Janus Kinase Inhibition Ameliorates Ischemic Stroke Injury and Neuroinflammation Through Reducing NLRP3 Inflammasome Activation via JAK2/STAT3 Pathway Inhibition

Abstract

Background: Inflammatory responses play a multiphase role in the pathogenesis of cerebral ischemic stroke (IS). Ruxolitinib (Rux), a selective oral JAK 1/2 inhibitor, reduces inflammatory responses via the JAK2/STAT3 pathway. Based on its anti-inflammatory and immunosuppressive effects, we hypothesized that it may have a protective effect against stroke. The aim of this study was to investigate whether inhibition of JAK2 has a neuroprotective effect on ischemic stroke and to explore the potential molecular mechanisms.

Methods: Rux, MCC950 or vehicle was applied to middle cerebral artery occlusion (MCAO) mice in vivo and an oxygen-glucose deprivation/reoxygenation (OGD/R) model in vitro. After 3 days of reperfusion, neurological deficit scores, infarct volume and brain water content were assessed. Immunofluorescence staining and western blots were used to measure the expression of NLRP3 inflammasome components. The infiltrating cells were investigated by flow cytometry. Proinflammatory cytokines were assessed by RT-qPCR. The expression of the JAK2/STAT3 pathway was measured by western blots. Local STAT3 deficiency in brain tissue was established with a lentiviral vector carrying STAT3 shRNA, and chromatin immunoprecipitation (ChIP) assays were used to investigate the interplay between NLRP3 and STAT3 signaling.

Results: Rux treatment improved neurological scores, decreased the infarct size and ameliorated cerebral edema 3 days after stroke. In addition, immunofluorescence staining and western blots showed that Rux application inhibited the expression of proteins related to the NLRP3 inflammasome and phosphorylated STAT3 (P-STAT3) in neurons and microglia/macrophages. Furthermore, Rux administration inhibited the expression of proinflammatory cytokines, including TNF-α, IFN-γ, HMGB1, IL-1β, IL-2, and IL-6, suggesting that Rux may alleviate IS injury by inhibiting proinflammatory reactions via JAK2/STAT3 signaling pathway regulation. Infiltrating macrophages, B, T, cells were also reduced by Rux. Local STAT3 deficiency in brain tissue decreased histone H3 and H4 acetylation on the NLRP3 promoter and NLRP3 inflammasome component expression, indicating that the NLRP3 inflammasome may be directly regulated by STAT3 signaling. Rux application suppressed lipopolysaccharide (LPS)-induced NLRP3 inflammasome secretion and JAK2/STAT3 pathway activation in the OGD/R model in vitro.

Conclusion: JAK2 inhibition by Rux in MCAO mice decreased STAT3 phosphorylation, thus inhibiting the expression of downstream proinflammatory cytokines and the acetylation of histones H3 and H4 on the NLRP3 promoter, resulting in the downregulation of NLRP3 inflammasome expression.

Keywords: JAK2/STAT3; NLRP3 inflammasome; ischemic stroke; neuroinflammation; ruxolitinib.

Figure 1

Rux reduced infarct size, neurological deficits and cerebral edema in mice after MCAO. (A) Representative TTC-stained slices showing infarction in vehicle- and Rux-treated mice. (B) Quantitative analysis of the infarct size is presented as a percentage of the contralateral hemisphere size 3 days after stroke. (C) Neurological scores were determined 3 days after MCAO. (D) Representative western blots showing that Rux application reduced AQP4 expression. (E) Quantification of AQP4 expression. (F) Quantification of water content. Means ± SD. n = 5 in each group. ^aP < 0.05 versus the vehicle group; ^bP < 0.05 versus the 30 mg/kg Rux group; ^cP < 0.05 versus the 60 mg/kg Rux group.

Figure 2

Rux treatment decreased the number of NLRP3⁺ and CD68⁺ cells in the ischemic penumbra. (A) Representative images of NLRP3 and CD68 immunostaining in the ischemic penumbra of sham-, vehicle-, Rux- and MCC950-treated mice 3 days after stroke. (B, C) Statistical analysis of NLRP3⁺ and CD68⁺ cells. (D) Quantification of double-positive cells in the peri-ischemic region. Means ± SD. n = 5. ^aP < 0.01 versus sham; ^bP < 0.01 versus vehicle. Scale bar: 50 µm.

Figure 3

Rux treatment reduced the number of NLRP3⁺ cells and increased the number of NeuN⁺ cells in the ischemic penumbra 3 days after MCAO. (A) Representative immunostaining of NLRP3 and NeuN in the penumbra zone in sham-, vehicle-, Rux- and MCC950-treated mice. (B, C) Statistical analysis of NLRP3⁺ and CD68⁺ cells. (D) Quantification results of double-positive cells in the peri-ischemic region. Means ± SD. n = 5. ^aP < 0.01 versus sham; ^bP < 0.01 versus vehicle; ^cP < 0.05 versus Rux. Scale bar: 50 µm.

