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. 2018 Mar 15;15(1):83.
doi: 10.1186/s12974-018-1117-5.

Lentivirus-mediated overexpression of OTULIN ameliorates microglia activation and neuroinflammation by depressing the activation of the NF-κB signaling pathway in cerebral ischemia/reperfusion rats

Hongbei Xu  1   2 , Wenyi Qin  3 , Xiao Hu  4 , Song Mu  5 , Jun Zhu  1   2 , Wenhao Lu  1   2 , Yong Luo  6   7
Affiliations

Lentivirus-mediated overexpression of OTULIN ameliorates microglia activation and neuroinflammation by depressing the activation of the NF-κB signaling pathway in cerebral ischemia/reperfusion rats

Hongbei Xu et al. J Neuroinflammation. .

Abstract

Background: Ischemic stroke-induced neuroinflammation is mainly mediated by microglial cells. The nuclear factor kappa B (NF-κB) pathway is the key transcriptional pathway that initiates inflammatory responses following cerebral ischemia. OTULIN, a critical negative regulator of the NF-κΒ signaling pathway, exerts robust effects on peripheral immune cell-mediated inflammation and is regarded as an essential mediator for repressing inflammation in vivo. The effect of OTULIN on inflammatory responses in the central nervous system (CNS) was previously unstudied. This current study investigated the anti-inflammatory effect of OTULIN both in vitro and in vivo in ischemic stroke models.

Methods: Sprague-Dawley (SD) rats were subjected to transient middle cerebral artery occlusion (tMCAO) or an intraperitoneal injection of lipopolysaccharide (LPS). Overexpression of the OTULIN gene was utilized to observe the effect of OTULIN on ischemic stroke outcomes. The effect of OTULIN overexpression on microglia-mediated neuroinflammation was examined in rat primary microglia (PM) and in the microglial cell line N9 after induction by oxygen-glucose deprivation (OGD)-treated neuronal medium. The activation and inflammatory responses of microglia were detected using immunofluorescence, ELISA, and qRT-PCR. The details of molecular mechanism were assessed using Western blotting.

Results: In the tMCAO rats, the focal cerebral ischemia/reperfusion injury induced a continuous increase in OTULIN expression within 72 h, and OTULIN expression was increased in activated microglial cells. OTULIN overexpression obviously decreased the cerebral infarct volume, improved the neurological function deficits, and reduced neuronal loss at 72 h after reperfusion, and it also inhibited the activation of microglia and attenuated the release of TNF-α, IL-1β, and IL-6 by suppressing the NF-κB pathway at 24 h after tMCAO. In vitro, OTULIN overexpression inhibited the microglia-mediated neuroinflammation by reducing the production of TNF-α, IL-1β, and IL-6 via depressing the NF-κB pathway in both PM and N9 cells.

Conclusions: OTULIN provides a potential therapeutic target for ischemic brain injury by ameliorating the excessive activation of microglial cells and neuroinflammation through repressing the NF-κB signaling pathway.

Keywords: Cerebral ischemia/reperfusion; Microglia; NF-κB signaling pathway; Neuroinflammation; OTULIN.

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Conflict of interest statement

