MiRNA-210 induces microglial activation and regulates microglia-mediated neuroinflammation in neonatal hypoxic-ischemic encephalopathy

Abstract

Neuroinflammation is a major contributor to secondary neuronal injury that accounts for a significant proportion of final brain cell loss in neonatal hypoxic-ischemic encephalopathy (HIE). However, the immunological mechanisms that underlie HIE remain unclear. MicroRNA-210 (miR-210) is the master "hypoxamir" and plays a key role in hypoxic-ischemic tissue damage. Herein, we report in an animal model of neonatal rats that HIE significantly upregulated miR-210 expression in microglia in the neonatal brain and strongly induced activated microglia. Intracerebroventricular administration of miR-210 antagomir effectively suppressed microglia-mediated neuroinflammation and significantly reduced brain injury caused by HIE. We demonstrated that miR-210 induced microglial M1 activation partly by targeting SIRT1, thereby reducing the deacetylation of the NF-κB subunit p65 and increasing NF-κB signaling activity. Thus, our study identified miR-210 as a novel regulator of microglial activation in neonatal HIE, highlighting a potential therapeutic target in the treatment of infants with hypoxic-ischemic brain injury.

Keywords: SIRT1; microRNA-210; microglial activation; neonatal hypoxic-ischemic encephalopathy; neuroinflammation.

Fig. 1

MiR-210 is upregulated in activated microglia in HIE. The experiments were repeated three times. Representative results are presented. a Schematic representation of the experimental design to induce sham and HIE in P7 Sprague-Dawley rat pups. b Bar graph showing the FACS quantitative analysis of the various populations of immune cells in ipsilateral hemisphere harvested from sham or HIE animals at the indicated time points post-HIE induction. Data are presented as the mean ± SEM (n = 7–8). c Representative FACS plots showing the staining for CD11b/c and CD45 in mononuclear cells isolated from the ipsilateral hemisphere of sham or HIE animals at the indicated time points post-HIE induction. d Dot plots showing the RT-qPCR analysis of proinflammatory molecules in the ipsilateral hemisphere harvested from sham or HIE animals at the indicated time points post-HIE induction. Data are presented as the mean ± SEM (n = 5). e Bar graphs showing the qPCR analysis of global miR-210 expression in the ipsilateral hemisphere of sham or HIE animals. Data are presented as the mean ± SEM (n = 4). f Representative images showing the in situ hybridization staining of brain sections of sham or 12 h HIE animals (n = 3). Scale bar: 400 μm. g Bar graphs showing the qPCR analysis of miR-210 expression in microglia sorted from the ipsilateral hemisphere of sham or HIE animals at the indicated time points post-HIE induction. Data are presented as the mean ± SEM (n = 5). h Bar graphs showing the qPCR analysis of miR-210 expression in other neural cells sorted from the ipsilateral hemisphere of sham or HIE animals at 6 h post-HIE induction. Data are presented as the mean ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 1

