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Heme activates TLR4-mediated inflammatory injury via MyD88/TRIF signaling pathway in intracerebral hemorrhage.

Lin S, Yin Q, Zhong Q, Lv FL, Zhou Y, Li JQ, Wang JZ, Su BY, Yang QW - J Neuroinflammation (2012)

Bottom Line: In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR.Our findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately increasing cytokine expression and inflammatory injury in ICH.Targeting TLR4 signaling may be a promising therapeutic strategy for ICH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Daping Hospital, Third Military Medical University, Yuzhong District, Chongqing, China.

ABSTRACT

Background: Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced neurological deficits; however, the signaling pathways are not apparent by which the upstream cellular events trigger innate immune and inflammatory responses that contribute to neurological impairments. Toll-like receptor 4 (TLR4) plays a role in inflammatory damage caused by brain disorders.

Methods: In this study, we investigate the role of TLR4 signaling in ICH-induced inflammation. In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR. Activation of microglia was detected by immunohistochemistry, cytokines were measured by ELISA, MyD88, TRIF and NF-κB were measured by Western blot and EMSA, animal behavior was evaluated by animal behavioristics.

Results: Compared to WT mice, TLR4(-/-) mice had restrained ICH-induced brain damage showing in reduced cerebral edema and lower neurological deficit scores. Quantification of cytokines including IL-6, TNF-α and IL-1β and assessment of macrophage infiltration in perihematoma tissues from TLR4(-/-), MyD88(-/-) and TRIF(-/-) mice showed attenuated inflammatory damage after ICH. TLR4(-/-) mice also exhibited reduced MyD88 and TRIF expression which was accompanied by decreased NF-κB activity. This suggests that after ICH both MyD88 and TRIF pathways might be involved in TLR4-mediated inflammatory injury possibly via NF-κB activation. Exogenous hemin administration significantly increased TLR4 expression and microglial activation in cultures and also exacerbated brain injury in WT mice but not in TLR4(-/-) mice. Anti-TLR4 antibody administration suppressed hemin-induced microglial activation in cultures and in the mice model of ICH.

Conclusions: Our findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately increasing cytokine expression and inflammatory injury in ICH. Targeting TLR4 signaling may be a promising therapeutic strategy for ICH.

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TLR4 mRNA and protein expression after ICH. A) Real-time RT-PCR shows an upregulation of TLR4 mRNA expression in perihematoma tissues in WT mice (n = 6) on days 1, 2, 3, and 5 post ICH. B) TLR4 co-labeling with tubulin-positive neuron (arrows, n = 6). C) TLR4 co-labeling with GFAP-positive astrocytes (arrows, n = 6). D) TLR4 co-labeling with CD11b-positive microglia. Compared to sham control mice (n = 6), ICH induced significant increase in TLR4 protein. *P < 0.05, **P < 0.01 vs. sham control. Values (mean ± SD) are representative of two independent experiments. Bar = 20 μM.
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Figure 1: TLR4 mRNA and protein expression after ICH. A) Real-time RT-PCR shows an upregulation of TLR4 mRNA expression in perihematoma tissues in WT mice (n = 6) on days 1, 2, 3, and 5 post ICH. B) TLR4 co-labeling with tubulin-positive neuron (arrows, n = 6). C) TLR4 co-labeling with GFAP-positive astrocytes (arrows, n = 6). D) TLR4 co-labeling with CD11b-positive microglia. Compared to sham control mice (n = 6), ICH induced significant increase in TLR4 protein. *P < 0.05, **P < 0.01 vs. sham control. Values (mean ± SD) are representative of two independent experiments. Bar = 20 μM.

Mentions: To explore the role of TLR4 in inflammation after ICH, we first analyzed TLR4 mRNA expression on day 1, 2, 3, and 5 post-ICH using a real-time RT-PCR. The result showed that TLR4 mRNA was significantly upregulated in perihematoma brain tissues following ICH at all tested time-points when compared to sham-operated control mice (Figure 1A). TLR4 mRNA expression peaked on day 3 (P < 0. 01) and started decreasing on day 5 (P 0. 01). We next performed double-immunofluorescence staining to assess cellular expression of TLR4. Anti-tubilin, anti-GFAP, and anti-CD11b, respectively, were used as cell marker for neuron (Figure 1B), astrocyte (Figure 1C) and microglial cell (Figure 1D), respectively. We found that TLR4 expressed in all these cell types and the protein expression was significantly upregulated when compared to that of the sham group (P < 0.01; Figure 1B-D. Consistent with the TLR4 mRNA upregulation, TLR4 expression also peaked on day 3 and began decreasing on day 5 following ICH. Although both neuron and reactive astrocyte exhibited TLR4 expression, double-fluorescent staining showed that TLR4 predominantly expressed in CD11b-positive cells.