Figure 4

Rux treatment suppressed NLRP3 inflammasome-associated proteins. (A) Representative western blots showing that administration of Rux reduced the expression of NLRP3, ASC, CL-caspase-1, IL-18 and IL-1β in the peri-ischemic region 3 days after MCAO. (B–F) Quantification results of NLRP3, ASC, CL-caspase-1, IL-18 and IL-1β protein levels in the ischemic cortex. Means ± SD. n = 5/group. ^aP < 0.01 versus sham; ^bP < 0.01 versus vehicle; ^cP < 0.05 versus Rux.

Figure 5

Rux administration reduced the infiltrated macrophages, B, and T cells. (A, B) Gating strategy to identify CD45^interCD11b⁺ (MiDM) and CD45^highCD11b⁺ (MDMs) cells. (D–F) Bar graphs showing the results for CD45^interCD11b⁺ (MiDM) and CD45^highCD11b⁺ (MDMs) cells. (C, G, H) Gating strategies for identifying B220⁺ B cells, and CD3⁺ T cells. (I, J) Bar graphs showing the results for B lymphocytes and T lymphocytes. n = 3 to 5 per group. ^aP < 0.01 versus sham; ^bP < 0.01 versus vehicle.

Figure 6

Rux treatment decreased the number of GFAP⁺ and MPO⁺ cells in the ischemic penumbra 3 days after MCAO. (A, C) Representative immunostaining of GFAP and MPO in the penumbra region. (C, D) Quantification results of GFAP⁺ and MPO⁺ cells in the peri-ischemic region. Means ± SD. n = 5. ^aP < 0.01 versus sham; ^bP < 0.01 versus vehicle; Scale bar: 50 µm.

Figure 7

Rux treatment reduced the mRNA expression of proinflammatory cytokines 3 days after MCAO. (A–F) mRNA expression of IL-1β, IL-2, IL-6, HMGB1, TNF-α, and IFN-γ. (G, H) mRNA expression of IL-4 and IL-10. The data are shown as fold changes compared to the sham. n = 5. ^aP < 0.001 versus sham; ^bP < 0.01 versus vehicle; ^cP < 0.05 versus Rux.

Figure 8

The administration of Rux inhibited the activation of P-JAK2 and P-STAT3 3 days after MCAO. (A) Representative western blots showing that Rux inhibited P-JAK2/P-STAT3 expression in the peri-ischemic region 3 days after MCAO. (B, C) Quantification of P-JAK2/P-STAT3 expression in the ischemic penumbra. Means ± SD, n = 5/group. ^aP < 0.01 versus sham; ^bP < 0.05 versus vehicle; ^cP < 0.05 versus Rux.

Figure 9

Local STAT3 deficiency decreased histone H3 and H4 acetylation and NLRP3 expression. (A) Representative immunostaining of NLRP3 and P-STAT3 in the penumbra of the ischemic cortex 3 days after MCAO. (B) ChIP assay indicating that P-STAT3 bound to the NLRP3 promotor. (C, D) ChIP-qPCR assays showing that Ac-H3 and Ac-H4 on the NLRP3 promotor were decreased by local STAT3 deficiency. (E) Representative western blots showing that STAT3 knockout restrained the expression of P-STAT3, NLRP3, and global Ac-H3 and Ac-H4 in the peri-ischemic region 3 days after MCAO. (F–J) Quantification of STAT3, P-STAT3, NLRP3, and global Ac-H3 and Ac-H4 levels in the ischemic penumbra. Means ± SD. n = 5/group. ^aP < 0.001 versus sham; ^bP < 0.05 versus lenti-GFP. Scale bar: 50 µm.

Figure 10

Administration of Rux reduced NLRP3 inflammasome activation in HT22 cells after OGD/R. (A) Representative western blots showing NLRP3, ASC, CL-caspase-1, IL-1β and IL-18 at the protein level. (B–F) Quantification results of NLRP3, ASC, CL-caspase-1, IL-1β and IL-18 expression at the protein level. Means ± SD. n = 5/group. ^aP < 0.01 versus control; ^bP < 0.05 versus OGD/R + vehicle; ^cP < 0.05 versus OGD/R + LPS.

Figure 11

Rux treatment inhibited LPS-induced JAK2/STAT3 activation in HT22 cells after OGD/R. (A) Representative western blots showing that Rux administration downregulated P-JAK2/P-STAT3 expression in HT22 cells after OGD/R. (B, C) Quantification of P-JAK2 and P-STAT3 presented as fold changes. Means ± SD. n = 5/group. ^aP < 0.01 versus control; ^bP < 0.05 versus OGD/R + vehicle; ^cP < 0.01 versus OGD/R + LPS.