MiR-210 is upregulated in activated microglia in HIE. The experiments were repeated three times. Representative results are presented. a Schematic representation of the experimental design to induce sham and HIE in P7 Sprague-Dawley rat pups. b Bar graph showing the FACS quantitative analysis of the various populations of immune cells in ipsilateral hemisphere harvested from sham or HIE animals at the indicated time points post-HIE induction. Data are presented as the mean ± SEM (n = 7–8). c Representative FACS plots showing the staining for CD11b/c and CD45 in mononuclear cells isolated from the ipsilateral hemisphere of sham or HIE animals at the indicated time points post-HIE induction. d Dot plots showing the RT-qPCR analysis of proinflammatory molecules in the ipsilateral hemisphere harvested from sham or HIE animals at the indicated time points post-HIE induction. Data are presented as the mean ± SEM (n = 5). e Bar graphs showing the qPCR analysis of global miR-210 expression in the ipsilateral hemisphere of sham or HIE animals. Data are presented as the mean ± SEM (n = 4). f Representative images showing the in situ hybridization staining of brain sections of sham or 12 h HIE animals (n = 3). Scale bar: 400 μm. g Bar graphs showing the qPCR analysis of miR-210 expression in microglia sorted from the ipsilateral hemisphere of sham or HIE animals at the indicated time points post-HIE induction. Data are presented as the mean ± SEM (n = 5). h Bar graphs showing the qPCR analysis of miR-210 expression in other neural cells sorted from the ipsilateral hemisphere of sham or HIE animals at 6 h post-HIE induction. Data are presented as the mean ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Inhibition of miR-210 prevents microglial activation in vivo and suppresses HIE. The experiments were repeated three times. Representative results are presented. a Schematic representation of the experimental design to study the role of miR-210 as an autonomous factor in regulating activated microglia induction of HIE in P7 Sprague-Dawley rat pups. ICV intracerebroventricular, MRI magnetic resonance imaging. b Bar graphs showing the qPCR analysis of global miR-210 expression in the ipsilateral hemisphere of 24 h HIE animals. Data are presented as the mean ± SEM (n = 4–6). c Representative MRI images showing the infarct size of the ipsilateral hemisphere of 48 h HIE animals (n = 6). Note that miR-210 inhibition strongly reduced the infarct size in the right ipsilateral brain (areas in white). d Bar graph showing the quantification of MRI images presented in c. Data are presented as the mean ± SEM (n = 6). e Bar graphs showing the RT-qPCR analysis of proinflammatory molecules in the ipsilateral hemisphere harvested from 12 h HIE animals. Data are presented as the mean ± SEM (n = 6–8). f Representative FACS plots showing the staining for CD11b/c and CD45 in mononuclear cells isolated from the ipsilateral hemisphere of HIE animals at the indicated time points post-HIE induction. g Bar graph showing the quantification of the FACS plots presented in f. Data are presented as the mean ± SEM (n = 6). h Bar graphs showing the qPCR analysis of miR-210 expression in microglia sorted from the ipsilateral hemisphere of 12 h HIE animals. Data are presented as the mean ± SEM (n = 6–8). i Bar graphs showing the RT-qPCR analysis of proinflammatory molecule expression in microglia sorted from the ipsilateral hemisphere of 12 h HIE animals. Data are presented as the mean ± SEM (n = 6–8). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Inhibition of miR-210 prevents microglial activation in vivo and suppresses HIE. The experiments were repeated three times. Representative results are presented. a Schematic representation of the experimental design to study the role of miR-210 as an autonomous factor in regulating activated microglia induction of HIE in P7 Sprague-Dawley rat pups. ICV intracerebroventricular, MRI magnetic resonance imaging. b Bar graphs showing the qPCR analysis of global miR-210 expression in the ipsilateral hemisphere of 24 h HIE animals. Data are presented as the mean ± SEM (n = 4–6). c Representative MRI images showing the infarct size of the ipsilateral hemisphere of 48 h HIE animals (n = 6). Note that miR-210 inhibition strongly reduced the infarct size in the right ipsilateral brain (areas in white). d Bar graph showing the quantification of MRI images presented in c. Data are presented as the mean ± SEM (n = 6). e Bar graphs showing the RT-qPCR analysis of proinflammatory molecules in the ipsilateral hemisphere harvested from 12 h HIE animals. Data are presented as the mean ± SEM (n = 6–8). f Representative FACS plots showing the staining for CD11b/c and CD45 in mononuclear cells isolated from the ipsilateral hemisphere of HIE animals at the indicated time points post-HIE induction. g Bar graph showing the quantification of the FACS plots presented in f. Data are presented as the mean ± SEM (n = 6). h Bar graphs showing the qPCR analysis of miR-210 expression in microglia sorted from the ipsilateral hemisphere of 12 h HIE animals. Data are presented as the mean ± SEM (n = 6–8). i Bar graphs showing the RT-qPCR analysis of proinflammatory molecule expression in microglia sorted from the ipsilateral hemisphere of 12 h HIE animals. Data are presented as the mean ± SEM (n = 6–8). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Inhibition of miR-210 reduces the loss of neural cells and oligodendrocytes in HIE. The experiments were repeated three times. Representative results are presented. a Representative FACS plots showing the intracellular NeuN staining of mononuclear cells isolated from the ipsilateral hemisphere of scramble control or LNA-anti-miR-210-treated animals at 48 h post-HIE induction. b Bar graph showing the quantification of the FACS plots presented in a. Data are presented as the mean ± SEM (n = 7–8). c Representative FACS plots showing A2B5 staining of mononuclear cells isolated from the ipsilateral hemisphere at 48 h post-HIE induction. d Bar graph showing the quantification of the FACS plots presented in c. Data are presented as the mean ± SEM (n = 7–8). e Representative FACS plots showing the staining for oligodendrocyte O4 in mononuclear cells isolated from the ipsilateral hemisphere at 48 h post-HIE induction. f Bar graph showing the quantification of the FACS plots presented in e. Data are presented as the mean ± SEM (n = 7–8). g Representative FACS plots showing the intracellular MBP staining of mononuclear cells isolated from the ipsilateral hemisphere at 48 h post-HIE induction. h Bar graph showing the quantification of the FACS plots presented in g. Data are presented as the mean ± SEM (n = 7–8). *P < 0.05, ***P < 0.001

Fig. 4

MiR-210 activates neonatal rat microglia in vitro. a–e Microglia were isolated from neonatal rat primary microglia culture and treated with negative mimic or miR-210 mimic for 48 h (100 nM). Cells were collected for analysis at the indicated time points. The experiments were repeated three times, and representative results are presented. a Bar graphs showing the qPCR analysis of miR-210 expression in microglia at 24 h post transfection. Data are presented as the mean ± SEM (n = 6). b Representative FACS plots showing the staining for CD11b/c, CD45, CD80, and CD86 in microglia at 24 h post transfection. c Bar graph showing the quantification of the FACS plots presented in b. Data are presented as the mean ± SEM of triplicate cultures. d, e Bar graphs showing the RT-qPCR analysis of proinflammatory M1 markers (d) or antiinflammatory M2 markers (e) of microglia at the indicated time points post transfection. Data are presented as the mean ± SEM (n = 6). f–h Microglia were isolated from neonatal rat primary microglia culture, transfected with LNA scramble control or LNA-anti-miR-210 (100 nM), and recovered overnight (12 h) prior to being treated with or without LPS (0.5 ng/ml) and IFN-γ (0.1 ng/ml) for 12 h. Cells were collected for expression analysis. f Bar graphs showing the qPCR analysis of miR-210 expression in microglia at 24 h post transfection of miR-210 inhibitor. Data are presented as the mean ± SEM (n = 4). g, h Bar graphs showing the RT-qPCR analysis of proinflammatory cytokines (TNF-α, IL-1β, IL-6) in microglia without g or with h M1 stimulation. Data are presented as the mean ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

