title: LPS preconditioning redirects TLR signaling following stroke: TRIF-IRF3 plays a seminal role in mediating tolerance to ischemic injury.
abstract: BACKGROUND
  Toll-like receptor 4 (TLR4) is activated in response to cerebral ischemia leading to substantial brain damage. In contrast, mild activation of TLR4 by preconditioning with low dose exposure to lipopolysaccharide (LPS) prior to cerebral ischemia dramatically improves outcome by reprogramming the signaling response to injury. This suggests that TLR4 signaling can be altered to induce an endogenously neuroprotective phenotype. However, the TLR4 signaling events involved in this neuroprotective response are poorly understood. Here we define several molecular mediators of the primary signaling cascades induced by LPS preconditioning that give rise to the reprogrammed response to cerebral ischemia and confer the neuroprotective phenotype.
  
  
  METHODS
  C57BL6 mice were preconditioned with low dose LPS prior to transient middle cerebral artery occlusion (MCAO). Cortical tissue and blood were collected following MCAO. Microarray and qtPCR were performed to analyze gene expression associated with TLR4 signaling. EMSA and DNA binding ELISA were used to evaluate NFκB and IRF3 activity. Protein expression was determined using Western blot or ELISA. MyD88-/- and TRIF-/- mice were utilized to evaluate signaling in LPS preconditioning-induced neuroprotection.
  
  
  RESULTS
  Gene expression analyses revealed that LPS preconditioning resulted in a marked upregulation of anti-inflammatory/type I IFN-associated genes following ischemia while pro-inflammatory genes induced following ischemia were present but not differentially modulated by LPS. Interestingly, although expression of pro-inflammatory genes was observed, there was decreased activity of NFκB p65 and increased presence of NFκB inhibitors, including Ship1, Tollip, and p105, in LPS-preconditioned mice following stroke. In contrast, IRF3 activity was enhanced in LPS-preconditioned mice following stroke. TRIF and MyD88 deficient mice revealed that neuroprotection induced by LPS depends on TLR4 signaling via TRIF, which activates IRF3, but does not depend on MyD88 signaling.
  
  
  CONCLUSIONS
  Our results characterize several critical mediators of the TLR4 signaling events associated with neuroprotection. LPS preconditioning redirects TLR4 signaling in response to stroke through suppression of NFκB activity, enhanced IRF3 activity, and increased anti-inflammatory/type I IFN gene expression. Interestingly, this protective phenotype does not require the suppression of pro-inflammatory mediators. Furthermore, our results highlight a critical role for TRIF-IRF3 signaling as the governing mechanism in the neuroprotective response to stroke.
authors: Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, 97239, USA.
authors: Keri B
authors: KB
authors: Vartanian
authors: Susan L
authors: SL
authors: Stevens
authors: Brenda J
authors: BJ
authors: Marsh
authors: Rebecca
authors: R
authors: Williams-Karnesky
authors: Nikola S
authors: NS
authors: Lessov
authors: Mary P
authors: MP
authors: Stenzel-Poore
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000276:immunology*; D000818:Animals; D002545:Brain Ischemia / Q000097:blood / Q000276:immunology / Q000473:pathology; D018925:Chemokines / Q000097:blood / Q000276:immunology; D016207:Cytokines / Q000097:blood / Q000276:immunology; D020869:Gene Expression Profiling; D006801:Humans; D020244:Infarction, Middle Cerebral Artery; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000276:immunology*; D019194:Ischemic Preconditioning; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D046228:Microarray Analysis; D016328:NF-kappa B / Q000276:immunology; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D020521:Stroke / Q000097:blood / Q000276:immunology / Q000473:pathology; D051197:Toll-Like Receptor 4 / Q000276:immunology*
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D018925:Chemokines; D016207:Cytokines; D050838:Interferon Regulatory Factor-3; D008070:Lipopolysaccharides; D016328:NF-kappa B; D051197:Toll-Like Receptor 4; C494233:Irf3 protein, mouse; C476286:Ticam1 protein, mouse