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

J Neuroinflammation. 2012 Mar 6;9:46. doi: 10.1186/1742-2094-9-46.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Vesicular Transport / deficiency
  • Adaptor Proteins, Vesicular Transport / metabolism*
  • Animals
  • Animals, Newborn
  • Antibodies, Monoclonal / therapeutic use
  • Cells, Cultured
  • Cerebral Cortex / cytology
  • Cerebral Hemorrhage / complications*
  • Cerebral Hemorrhage / drug therapy
  • Cytokines / metabolism
  • Electrophoretic Mobility Shift Assay
  • Enzyme-Linked Immunosorbent Assay
  • Heme / pharmacology*
  • Inflammation / etiology
  • Lipopolysaccharides / pharmacology
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Microglia / drug effects
  • Myeloid Differentiation Factor 88 / deficiency
  • Myeloid Differentiation Factor 88 / metabolism*
  • NF-kappa B / metabolism
  • Neurologic Examination
  • RNA, Messenger / metabolism
  • Signal Transduction / physiology*
  • Statistics, Nonparametric
  • Time Factors
  • Toll-Like Receptor 4 / deficiency
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / immunology
  • Toll-Like Receptor 4 / metabolism*
  • Up-Regulation / drug effects*
  • Up-Regulation / genetics
  • Up-Regulation / physiology


  • Adaptor Proteins, Vesicular Transport
  • Antibodies, Monoclonal
  • Cytokines
  • Lipopolysaccharides
  • Myd88 protein, mouse
  • Myeloid Differentiation Factor 88
  • NF-kappa B
  • RNA, Messenger
  • TICAM-1 protein, mouse
  • Tlr4 protein, mouse
  • Toll-Like Receptor 4
  • Heme