Ozone (O3)-induced Pulmonary Injury and Inflammation
Although regulatory actions have reduced levels in recent years, air pollution remains a significant public health concern worldwide. One of the major components of air pollution is the potent oxidant O3. O3 has important detrimental health effects on humans, although the mechanisms of its effects on cardiopulmonary health are not completely understood. Acute O3 exposures cause pulmonary function decrements, school absenteeism in children, injury and inflammation, cardiovascular events, and disease exacerbation, while chronic exposures to O3 have been associated with increased incidence of asthma, diminished lifespan, and increased mortality due to cardiovascular and pulmonary disease, as well as reproductive effects. Moreover, the numbers of people estimated to be exposed to potentially harmful levels of O3 in the U.S. have reached as high as 40 percent of the total population, thus underscoring the importance of understanding the mechanisms of O3 toxicity and developing strategies to prevent exposure and disease.
Importantly, some subpopulations are thought to be at increased risk to the harmful effects of O3. Preexisting cardiopulmonary disease (e.g., asthma, COPD, atrial fibrillation), age (children and the elderly), immune deficits, nutrition, obesity, and genetic background are thought to contribute to enhanced response to toxic effects of O3. Using genome-wide analyses, we found linkage of O3-induced lung hyperpermeability and inflammation QTLs to chromosomes 4 and 17, respectively ( Kleeberger et al, 1997 ; Kleeberger et al, 2000 ).
Ongoing research in the EGG has continued to focus on the hypotheses that gene(s) within the chromosome 4 QTL [e.g. toll-like receptor 4 (Tlr4)] and the chromosome 17 QTL [e.g. tumor necrosis factor-α (Tnf)] modulate differential susceptibility to O3-induced injury and inflammation. The specific aims of this project are to:
- confirm and reduce the length of chromosome 17 QTL (Inf2) to further characterize its genetic contribution to inflammation induced by subacute O3 (0.3 ppm) exposure
- identify signaling mechanisms through which Tlr4 controls O3-induced inflammation
- test whether functional polymorphisms in validated, candidate susceptibility genes associate with O3-induced phenotypes in human subjects
Ongoing Projects in the Laboratory:
Role of Inf2 in O3-induced inflammation
Previous studies in our group used positional cloning to identify a QTL on chromosome 17 (Inf2) that accounts for a significant portion of the genetic variance in susceptibility to O3-induced inflammation in inbred mice ( Kleeberger et al, 1997 ). In subsequent studies that used strains of mice that were congenic for overlapping lengths of Inf2, we confirmed the significance of Inf2, and found that a reduced length of Inf2 (34.38–35.34 Mbp) accounts for a major portion of O3-induced inflammation ( Bauer et al, 2009 ; Figure 1). Genes within the reduced Inf2 included H2-Eb1, H2-Eb2, Hspa1b, Btnl1, Tnf, and Lta. Because of the prevalence of MHC class II genes in Inf2, we tested their role as candidate susceptibility genes for O3-induced inflammation. Relative to wild-type mice, we found significantly greater O3-induced inflammation and mRNA expression of Tnf and Lta in H2Abl-Ea-/- mice which are deficient in MHC class II genes H2-Ab1, -Aa, -Eb1, -Eb2 and -Ea. These studies implicate histocompatibility genes in modulation of inflammation induced by O3.
Our laboratory also investigated the signaling pathway through which TNF-α modulates O3-induced pulmonary inflammation ( Cho et al, 2007 ; Figure 2). We subsequently designed another study to further investigate the role of the Tnf cluster in the model and found that inflammation was significantly reduced in Lta-/- and Lta/Tnf/Ltb-/- cluster mice compared with wild-type controls following O3. MHC class II gene expression was also significantly different between strains deficient in the Tnf cluster. These are the first studies to conclusively demonstrate a role for MHC class II genes and the entire Tnf cluster in oxidant-induced lung inflammation and provide evidence supporting a susceptibility ''superlocus'' on chromosome 17 (i.e. Inf2) that may have implications in other oxidant stress-induced diseases. Future investigations will be directed to understand the mechanisms through which the MHC class II genes modulate inflammation and interact with other genes in the Inf2 QTL. Other genes within Inf2 (e.g. Lta and Ltb) will also be investigated for their roles in susceptibility to O3-induced inflammation.
