficacy of TWS119 in microglial polarization was largely reversed by selective Wnt/β-catenin pathway blockade in vitro, suggesting that TWS119-enabled pro-inflammatory to anti-inflammatory phenotype switch of microglia was possibly mediated by Wnt/β-catenin signaling.

Conclusions: Wnt/β-catenin pathway activator TWS119 ameliorated neuroinflammatory microenvironment following chronic cerebral ischemia via modulating microglia towards anti-inflammatory phenotype, and facilitates neurological recovery in an anti-inflammatory phenotype polarization-dependent manner. Activation of Wnt/β-catenin pathway following ischemic stroke might be a potential restorative strategy targeting microglia-mediated neuroinflammation.

Keywords: Cerebral ischemia; Microglial polarization; Neuroinflammation; Neurological recovery; TWS119; WNT/β-catenin.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Experimental outline and schematic diagram of brain section. a Experimental outline: TWS119 or BrdU were administrated intraperitoneally once daily from day 1 to 14 or from day 7 to 14 after stroke. Neurobehavioral tests were performed at days 1, 7, 14, and 21 after stroke. Angiogenesis, neural plasticity, microglial polarization and inflammatory cytokines were detected at the indicated time point. Histological assessment was assessed at day 21. The number of mice in each group for each test were shown in parentheses. b Schematic diagram of brain section. Green squares indicated the region of interest in the ipsilateral peri-infarct cortex, in which immunofluorescence images were collected. Yellow strip (0.5 mm wide) indicated peri-infarct region, in which brain samples for qRT-PCR and ELISA were harvested. stroke, focal cerebral ischemia; IF, immunofluorescence; qRT-PCR, quantitative real time polymerase chain reaction; ELISA, enzyme linked immunosorbent assay

Fig. 2

TWS119 improved post-stroke neurological function. CatWalk test (a–c), Adhesive Removal test (d, e), Accelerated Rotarod test (f) and mNSS (g) were performed between vehicle group and TWS119 group at day 1, 7, 14 and 21 after stroke. a-e TWS119 treatment, as opposed to saline injection, accelerated LF functional recovery. f TWS119 mice displayed better performance in Accelerated Rotarod test compared with vehicle mice. g TWS119 mice had lower mNSS compared with vehicle mice. LF, left forelimb, mNSS, modified neurological severity scores. (n = 8 per group, * P < 0.05, ** P < 0.01, *** P < 0.001, N.S = no statistical difference)

Fig. 3

TWS119 facilitated post-stroke angiogenesis. a Representative images of coronal sections labeled with CD31 (cerebral microvascular endothelial cells marker). b Area of CD31-positive microvessels were increased in ischemic mice at day 14 and 21 after stroke, and further upregulated by TWS119 treatment at day 21. c Cerebral ischemia enhanced the density of CD31-positive microvessels in peri-infarct cortex at day 14 and 21 after stroke, this change was more significant in TWS119 mice. d Representative images of coronal sections labeled with CD31 or/and BrdU (cell proliferation marker). e TWS119 increased the percentage of CD31+BrdU+ cells in total CD31+ cells at day 14 after stroke. (n = 6 per group, * P < 0.05, ** P < 0.01)

Fig. 4

TWS119 stimulated post-stroke neural plasticity. a Representative image of coronal sections labeled with GAP43 (axon marker), and PSD95 (postsynaptic structure marker). b TWS119 mice showed a higher density of GAP43+ puncta and PSD95+ puncta at day 21 after stroke compared with vehicle mice. c Representative image of coronal sections labeled with SMI312 (neurofilament marker), and Synaptophysin (presynaptic structure marker). d TWS119 also upregulated the density of SMI312+ puncta or Synaptophysin+ puncta at day 21 after stroke. (n = 6 per group, * P < 0.05, ** P < 0.01)

