Identification of Candidate Genes Downstream of TLR4 Signaling after Ozone Exposure in Mice: A Role for Heat-Shock Protein 70

BACKGROUND: Toll-like receptor 4 (TLR4) is involved in ozone (O-3)-induced pulmonary hyperpermeability and inflammation, although the downstream signaling events are unknown. OBJECTIVES: The aims of our study were to determine the mechanism through which TLR4 modulates O-3-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 O-3 or filtered air for 6, 24, 48, or 72 hr. We assessed inflammation using bronchoalveolar lavage and molecular analysis by mRNA micro array, quantitative RT-PCR (real-time polymerase chain reaction), immuno blots, 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: O-3-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 O-3-exposed HeJ and OuJ mice identified a cluster of genes that were significantly up-regulated in O-3-exposed OuJ mice compared with O-3-exposed HeJ mice or air-exposed controls of both strains; this cluster included genes for heat-shock proteins (e.g., Hspa1b, Hsp70). Moreover, O-3-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/Hspa1b(tm1Dix) (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 O-3-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.