The miR-451a facilitates natural killer cell-associated immune deficiency after ischemic stroke

Abstract

Ischemic stroke is a devastating neurological disease. Brain ischemia impairs systemic immune responses and heightens susceptibility to infections, though the underlying mechanisms remain incompletely understood. Natural killer (NK) cells exhibited decreased frequency and compromised function after acute stage of stroke, resulting in NK cell-associated immune deficiency and increased risk of infection. MicroRNAs (miRNAs) are post-transcriptional molecular modulators. Our previous study revealed a significant upregulation of miR-451a in circulating NK cells from patients with ischemic stroke, but its effects and precise mechanism on immune defense remain elusive. In this study, we observed a substantial elevation of miR-451a level in brain and splenic NK cells in murine model of ischemic stroke miR-451a mimics suppressed NK cell activation and cytotoxicity within the ischemic brain and periphery, including a downregulation of activation marker CD69, and reduced production of effector molecules IFN-γ and perforin. Conversely, miR-451a inhibitor preserved NK cell activation and cytotoxicity, rescuing local inflammation, and reducing bacterial burden in the lung. Pharmacological inhibition of Akt-mTOR pathway with AZD8055 effectively blocked the impacts of miR-451a on NK cell functions. Collectively, these findings suggest miR-451a negatively regulates NK cell cytotoxicity in both the brain and periphery, which could be re-addressed by modulating the Akt-mTOR signaling pathway.

Keywords: Brain ischemia; immune suppression; miR-451a; natural killer cells; poststroke infection.

Figure 1.

miR-451a down-regulates the phenotype and function of CNS-infiltrating NK cells after brain ischemia and reperfusion. (a) Validation of miR-451a expression in NK cells from MCAO and sham models in wild-type mice. n = 3 per group, error bars represent mean ± SD. *P < 0.05. (b) The schematic graph of the experimental design. NK cells sorted from pooled splenocytes of wild-type mice were transfected with miR-451a mimics, negative control or inhibitor. After being transfected for 24 h, NK cells were transferred intravenously (3 × 10⁶ per mouse) into NOD/Prkdc^scid/IL-2rg^null (NPG) mice prior to MCAO. On day 1 or day 3 after MCAO and reperfusion, mice were subjected to neurological assessment, MRI scan, flow cytometry and pathology staining. (c–d) Flow cytometry plots and bar graphs show the effect of miR-451a on the phenotype and function of NK cells in the ischemic brain at indicated time points, including NK cell counts (CD3⁻NK1.1⁺), inhibitory receptors on NK cell (KLRG1 and NKG2A), activating receptors on NK cell (NKG2D and Ly49H), activation markers of NK cell (CD69), and cytotoxic function of NK cell (IFN-γ and perforin). Day 1 after reperfusion, n = 5–7 mice per group. Day 3 after reperfusion, n = 11–12 mice per group. The data were representative of three independent experiments and calculated as mean ± SD; *P < 0.05, **P < 0.01 by two-way ANOVA and (e) The expression of IFN-γ, TNF-α, IL-1β and IL-6 in the ipsilateral brain at day 3 after MCAO in NPG mice receiving transfected NK cells was analyzed by ELISA. n = 7 mice per group. Data were presented as mean ± SD; *P < 0.05, **P < 0.01 by one-way ANOVA.

Figure 2.

MiR-451a is involved in the alteration of peripheral NK cells after MCAO. (a) After treatment with miR-451a mimics, negative control or inhibitor, NK cells were transferred intravenously into NPG mice before MCAO. Splenocytes were isolated at the indicated time points after surgery. Flow cytometry plots show the NK cell counts (CD3⁻NK1.1⁺), the expression of KLRG1, NKG2A, NKG2D, Ly49H, CD69, IFN-γ and perforin and (b) Summarized results show the suppression of miR-451a on peripheral NK cells at the acute stage of ischemic stroke. Day 1 after stroke, n = 5–8 mice per group; Day 3 after stroke, n = 10–12 mice per group. Data were collected from three independent experiments and expressed as mean ± SD; *P < 0.05, **P < 0.01 by two-way ANOVA.