Fig. 5

TWS119 modulated microglia/Macrophages to anti-inflammatory phenotype after ischemic stroke. a Pro-inflammatory-type marker (CD-16, TNF-α, iNOS, IL-1β and IL-6) and anti-inflammatory-type marker (CD206, IL-10, TGF-β, Arg-1 and YM1/2) of microglia were analyzed by qRT-PCR 14 days after stroke. The mRNA levels of CD-16, TNF-α, iNOS, IL-1β, IL-6 and IL-10 were increased in vehicle mice compared with sham mice, TWS119 treatment decreased the level of CD-16, TNF-α and iNOS, and upregulated the level of CD206, IL-10, TGF-β, Arg-1 and YM1/2 compared with saline treatment. b Representative images of coronal sections labeled with Iba1 (microglia marker), and CD16/32. c TWS119 reduced the ratio of CD16/32+Iba1+ cells at 14 days after stroke. d Representative images of coronal sections labeled with Iba1, and CD206. e TWS119 increased the ratio of CD206+Iba1+ cells at 14 and 21 days after stroke. (n = 6 per group, * P < 0.05, ** P < 0.01)

Fig. 6

TWS119 ameliorated local inflammatory microenvironment in peri-infarct cortex. a TNF-α (pro-inflammatory cytokine) and IL-10 (anti-inflammatory cytokine) in per-infarct region ware measured by ELISA. The production of TNF-α was obviously increased at day 14 and 21 after stroke. TWS119 significantly reduced the production of TNF-α at day 14, and increased the production of IL-10 at day 14 and 21. b The production of IL-10 had a positive correlation with the residence time of Accelerated Rotarod test, while the production of IL-10 had a negative correlation with the LF-removal time in Adhesive Removal test and the mNSS at day 21 after stroke. LF, left forelimb; mNSS, modified neurological severity scores. (n = 8 per group, * P < 0.05, ** P < 0.01)

Fig. 7

TWS119 promoted pro-inflammatory to anti-inflammatory phenotype switch of microglia probably via Wnt/β-catenin pathway. Pro-inflammatory polarization was stimulated by LPS plus IFN-γ for 24 h. a CCK-8 assay was performed to determine the cytotoxicity of TWS119, TWS119 with the doses (≥ 100 μM) exerted cytotoxic effect on microglia cells. b Using ELISA, 10 μM was determined the effective dose. TWS119 with doses (10 μM, 20 μM) increased the secretion of IL-10 in microglia cells stimulated with LPS + IFN-γ, while TWS119 with doses (5 μM, 40 μM) had no similar effect. c mRNA expression of CD16 was enhanced in LPS + IFN-γ-stimulated microglia cells, this enhancement was corrected by TWS119 treatment, IWR-1 reversed the effect of TWS119 on CD16. TWS119 increased the level of CD206, which was reversed by IWR-1. d TWS119 reduced the percentage of CD16/32 positive cells and raised CD206 positive cells in Flow Cytometry, which was reversed by IWR-1. e TWS119 increased the level of β-catenin, which was reversed by IWR-1. IFN-γ, interferon-γ; IWR-1, a reversible inhibitor of Wnt/β-catenin signaling pathway; LPS, lipopolysaccharide. (n = 5 per group, 5 independent experiments from 5 different microglia preps. * P < 0.05, ** P < 0.01, *** P < 0.001, N.S means no statistical significance)

Fig. 8

TWS119 promoted angiogenesis in vitro via an amelioration of inflammatory microenvironment by altering microglia polarization. a Tube formation of bEend3 cells in conventional medium (5 × magnification). OGD injury reduced vascular tubular structures, which was not corrected by TWS119 treatment. b Supernatants of each microglia culture were used as CM. Proinflammatory cytokines (TNF-α, Nitrite, IL-1β) and anti-inflammatory cytokines (IL-10, TGF-β) were detected in each CM. c Representative images of tube formation in OGDbEend.3 cells incubated with CM (5 × magnification). Interestingly, only CM co-treated with LPS plus IFN-γ and TWS119 significantly increased the formation of pro-angiogenic structures in OGDbEnd.3 cells. Other CM had no similar function. bEend3, mouse-derived brain microvascular cell line; IFN-γ, interferon-γ; CM, conditioned media; LPS, lipopolysaccharide; OGD, oxygen-glucose deprivation. (n = 5, *P < 0.05, ***P < 0.05, N.S means no statistical significance)