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 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.

Ozone Enhances Pulmonary Innate Immune Response to a Toll-Like Receptor–2 Agonist

“…Williams and colleagues identified 400 differentially expressed genes (greater than twofold) in BALB/c mice 3 hours after ozone exposure, and also identified a number of markers of inflammation as the most affected genes (44). Bauer and colleagues profiled C3H/ HeJ and C3H/HeOuJ mice to identify ozone candidate genes downstream of TLR4, and demonstrated a role for heat shock protein-70 (32). Our analysis of gene expression in the lung after ozone exposure differs from these previously published studies because it focused on the priming effect of ozone on innate immunity.…”

“…Previously, the increase in the response of TLR4 to ozone alone was determined to be a result of the ozone-induced release of endogenous ligands (hyaluronan and heat-shock proteins) that stimulate TLR4, resulting in the migration of TLR4 to the surface of the cell (31). A subsequent gene expression profiling study by another group showed that heat-shock protein-70 is an effector molecule downstream of TLR4, and is involved in the regulation of ozone-induced lung inflammation by triggering pathways similar to those of TLR4 (32). This is likely a general mechanism that acts on all TLRs and accounts for the increases of TLR1 and TLR2 in response to ozone alone in the present study.…”

“…Four published studies assessed the genomic transcription profiles of murine lung tissue after ozone exposure (26,32,44). Park and colleagues profiled lung tissue from C57BL/6 mice exposed to 2 ppm ozone for 3 hours, either 4 or 24 hours after exposure, but focused their analysis primarily on inflammatory markers (26).…”

Impact of air pollution on lung inflammation and the role of Toll-like receptors

“…These two transcription factors induce upregulation of effector molecules and interferon (IFN)-inducible genes, such as IFN-gamma-induced protein 1 and IFN-β. 81,84,87 TLR signaling can be initiated through the interaction of numerous exogenous and endogenous ligands specific to a wide variety of TLRs. As detailed in Table 1, exogenous ligands include viruses and cell wall components of Grampositive and Gram-negative bacteria such as bacterial endotoxin/lipopolysaccharide, a well known inflammatory agent.…”

“…87,94 However, the gaseous nature of O 3 means that an alternate mechanism is likely responsible for TLR involvement in O 3 -induced inflammation. Although TLR2 and TLR4 have been studied as noted in the aforementioned studies, future research is needed to define the role of the remaining TLRs (TLR1, TLR3, TLR5-12) in air pollution-induced injury and disease.…”