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. 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 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.R esults: 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.