TLR4 signaling in response to O3.
We found significantly greater mean total protein (at 24, 48, and 72 hr) and numbers of BALF PMNs (at 48 hr) in O3
-exposed OuJ mice compared with OuJ controls and both air- and O3
-exposed HeJ mice (), as described previously (Kleeberger et al. 2000
). Epithelial cell numbers in BALF were also significantly different (24 and 72 hr) between OuJ and HeJ strains [see Supplemental Material, Table 3 (doi:10.1289/ehp.1003326)]. After 24 hr O3
exposure, transcript levels of the adaptor molecules Myd88
were up-regulated in OuJ mice compared with the three other treatment groups (OuJ controls and both air- and O3
-exposed HeJ mice; ), similar to changes found previously for Tlr4
mRNA expression (Kleeberger et al. 2000
). Transcription factors NFκB (p65 subunit) and AP-1 (p-c-Jun) were likewise significantly higher after 24 hr O3
exposure in OuJ mice compared with O3
-exposed HeJ mice or air-exposed controls of both strains(; p
Figure 1 Differential response (mean ± SE) to air or 0.3 ppm O3 in lungs of OuJ and HeJ mice. Total protein concentration (a marker of lung permeability; A) and number of PMNs recovered (B) from the BALF (n = 3–7 mice/group, repeated once). Expression (more ...)
Three primary MAPKs—ERK1/2 (extracellular-signal–related kinase-1/2), JNK (c-Jun N-terminal kinase), and p38—are involved in response to O3
(Cho et al. 2007
) and LPS-mediated TLR4 signaling in the lung (Chanteux et al. 2007
; Fang et al. 2007
). After 24 hr O3
exposure, ERK1/2 and p38 proteins were significantly elevated in OuJ mice compared with OuJ controls and both air- and O3
-exposed HeJ mice, but after 48 hr of exposure ERK1/2 and p38 were significantly increased in O3
-exposed HeJ mice compared with O3
-exposed OuJ mice or controls of both strains (). JNK activity was unchanged in both strains after O3
exposure (data not shown). Furthermore, neutrophil chemoattractant KC (CXCL1) protein levels were significantly elevated in OuJ compared with HeJ mice after 24 and 48 hr O3
exposure (); Kc
transcript levels were also elevated after 24 hr O3
(data not shown). We found no effects of O3
on protein levels of MIP-2, another neutrophil chemoattractant, in either strain (data not shown).
Figure 2 MAPKs and chemokine pathways downstream of TLR4 in OuJ and HeJ mice in response to air or 0.3 ppm O3. Phosphorylated MAPK activity [pERK1/2 (A) and pp38 (B)] detected by immunoblotting; proteins were normalized to the unphosphorylated form of the same (more ...) Transcriptomic analysis to identify TLR4 effector genes. k
-Means clustering identified five clusters after the initial filtering, which determined 200 transcripts with significant interactions for strain and time with 2-fold changes in gene expression (p
< 0.05; data not shown). We focused subsequent analyses on 39 genes that were distributed in three distinct cluster patterns [clusters 2, 4, and 5; see Supplemental Material, Excel Table 1A–C (doi:10.1289/ehp.1003326)]. In cluster 2 (24 genes; see Supplemental Material, Table 4A and Supplemental Material, ), expression of transcripts was significantly greater in OuJ mice after 24 and 48 hr O3
exposure compared with air-exposed controls of both strains and O3
-exposed HeJ mice. Analysis using the Database for Annotation, Visualization, and Integrated Discovery [DAVID (Huang et al. 2009a
); see Supplemental Material, Excel Table 1C] identified protein folding, response to heat and stress, response to temperature stimulus, chaperone, and response to protein stimulus (p
-values ranged from 4.35 × 10–9
to 4.0 × 10–5
) as major functional categories. Five heat-shock proteins in the antigen processing and presentation KEGG pathway [Hspa1b
U; (Kyoto Encyclopedia of Genes and Genomes; Kanehisa Laboratories 2010
)] were particularly notable (see Supplemental Material, ).
In HeJ mice, cluster 4 transcripts [10 genes; see Supplemental Material, Table 4B (doi:10.1289/ehp.1003326)] were expressed at significantly higher levels in mice exposed to O3 for 24 hr compared with air-exposed controls, and most genes were decreased after 48 hr. In contrast, we found minimal changes in OuJ mice (data not shown). DAVID analysis categorized 6 of the 10 genes in this cluster (e.g., Cdkn1, Mt2, Mt1) into metal-binding, transition-metal ion-binding, zinc ion-binding, and cation-binding functions (p-values ranged from < 0.007 to < 0.044; for complete analysis, see Supplemental Material, Excel Table 1D). Cluster 5 (5 genes; see Supplemental Material, Table 4C) contained transcripts that were significantly greater in HeJ than in OuJ mice after 24 and 48 hr O3 exposure. Four of five of these genes are related to inflammation and immune response. DAVID analysis for cluster 5 (see Supplemental Material, Excel Table 1E) identified one functional category (secretion; p < 0.03) for three of these genes (Saa3, Cxcl5, and Timp1).
