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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−/− mice displayed attenuated brain injury and decreased neuroinflammation. TLR4−/− had decreased brain water content (A, n = 3) and decreased NDS (B, n = 3). ELISA showed that TLR4−/− mice had a marked decrease in release of IL-6, TNF-α, and IL-1β (C, n = 3) on day 3 post-ICH. D) Immunohistochemistry of CD68 showed decreased macrophage infiltration in TLR4−/− mice on day 3 post-ICH (n = 3). **P < 0.01 vs. sham group; ##P < 0. 01 vs. WT group; Bar = 50 μM in D. Values (mean ± SD) are representative of three independent experiments.
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Figure 2: TLR4−/− mice displayed attenuated brain injury and decreased neuroinflammation. TLR4−/− had decreased brain water content (A, n = 3) and decreased NDS (B, n = 3). ELISA showed that TLR4−/− mice had a marked decrease in release of IL-6, TNF-α, and IL-1β (C, n = 3) on day 3 post-ICH. D) Immunohistochemistry of CD68 showed decreased macrophage infiltration in TLR4−/− mice on day 3 post-ICH (n = 3). **P < 0.01 vs. sham group; ##P < 0. 01 vs. WT group; Bar = 50 μM in D. Values (mean ± SD) are representative of three independent experiments.

Mentions: To further assess the role of TLR4 in the inflammatory cascade in response to ICH, we employed TLR4 knockout mice (TLR4−/−). We induced ICH in TLR4−/− mice and then evaluated brain damage and neurological impairments 3 days after ICH when we observed the maximum upregulation of TLR4 in WT mice with ICH. Compared to WT mice, TLR4−/− mice showed a significantly lower brain water content (P < 0.01, n = 3, Figure 2A) and neurological deficit scores (NDS) (P < 0.01, n = 3, Figure 2B). These suggested that TLR4−/− mice had less extent of neurological impairments after ICH. Next, we detected inflammatory cytokines expression in TLR4−/− mice after ICH. ELISA showed that TLR4−/− mice exhibited significantly lower (P < 0.01, n = 3) expression of IL-6, TNF-α, and IL-1β in perihematoma tissues 3 days after ICH (Figure 2C). Finally, we examined macrophage infiltration in perihematoma tissues and found that the extent of macrophage infiltration is dramatically decreased (P < 0.01, n = 3, Figure 2D) as shown in decreased CD68-positive cells (arrows in Figure 2D) in 20 consecutive high-power fields (20 HPFs) obtained from the perihematoma region.


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−/− mice displayed attenuated brain injury and decreased neuroinflammation. TLR4−/− had decreased brain water content (A, n = 3) and decreased NDS (B, n = 3). ELISA showed that TLR4−/− mice had a marked decrease in release of IL-6, TNF-α, and IL-1β (C, n = 3) on day 3 post-ICH. D) Immunohistochemistry of CD68 showed decreased macrophage infiltration in TLR4−/− mice on day 3 post-ICH (n = 3). **P < 0.01 vs. sham group; ##P < 0. 01 vs. WT group; Bar = 50 μM in D. Values (mean ± SD) are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: TLR4−/− mice displayed attenuated brain injury and decreased neuroinflammation. TLR4−/− had decreased brain water content (A, n = 3) and decreased NDS (B, n = 3). ELISA showed that TLR4−/− mice had a marked decrease in release of IL-6, TNF-α, and IL-1β (C, n = 3) on day 3 post-ICH. D) Immunohistochemistry of CD68 showed decreased macrophage infiltration in TLR4−/− mice on day 3 post-ICH (n = 3). **P < 0.01 vs. sham group; ##P < 0. 01 vs. WT group; Bar = 50 μM in D. Values (mean ± SD) are representative of three independent experiments.
Mentions: To further assess the role of TLR4 in the inflammatory cascade in response to ICH, we employed TLR4 knockout mice (TLR4−/−). We induced ICH in TLR4−/− mice and then evaluated brain damage and neurological impairments 3 days after ICH when we observed the maximum upregulation of TLR4 in WT mice with ICH. Compared to WT mice, TLR4−/− mice showed a significantly lower brain water content (P < 0.01, n = 3, Figure 2A) and neurological deficit scores (NDS) (P < 0.01, n = 3, Figure 2B). These suggested that TLR4−/− mice had less extent of neurological impairments after ICH. Next, we detected inflammatory cytokines expression in TLR4−/− mice after ICH. ELISA showed that TLR4−/− mice exhibited significantly lower (P < 0.01, n = 3) expression of IL-6, TNF-α, and IL-1β in perihematoma tissues 3 days after ICH (Figure 2C). Finally, we examined macrophage infiltration in perihematoma tissues and found that the extent of macrophage infiltration is dramatically decreased (P < 0.01, n = 3, Figure 2D) as shown in decreased CD68-positive cells (arrows in Figure 2D) in 20 consecutive high-power fields (20 HPFs) obtained from the perihematoma region.

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