Ethics approval

Our manuscript data were collected from animals and the study was approved by the Ethics Committee for Animal Experimentation of Chongqing Medical University. The reference number includes number of permit SYXK (渝) 2012–0001 and number of animal qualitative qualification 0001815.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Cerebral ischemia/reperfusion increased the levels of OTULIN mRNA and protein at the indicated time points (6, 12, 24, 48, and 72 h) after reperfusion following 2 h of ischemia. a A coronal brain section (stained with TTC) is labeled with black boxes to show the regions analyzed (asterisk represents ischemic core area; boxes represent the ischemic penumbra proximal to the ischemic core area). b OTULIN mRNA levels in the Sham and tMCAO groups at each time point were detected by qRT-PCR (n = 5). c OTULIN protein levels in the Sham and tMCAO groups at each time point were examined with Western blot. d The histogram presents the quantitative analysis of OTULIN protein levels at the indicated time points in the Sham and tMCAO groups (n = 3). All values are presented as the means ± SEMs; ***P < 0.001, **P < 0.01, and *P < 0.05 versus the Sham group
Fig. 2
Fig. 2
Lentivirus-mediated OTULIN overexpression exerted a neuroprotective role at 72 h after reperfusion in focal cerebral ischemia/reperfusion rats. a Schematic representation of the animal experiments over time. OTULIN mRNA (b, n = 5) and protein (c and d, n = 3) levels were elevated effectively by LV-OTULIN. The Longa score (e, n = 6), Bederson core (f, n = 6), and Garcia score (g, n = 6) were applied to assess neurological function deficits. i Quantification of infarction volumes was calculated based on TTC staining (h, n = 5). MAP2 (j, n = 6) and NeuN (j, n = 6) antibodies were used to stain neurons in the ischemic penumbra; scale bar = 75 μm. Quantification of the number of MAP2+/NeuN+ neurons was presented in panel k. All values are presented as the means ± SEMs; ###P < 0.001, ##P < 0.01, and #P < 0.05 versus the tMCAO group; &&&P < 0.001, &&P < 0.01, and &P < 0.05 versus the tMCAO+LV-Scramble group
Fig. 3
Fig. 3
OTULIN overexpression reduced the number of Iba-1+ microglial cells in the cortical ischemic penumbra in focal cerebral ischemia/reperfusion rats. A Representative images of Iba-1+ microglial cells that were morphologically classified into three types; scale bar = 10 μm. B Iba-1+ microglial cells in the ischemic penumbra and contralateral cortex of each group were detected by confocal immunofluorescence at 24 h, 72 h, and 7 days; scale bar = 75 μm. C The histogram represents the quantitative analysis of mean Iba-1 immunofluorescence intensity in each group (n = 6). Data are presented as the means ± SEMs; ###P < 0.001 versus the tMCAO group; &&&P < 0.001 versus the tMCAO+LV-Scramble group
Fig. 4
Fig. 4
OTULIN overexpression decreased LPS-induced microglial activation. Pure brain inflammation without neuronal death was induced by IP injection of LPS, and tissues were collected 24 h later. B The mean Iba-1 immunofluorescence intensities in the A LPS(−) group (a), the LPS(+) group (b), the LPS(+)+LV-Scramble group (c), and the LPS (+)+LV-OTULIN group (d) were quantitatively analyzed (n = 6); scale bar = 75 μm. All data are presented as the means ± SEMs; ##P < 0.01 versus the LPS(+) group; &P < 0.05 versus the LPS(+)+LV-Scramble group
Fig. 5
Fig. 5
OTULIN was markedly increased in activated microglial cells. a Analysis of immunofluorescent confocal images showed that OTULIN was increased in activated microglial cells after ischemic stroke (OTULIN, red; Iba-1, green; DAPI, blue; scale bar = 75 μm; n = 6). The red boxes indicate enlarged areas. The levels of OTULIN mRNA (b, n = 6) and protein (c, d; n = 3) in PM and N9 cells were detected at 24 h after exposure to regular microglia medium, conditioned medium (CM) collected from OGD-treated neuronal cultures (OGD[+]CM), or OGD-untreated (OGD[−])CM. All values are presented as the means ± SEMs; ###P < 0.001 versus the NC group; &&&P < 0.001, &&P < 0.01 versus the OGD(−)CM group
Fig. 6
Fig. 6
OTULIN overexpression reduced TNF-α, IL-1β, and IL-6 expression via the NF-κB signaling pathway at 24 h after reperfusion in tMCAO rats. The levels of TNF-α (a), IL-1β (b), and IL-6 (c) in the Sham, tMCAO, tMCAO+LV-Scramble, and tMCAO+LV-OTULIN groups were measured by ELISA (n = 6). e Quantitative analysis of the levels of p-IκBα and IκBα proteins in whole-cell preparations and p65 proteins in the cytoplasm and nucleus was detected by Western blot (d; n = 3). Data are presented as the means ± SEMs; ##P < 0.01, #P < 0.05 versus the tMCAO group; &&&P < 0.001, &&P < 0.01, &P < 0.05 versus the tMCAO+LV-Scramble group
Fig. 7
Fig. 7
Overexpression of OTULIN suppressed the levels of the pro-inflammatory cytokines produced by activated PM and N9 cells through depressing the NF-κB pathway. ELISA was used to measure the levels of TNF-α (a; n = 6), IL-1β (b; n = 6), and IL-6 (c; n = 6) in the NC, OGD(−)CM, and OGD(+)CM groups of PM and N9 cells. Each group was transfected with the LV-OTULIN or LV-Scramble. OTULIN and NF-κB pathway-associated proteins including p-IκBα, IκBα, and cytoplasm/nucleus NF-κB p65 in PM (d, e; n = 3) and N9 (d, f; n = 3) cells were detected by Western blot and analyzed quantitatively. All values are presented as the means ± SEMs. ***P < 0.001, **P < 0.01, and *P < 0.05 versus the OGD(−)+LV-OTULIN group; &&&P < 0.001, &&P < 0.01, and &P < 0.05 versus the OGD(+)+LV-Scramble group
Fig. 8
Fig. 8
Schematic representation of OTULIN regulating the NF-κB pathway in ischemic stroke. Ischemic stroke induced rapid activation of the NF-κB pathway, mainly shown by the activation of IKKs, the degradation and phosphorylation of IκBα, and the increased nuclear translocation of NF-κB p65. The activated NF-κB pathway leads to microglial activation along with a sharp increase in the production of pro-inflammatory cytokines and the occurrence of inflammatory responses in the brain. LUBAC, which consists of HOIP, HOIL-1, and SHARPIN, participates in the assembly of ubiquitin chains on IKK complexes. The deubiquitinase OTULIN interacts with HOIP to cleave the Met-1 Ub on IKKγ, thus resulting in depression of the NF-κB pathway. OTULIN overexpression obviously inhibited the excessive activation of microglia and the production of inflammatory responses through suppressing the phosphorylation and degradation of IκBα and subsequently attenuating the nuclear translocation of NF-κB p65
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