title: Identification of candidate genes downstream of TLR4 signaling after ozone exposure in mice: a role for heat-shock protein 70.
abstract: BACKGROUND
  Toll-like receptor 4 (TLR4) is involved in ozone (O3)-induced pulmonary hyperpermeability and inflammation, although the downstream signaling events are unknown.
  
  
  OBJECTIVE
  The aims of our study were to determine the mechanism through which TLR4 modulates O3-induced pulmonary responses and to use transcriptomics to determine potential TLR4 effector molecules.
  
  
  METHODS
  C3H/HeJ (HeJ; Tlr4 mutant) and C3H/HeOuJ (OuJ; Tlr4 normal) mice were exposed continuously to 0.3 ppm O3 or filtered air for 6, 24, 48, or 72 hr. We assessed inflammation using bronchoalveolar lavage and molecular analysis by mRNA microarray, quantitative RT-PCR (real-time polymerase chain reaction), immunoblots, immunostaining, and ELISAs (enzyme-linked immunosorbent assays). B6-Hspa1a/Hspa1btm1Dix/NIEHS (Hsp70-/-) and C57BL/6 (B6; Hsp70+/+ wild-type control) mice were used for candidate gene validation studies.
  
  
  RESULTS
  O3-induced TLR4 signaling occurred through myeloid differentiation protein 88 (MyD88)-dependent and -independent pathways in OuJ mice and involved multiple downstream pathways. Genomewide transcript analyses of lungs from air- and O3-exposed HeJ and OuJ mice identified a cluster of genes that were significantly up-regulated in O3-exposed OuJ mice compared with O3-exposed HeJ mice or air-exposed controls of both strains; this cluster included genes for heat-shock proteins (e.g., Hspa1b, Hsp70). Moreover, O3-induced inflammation, MyD88 up-regulation, extracellular-signal-related kinase-1/2 (ERK1/2) and activator protein-1 (AP-1) activation, and kerotinocyte-derived chemokine (KC) protein content were significantly reduced in Hspa1a/Hspa1btm1Dix (Hsp70-/-) compared with Hsp70+/+ mice (p < 0.05).
  
  
  CONCLUSIONS
  These studies suggest that HSP70 is an effector molecule downstream of TLR4 and is involved in the regulation of O3-induced lung inflammation by triggering similar pathways to TLR4. These novel findings may have therapeutic and preventive implications for inflammatory diseases resulting from environmental exposures.
authors.affiliation: Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA. alison.bauer@ucdenver.edu
authors.forename: Alison K
authors.initials: AK
authors.lastname: Bauer
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Rondini
authors.forename: Kristin A
authors.initials: KA
authors.lastname: Hummel
authors.forename: Laura M
authors.initials: LM
authors.lastname: Degraff
authors.forename: Christopher
authors.initials: C
authors.lastname: Walker
authors.forename: Anne E
authors.initials: AE
authors.lastname: Jedlicka
authors.forename: Steven R
authors.initials: SR
authors.lastname: Kleeberger
mesh_terms: D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D010126:Ozone / Q000633:toxicity*; D011014:Pneumonia / Q000139:chemically induced / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000378:metabolism*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D053594:Myeloid Differentiation Factor 88; D051197:Toll-Like Receptor 4; D010126:Ozone