SIRT1 is a bona fide target of miR-210 in neonatal rat microglia. a. Schematic representation of alignment of the predicted miR-210 binding site to the SIRT1 3′UTR is shown for Rattus norvegicus. b The rat PC12 cell line was cotransfected with the indicated constructs (500 ng/well) containing the wild-type or mutant 3′UTR of SIRT1 or the wild-type 3′UTR of TET2 (positive control) in the presence of miR-210 mimic or negative mimic (100 nM, 200 nM, or 500 nM) for 48 h. Bar graph showing the normalized levels of luciferase activity in the transfected PC12 cells. Data are presented as the mean ± SEM of four replicate cultures. c Immortalized rat microglia were transfected with negative mimic or miR-210 mimic (100 nM) and collected for analysis 24 h post transfection. Bar graph showing the RISC-IP analysis of abundance of SIRT1 3′UTR pulled down by Ago1/2/3 antibody. Data are presented as the mean ± SEM (n = 3). d Neonatal rat microglia were transfected with negative mimic or miR-210 mimic (100 nM) and collected for analysis 24 h post transfection. Western blots showing the analysis of SIRT1 protein levels. e Bar graph showing the quantification of the western blots presented in d. Data are presented as the mean ± SEM of triplicate cultures. f Neonatal rat microglia were transfected with LNA scramble control or LNA-anti-miR-210 (100 nM) and collected for analysis 24 h post transfection. Western blots showing the analysis of SIRT1 protein levels. g Bar graph showing the quantification of the western blots presented in f. Data are presented as the mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

SIRT1 is a possible mediator of miR-210 in its regulation of M1 activation in neonatal rat microglia. a, b Neonatal rat primary microglia were treated with nonsilencing control siRNA (siCtrl) or siRNAs specific for rat SIRT1 (siSIRT1) (100 nM) and collected for analysis 24 h post transfection. Bar graph showing the RT-qPCR analysis of the expression of SIRT1 (a) or proinflammatory cytokines (TNF-α, IL-1β, IL-6) (b). Data are presented as the mean ± SEM (n = 6). c–e Neonatal rat microglia were transfected with siCtrl or siSIRT1 (100 nM), recovered overnight (12 h), and then treated with LPS (0.5 ng/ml) and IFN-γ (0.1 ng/ml) for 12 h. Cells were collected for analysis. c Bar graphs showing the RT-qPCR analysis of proinflammatory cytokines (TNF-α, IL-1β, IL-6) in siRNA-transfected microglia. Data are presented as the mean ± SEM (n = 6). d Representative FACS plots showing staining for CD11b/c, CD45, CD80, and CD86 in siRNA-transfected microglia. e Bar graph showing the quantification of the FACS plots presented in d. Data are presented as the mean ± SEM (n = 6). f, g Neonatal rat primary microglia were treated with siCtrl or siSIRT1 or LNA-anti-miR-210 (100 nM) and collected for protein analysis 24 h post transfection. f Western blots showing the analysis of the indicated protein levels in transfected microglia. g Bar graph showing the quantification of the Western blots presented in f. Data are presented as the mean ± SEM of triplicates. h Neonatal rat primary microglia were treated with siCtrl or siSIRT1 (100 nM) and collected for analysis 24 h post transfection. Bar graph showing the ChIP analysis of nuclear binding of acetyl-NF-κB p65 to the IL-1β promoter. Data are presented as the mean ± SEM of triplicates. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

Schematic of a proposed working model wherein miR-210 positively regulates microglia-mediated neuroinflammation via activating the NF-κB signaling pathway by targeting SIRT1 and blocking p65 deacetylation. Upon recognition of DAMPs following acute hypoxic-ischemic insult, developing microglia initiate the TLR signaling pathway that leads to NF-κB activation. NF-κB induces the production of proinflammatory cytokines (such as TNF-α, IL-1β, and IL-6), NO, and ROS, which promotes the apoptotic death of neural cells and causes secondary neurotoxicity. However, SIRT1, as a negative regulator, governs the excessive activation of microglia by deacetylating p65 at lysine 310 and suppressing NF-κB activity. As a HIF-1α-induced master hypoxamir, miR-210 is upregulated in microglia in response to neonatal HI, which rescues NF-κB activity by targeting the NF-κB signaling negative regulator SIRT1. Therefore, through upregulation of NF-κB activity, miR-210, as an autologous factor, promotes sustained activation of microglia and enhances microglia-mediated neuroinflammation in the developing brain