Figure 3.

Targeting NK cells with miR-451a is not sufficient to affect stroke outcomes. Splenic NK cells isolated from wild-type mice were transfected with miR-451a mimics, negative control or inhibitor, and 3 × 10⁶ NK cells were transfused intravenously into NPG mice, followed by the MCAO procedure. Mice were subjected to neurological assessment and MRI evaluation at the indicated time points. (a) Cumulative data illustrate the neurological assessment of NPG mice (without NK cells) and NPG mice receiving NK cells transfected with the indicated treatment, the neurological assessment includes mNSS scores, corner turning test and rota-rod test. NPG mice receiving NK cells transfected with miR-451a inhibitor versus NPG mice: *P < 0.05 and **P < 0.01 by two-way ANOVA; NPG mice receiving NK cells transfected with miR-451a control versus NPG mice: ^#P < 0.05 and ^##P < 0.01 by two-way ANOVA and (b) The representative MRI images and quantification of infarct lesion of the indicated groups at day 3 after brain ischemia and reperfusion. *P < 0.05 by one-way ANOVA. n = 6–7 mice per group. The data were representative of three independent experiments and were expressed as mean ± SD.

Figure 4.

miR-451a exacerbates lung bacterial burden in MCAO mice, which is related to compromised function of NK cells in the lung. Isolated splenic wild-type NK cells were treated with miR-451a mimics, negative control or inhibitor and cultured for 24 hours, then transferred via intravenous injection to NPG transgenic mice, prior to MCAO. On day 3 after ischemia and reperfusion, lung tissues were collected for histological staining, bacteriological analysis, flowcytometry and ELISA analysis. (a) H&E-stained lung sections of NPG mice revealed the infiltration of inflammatory cells and the thickening of alveolar walls on day 3 after MCAO. Scale bras: 20 μm; insert: 10 μm. (b) Quantification analysis show the infiltrated cell counts from lung sections on day 3 after ischemia. n = 5–7 mice per group. (c–d) Representative image of bacterial culture and quantification of colony-forming units (CFU) in the lung of NPG mice on day 3 after surgery. n = 3–4 mice per group. (e) Bar graph show the effect of miR-451a on cell number and functional marker expression (CD69, IFN-γ and perforin) of lung NK cells on day 3 after MCAO. n = 5 mice per group and (f) ELISA measurement of IFN-γ, TNF-α, IL-1β and IL-6 protein levels in the lung on day 3 after MCAO. n = 7 mice per group. The results represented three independent experiments. Error bars represent mean ± SD. *P < 0.05; **P < 0.01 by one-way ANOVA.

Figure 5.

miR-451a modulates the phenotype and function of NK cells via the Akt-mTOR pathway. (a) Three databases were searched for target prediction of miR-451a, and 16 candidates appeared three times in these databases. (b) Splenic NK cells isolated from wild-type mice were treated with miR-451a mimics, negative control or inhibitor. After culturing for 24 hours, the expression of miR-451a, AKT, and mTOR were quantified by quantitative real-time PCR. n = 3 per group. (c) The schematic diagram delineates the acute mTOR inhibition with AZD8055 in vivo. After treatment with miR-451a inhibitor or negative control, NK cells were passively transferred by intravenous injection into NPG recipients, followed by MCAO surgery. Meanwhile, the MCAO mice were treated with AZD8055 or vehicle by oral gavage at a dose of 20 mg/kg immediately after reperfusion. Brain cells and splenocytes were measured using flow cytometry on day 1 after ischemia and reperfusion. (d–e) Cumulative data show the NK cell number and the expression of KLRG1, CD69, IFN-γ and perforin in the brain and spleen of MCAO mice receiving AZD8055 or vehicle. n = 3–5 mice per group and (f) The graphical abstract depicts that miR-451a contributed to stroke-mediated NK cell deficiency through the Akt-mTOR pathway. Data were collected from three independent experiments and were expressed as mean ± SD. *P < 0.05; **P < 0.01 by one-way ANOVA.