Heme activates TLR4-mediated inflammatory injury via MyD88/TRIF signaling pathway in intracerebral hemorrhage.

Lin S, Yin Q, Zhong Q, Lv FL, Zhou Y, Li JQ, Wang JZ, Su BY, Yang QW - J Neuroinflammation (2012)

TLR4 mRNA and protein expression after ICH. A) Real-time RT-PCR shows an upregulation of TLR4 mRNA expression in perihematoma tissues in WT mice (n = 6) on days 1, 2, 3, and 5 post ICH. B) TLR4 co-labeling with tubulin-positive neuron (arrows, n = 6). C) TLR4 co-labeling with GFAP-positive astrocytes (arrows, n = 6). D) TLR4 co-labeling with CD11b-positive microglia. Compared to sham control mice (n = 6), ICH induced significant increase in TLR4 protein. *P < 0.05, **P < 0.01 vs. sham control. Values (mean ± SD) are representative of two independent experiments. Bar = 20 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3344687&req=5

Figure 1: TLR4 mRNA and protein expression after ICH. A) Real-time RT-PCR shows an upregulation of TLR4 mRNA expression in perihematoma tissues in WT mice (n = 6) on days 1, 2, 3, and 5 post ICH. B) TLR4 co-labeling with tubulin-positive neuron (arrows, n = 6). C) TLR4 co-labeling with GFAP-positive astrocytes (arrows, n = 6). D) TLR4 co-labeling with CD11b-positive microglia. Compared to sham control mice (n = 6), ICH induced significant increase in TLR4 protein. *P < 0.05, **P < 0.01 vs. sham control. Values (mean ± SD) are representative of two independent experiments. Bar = 20 μM.
Mentions: To explore the role of TLR4 in inflammation after ICH, we first analyzed TLR4 mRNA expression on day 1, 2, 3, and 5 post-ICH using a real-time RT-PCR. The result showed that TLR4 mRNA was significantly upregulated in perihematoma brain tissues following ICH at all tested time-points when compared to sham-operated control mice (Figure 1A). TLR4 mRNA expression peaked on day 3 (P < 0. 01) and started decreasing on day 5 (P 0. 01). We next performed double-immunofluorescence staining to assess cellular expression of TLR4. Anti-tubilin, anti-GFAP, and anti-CD11b, respectively, were used as cell marker for neuron (Figure 1B), astrocyte (Figure 1C) and microglial cell (Figure 1D), respectively. We found that TLR4 expressed in all these cell types and the protein expression was significantly upregulated when compared to that of the sham group (P < 0.01; Figure 1B-D. Consistent with the TLR4 mRNA upregulation, TLR4 expression also peaked on day 3 and began decreasing on day 5 following ICH. Although both neuron and reactive astrocyte exhibited TLR4 expression, double-fluorescent staining showed that TLR4 predominantly expressed in CD11b-positive cells.

Bottom Line: In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR.Our findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately increasing cytokine expression and inflammatory injury in ICH.Targeting TLR4 signaling may be a promising therapeutic strategy for ICH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Daping Hospital, Third Military Medical University, Yuzhong District, Chongqing, China.

ABSTRACT

Background: Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced neurological deficits; however, the signaling pathways are not apparent by which the upstream cellular events trigger innate immune and inflammatory responses that contribute to neurological impairments. Toll-like receptor 4 (TLR4) plays a role in inflammatory damage caused by brain disorders.

Methods: In this study, we investigate the role of TLR4 signaling in ICH-induced inflammation. In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR. Activation of microglia was detected by immunohistochemistry, cytokines were measured by ELISA, MyD88, TRIF and NF-κB were measured by Western blot and EMSA, animal behavior was evaluated by animal behavioristics.

Results: Compared to WT mice, TLR4(-/-) mice had restrained ICH-induced brain damage showing in reduced cerebral edema and lower neurological deficit scores. Quantification of cytokines including IL-6, TNF-α and IL-1β and assessment of macrophage infiltration in perihematoma tissues from TLR4(-/-), MyD88(-/-) and TRIF(-/-) mice showed attenuated inflammatory damage after ICH. TLR4(-/-) mice also exhibited reduced MyD88 and TRIF expression which was accompanied by decreased NF-κB activity. This suggests that after ICH both MyD88 and TRIF pathways might be involved in TLR4-mediated inflammatory injury possibly via NF-κB activation. Exogenous hemin administration significantly increased TLR4 expression and microglial activation in cultures and also exacerbated brain injury in WT mice but not in TLR4(-/-) mice. Anti-TLR4 antibody administration suppressed hemin-induced microglial activation in cultures and in the mice model of ICH.

Conclusions: Our findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately increasing cytokine expression and inflammatory injury in ICH. Targeting TLR4 signaling may be a promising therapeutic strategy for ICH.

Show MeSH
Related in: MedlinePlus