Candidate gene validation. To confirm some of the genes identified using expression profiling, we performed qRTPCR on genes from all three focus clusters in the same samples used for the microarray analysis [Supplemental Material, (doi:10.1289/ehp.1003326)]. Hspa1b mRNA expression did not significantly change after 48 or 72 hr O3 exposure in HeJ mice, in contrast to the array results (see Supplemental Material, ). Because a significant number of genes were identified in the heat-shock protein functional category, we focused on HSP70 (the protein encoded by Hspa1b) for further validation. In HeJ mice, HSP70 protein expression was not changed after O3 exposure. However, HSP70 protein expression was significantly elevated in O3-exposed OuJ mice compared with OuJ controls and both air- and O3-exposed HeJ mice (). HSP70 immunostaining confirmed up-regulation and localization in alveolar macrophages and epithelial cells in OuJ mice ().
Figure 3 Immunodetection of 70 HSP70 protein in lungs of OuJ and HeJ mice in response to 48 hr exposure to air or 0.3 ppm O3. (A) HSP70 expression detected by immunoblotting; HSP70 protein was normalized to β-actin and is expressed as fold change relatative (more ...)
HSP70 involvement in O3-induced responses. To further investigate the role of HSP70 in this model, we exposed Hsp70–/– and Hsp70+/+ mice to air and 0.3 ppm O3. Relative to Hsp70+/+ mice, BALF total protein (at 24 and 48 hr), PMNs (at 24 and 48 hr), and macrophages (at 48 and 72 hr) were significantly reduced in Hsp70–/– mice after O3 exposure (). Epithelial cell numbers were not significantly different between strains [see Supplemental Material, Table 3 (doi:10.1289/ehp.1003326)]. Histopathology also demonstrated increased cellularity and thickening of the airways in Hsp70+/+ mice (data not shown). We found significantly increased Hspa1b gene expression in Hsp70+/+ mice after 48 hr of O3 compared with Hsp70+/+ controls (data not shown).
Figure 4 Inflammatory parameters measured in BALF from Hsp70–/– and Hsp+/+ mice after exposure to air or 0.3 ppm O3. (A) Total protein concentration. (B) Total number of PMNs. (C) Total number of macrophages in BAL from Hsp70–/– (more ...)
After 24 hr exposure, transcript levels
of Myd88 were significantly increased in O3-exposed Hsp70+/+ mice compared with O3-exposed Hsp70–/– mice and controls of both strains (). Trif mRNA expression was significantly higher in Hsp70+/+ mice than in Hsp70–/– mice (24 hr O3 exposure), but it was not significantly different from controls of either strain (). Importantly, Tlr4 was also significantly increased in the Hsp70+/+ but not Hsp70–/– mice after 6 and 24 hr O3 exposure (). NFκB p65 binding activity was significantly increased in both genotypes after 24 hr exposure to O3 compared with controls, but we found no significant differences between genotypes (). Binding activity of p-c-Jun was significantly increased in O3-exposed animals of both genotypes compared with controls, but was significantly higher in Hsp70+/+ mice than in Hsp70–/– mice (). ERK1/2 protein was also significantly elevated in O3-exposed Hsp70+/+ mice compared with Hsp70+/+ controls and both air- and O3-exposed Hsp70–/– mice (), whereas p38 was elevated in both strains after 24 hr exposure to O3 compared with controls (). JNK was unchanged in both strains (data not shown), similar to OuJ and HeJ mice. In the 24-hr exposure group, KC protein levels were significantly elevated in O3-exposed Hsp70+/+ mice compared with O3-exposed Hsp70–/– mice and air controls of both genotypes (; p < 0.05). In the 48-hr exposure group, KC levels in O3-exposed mice of both genotypes were significantly greater than those in respective air controls. We found a significant decrease in MIP-2 protein levels in O3-exposed Hsp70–/– mice compared with the corresponding controls in the 24-hr group, but MIP-2 levels were significantly increased in both genotypes after 48 hr of O3 exposure compared with air controls of both genotypes ().
Figure 5 TLR4 and adaptor mRNA expression and pathways downstream of TLR4, including transcription factors, MAPKs, and chemokines, in control and O3-exposed Hsp70+/+ and Hsp70–/– mice. Expression of Tlr4 (A), Myd88 (B), and Trif (C) transcripts (more ...)