Role of Tlr4 in O3-induced inflammation
Previous studies in our group identified Tlr4 as a susceptibility gene in a chromosome 4 QTL for O3-induced lung hyperpermeability ( Kleeberger et al, 2000 ). Further, relative to C3H/HeOuJ (OuJ) mice, inflammation and hyperpermeability responses to O3 were significantly reduced in HeJ mice, which differ from OuJ only at a missense mutation in Tlr4. This was the first study to implicate Tlr4 in O3-induced injury. Importantly, epidemiological investigations have subsequently found that Tlr4 influences susceptibility to adverse effects of traffic-related air pollution on childhood asthma.
To investigate mechanisms of Tlr4-mediated hyperpermeability responses to O3, we used genome-wide mRNA expression analyses to define gene expression profile differences between HeJ and OuJ mice exposed to 0.3 ppm O3 and air. A number of transcript profiles differed between strains after O3, but one remarkable profile included transcripts upregulated in HeJ compared to OuJ mice, and included the gene Marco (macrophage receptor with a collagenous structure), a class A scavenger receptor (SRA) which functions in innate immune defense against inhaled pathogens, bacteria, and oxidized lipids. With Les Kobzik, Ph.D., (Harvard), we found that O3 significantly up-regulated MARCO on the surface of alveolar macrophages (AM) from Marco+/+ mice, and caused greater lung injury in Marco-/- compared to Marco+/+ mice. Instillation (IT) of 5β,6β-epoxycholesterol or 1palmitoyl–2-(9'-oxo-nonanoyl)–glycerophosphocholine (oxidized, pro-inflammatory lipids generated by O3) into Marco-/- mice caused inflammation, but had no effect in Marco+/+ mice ( Dahl et al, 2007 ). We found greater AM uptake in vitro of 5β,6β-epoxycholesterol in Marco+/+ compared to Marco-/- mice, consistent with SRA function in binding oxidized lipids. Results thus identified a novel, Tlr4-dependent function for SRAs in innate immune defense by scavenging pro-inflammatory oxidized lipids from lung lining fluids and therefore decreasing inflammation (Figure 3).
Tlr4 is activated by binding of ligand(s), and recruits adaptor molecules. To identify pathways through which Tlr4 mediates O3-induced lung inflammation and injury, we found transcript levels of adaptor molecules MyD88 and TRIF, and transcription factors NFκB (p65 subunit) and AP-1 (phospho (p) c-jun) proteins were up-regulated in OuJ mice compared to HeJ mice after O3 ( Bauer et al, 2011 ). Furthermore, KC (CXCL1) transcript and protein levels were significantly elevated in OuJ compared to HeJ mice after O3. Analyses of mRNA array data identified a cluster of 24 gene transcripts that were significantly greater in OuJ mice compared to HeJ mice after O3. GO analysis identified major functional categories as protein folding, response to heat and stress, response to temperature stimulus, chaperone, and response to protein stimulus.
Five heat shock protein genes in the KEGG antigen processing and presentation pathway (Hspa1b, Hsp90aa1, Hsp90ab1, Hspa5, Hspa8) were particularly notable. Interestingly, Hspa1b is included in the reduced Inf2 locus (see above). Moverover, O3-induced expression of HSP70 protein was significantly increased in OuJ compared to HeJ mice. Moreover, O3-induced inflammation and MYD88 upregulation, ERK1/2 and AP-1 activation, and KC protein content were reduced in Hspa1a/Hspa1btm1Dix (Hsp70-/-) compared to Hsp70+/+ mice. Our studies demonstrate novel Tlr4 effector genes and suggest that these networks, including HSPs (e.g. Hspa1b, Hsp90ab1), should be investigated concerning their role in susceptibility to O3-induced lung inflammation in humans. We also provided in vivo evidence that HSP70 triggers activation of multiple signaling pathways downstream of Tlr4 (Figure 4).
- Role of pro-inflammatory (IL12) and anti-inflammatory (IL10) cytokines and chemokines (Cx3CR1) in O3-induced inflammation
- Mechanisms of O3-induced exacerbation of allergic responses in the lung
- Association of candidate susceptibility gene polymorphisms in O3-exposed human subjects (collaboration with David Peden, Ph.D., and colleagues, UNC)
- Genetic and genomic mechanisms of response to repeated O3 exposure in juvenile rhesus monkey (collaboration with Ed Postlethwait, Ph.D., and colleagues at U Alabama, Birmingham and UC, Davis)