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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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Declined neurologic deficits and inflammation in MyD88 −/− and TRIF−/− mice. Compared to WT mice, both MyD88−/− and TRIF−/− mice had decreased brain water content (A), NDS (B), (C) Both MyD88−/− and TRIF−/− mice showed decreased protein levels of IL-6, TNF-α, and IL-1β, and (D) immunohistochemistry of CD68 showed decreased macrophage infiltration in the perihematoma region of MyD88−/− and TRIF−/− brain on day3 post-ICH. **P < 0.01 vs. sham group; ##P < 0. 01 vs. WT group. $$P < 0.01 vs. WT group, Bar = 50 μM in D. Values (mean ± SD, n = 3 for each group) are representative of three independent experiments.
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Figure 3: Declined neurologic deficits and inflammation in MyD88 −/− and TRIF−/− mice. Compared to WT mice, both MyD88−/− and TRIF−/− mice had decreased brain water content (A), NDS (B), (C) Both MyD88−/− and TRIF−/− mice showed decreased protein levels of IL-6, TNF-α, and IL-1β, and (D) immunohistochemistry of CD68 showed decreased macrophage infiltration in the perihematoma region of MyD88−/− and TRIF−/− brain on day3 post-ICH. **P < 0.01 vs. sham group; ##P < 0. 01 vs. WT group. $$P < 0.01 vs. WT group, Bar = 50 μM in D. Values (mean ± SD, n = 3 for each group) are representative of three independent experiments.

Mentions: First, we evaluated the extent of ICH-induced brain damage and inflammatory responses in MyD88−/− and TRIF−/− mice. We found that compared to WT mice, both MyD88−/− and TRIF−/− mice had significantly less impairments after ICH, indicated by decreased cerebral water content (Figure 3A) and lower neurological deficit score (Figure 3B). Furthermore, we quantified inflammatory cytokine expression and macrophage infiltration in perihematoma tissues of these mice. ELISA showed that 3 days after ICH WT mice displayed dramatic increase in TNF-α, IL-1β, and IL-6 levels compared to the sham group. On the contrary, both MyD88−/− and TRIF−/− exhibited significant lower expression of these cytokines compared to WT mice, although significantly higher than the sham control (Figure 3C). This suggested that depletion of either MyD88 or TRIF dramatically suppressed ICH-induced cytokine expression. Reduced inflammation in these mice was also supported by the observation that MyD88−/− and TRIF−/− had significantly lower macrophage infiltration compared to WT mice, as shown in reduced CD68-positive cells (P < 0.01) in perihematoma tissues (Figure 3D). Taken together, these findings indicate that both MyD88 and TRIF signaling pathway might be involved in the TLR4-mediated inflammatory process following ICH.


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)

Declined neurologic deficits and inflammation in MyD88 −/− and TRIF−/− mice. Compared to WT mice, both MyD88−/− and TRIF−/− mice had decreased brain water content (A), NDS (B), (C) Both MyD88−/− and TRIF−/− mice showed decreased protein levels of IL-6, TNF-α, and IL-1β, and (D) immunohistochemistry of CD68 showed decreased macrophage infiltration in the perihematoma region of MyD88−/− and TRIF−/− brain on day3 post-ICH. **P < 0.01 vs. sham group; ##P < 0. 01 vs. WT group. $$P < 0.01 vs. WT group, Bar = 50 μM in D. Values (mean ± SD, n = 3 for each group) are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3344687&req=5

Figure 3: Declined neurologic deficits and inflammation in MyD88 −/− and TRIF−/− mice. Compared to WT mice, both MyD88−/− and TRIF−/− mice had decreased brain water content (A), NDS (B), (C) Both MyD88−/− and TRIF−/− mice showed decreased protein levels of IL-6, TNF-α, and IL-1β, and (D) immunohistochemistry of CD68 showed decreased macrophage infiltration in the perihematoma region of MyD88−/− and TRIF−/− brain on day3 post-ICH. **P < 0.01 vs. sham group; ##P < 0. 01 vs. WT group. $$P < 0.01 vs. WT group, Bar = 50 μM in D. Values (mean ± SD, n = 3 for each group) are representative of three independent experiments.
Mentions: First, we evaluated the extent of ICH-induced brain damage and inflammatory responses in MyD88−/− and TRIF−/− mice. We found that compared to WT mice, both MyD88−/− and TRIF−/− mice had significantly less impairments after ICH, indicated by decreased cerebral water content (Figure 3A) and lower neurological deficit score (Figure 3B). Furthermore, we quantified inflammatory cytokine expression and macrophage infiltration in perihematoma tissues of these mice. ELISA showed that 3 days after ICH WT mice displayed dramatic increase in TNF-α, IL-1β, and IL-6 levels compared to the sham group. On the contrary, both MyD88−/− and TRIF−/− exhibited significant lower expression of these cytokines compared to WT mice, although significantly higher than the sham control (Figure 3C). This suggested that depletion of either MyD88 or TRIF dramatically suppressed ICH-induced cytokine expression. Reduced inflammation in these mice was also supported by the observation that MyD88−/− and TRIF−/− had significantly lower macrophage infiltration compared to WT mice, as shown in reduced CD68-positive cells (P < 0.01) in perihematoma tissues (Figure 3D). Taken together, these findings indicate that both MyD88 and TRIF signaling pathway might be involved in the TLR4-mediated inflammatory process following ICH.

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